JP2002526034A - Growth differentiation factor-11 - Google Patents

Abstract

  (57) [Summary] Has a transgene integrated into a chromosome in germ cells of a species selected from the group consisting of birds, cattle, sheep and pigs, which disrupts the production and / or activity of growth differentiation factor-11 (GDF-11) A transgenic non-human animal is disclosed. Also disclosed are methods for producing such animals, and methods for treating animals, including humans, using antibodies or antisense to GDF-11. Animals treated in this manner exhibit characteristics of increased muscle and bone tissue.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application claims priority with US patent application Ser. No. 08/2, filed Jul. 8, 1994, now abandoned.
U.S. patent application Ser.No. 08/706, filed Sep. 3, 1996, which is a continuation of U.S. Pat.
No. 09 / 019,901 filed on Feb. 6, 1998, which is a continuation-in-part of U.S. patent application Ser. Partial continuation application.

[0002] Background of the Invention 1. FIELD OF THE INVENTION The present invention relates to growth factors, particularly a new member of the transforming growth factor beta (TGF-beta) superfamily, called growth differentiation factor-11 (GDF-11), and muscle cells, bone, kidney and adipose tissue. It relates to methods of use for modulating growth.

[0003] 2. Description of the Related Art The transforming growth factor beta (TGF-beta) superfamily encompasses a group of structurally related proteins that affect a wide range of differentiation processes during embryonic development. This family includes the Mullerian inhibitor (MIS) required for normal male development (Behringer et al., Nature, 345: 167, 1990), the Drosophila melanogaster required for dorsal-ventral axis formation and adult primordium morphogenesis. Pentapleg (decapentapleg
ic) (DPP) gene product (Padgett et al., Nature, 325: 81-84, 1987), Xenopus Vg-1 gene product located at the egg plant pole (Weeks et al., Cell, 51: 861-867, 1987).
), Can induce the formation of mesoderm and anterior lobe structures in Xenopus embryos (Thomsen et al., Cell, 63: 485, 1990) activin (Mason et al., Bio
Chem. Biophys. Res. Commun., 135: 957-964, 1986), and can induce cartilage and bone formation de novo (Sampath et al., J. Biol. Chem., 265: 13198, 1).
990) including bone morphogenetic proteins (BMP, ostogenin, OP-1). TG
F-βs can affect a variety of differentiation processes including adipose tissue formation, myogenesis, cartilage formation, hematopoiesis and epithelial cell differentiation (for a review, see Massague, Cell
49: 437, 1987).

The TGF-β family of proteins is first synthesized as a large precursor protein, which then undergoes hydrolytic cleavage at a cluster of basic residues about 110-140 amino acids away from the C-terminus. . The C-terminal, or mature, region of the protein is all structurally related, and different family members can be divided into distinct subgroups based on the degree of homology. Homology within a particular subgroup ranges in amino acid sequence identity from 70% to 90%, while homology between subgroups is significantly lower, generally only 20% to 50%. In each case, the active species appears to be a disulfide-linked dimer of the C-terminal fragment.
Studies have shown that when the pro-region of a member of the TGF-β family is co-expressed with the mature region of another member of the TGF-β family, the intracellular dimerization of a biologically active homodimer Have been shown to occur (Gray, A. and M.
aston, A., Science, 247: 1328, 1990). Hammonds et al. (Molec. Emdocrin, 5: 149,
Further studies by 1991) have shown that the use of the BMP-2 pre-region in combination with the BMP-4 mature region dramatically improves the expression of mature BMP-4. For most of the family members studied, homodimeric species were found to be biologically active, but inhibins (Ling et al., Nature, 321: 779, 1986) and TGF-βs (Cheifetz
Heterodimers were also detected for other family members, such as, for example, Cell, 48: 409, 1987). And they appear to have different biological properties than the individual homodimers.

In addition, livestock and game animals with relatively high muscle tissue and protein content and low fat content, such as cattle, sheep, pigs, chickens and turkeys,
It is desirable to produce fish. Although there are many drugs and diet controls that can help increase muscle and protein content and reduce undesirable high fat and / or cholesterol levels, such treatments are generally administered after that fact. And only after significant damage has occurred to the vasculature. Therefore, it would be desirable to produce genetically predisposed animals with higher muscle content without concomitantly increasing fat levels.

[0006] The food industry has made great efforts to increase the amount of muscle and protein in foods. This pursuit is relatively straightforward in the production of synthetic foods, but in animal food production, hitherto limited success has been achieved. For example, attempts have been made to reduce cholesterol levels in beef and poultry products by including cholesterol-lowering agents in animal feeds (eg, Elkin and Rogler,
J. Agric. Food Chem. 1990, 38, 1635-1641). However, it increases muscle levels and lowers fat and cholesterol levels in animal food products,
There is still a need for more effective methods.

SUMMARY OF THE INVENTION The present invention provides a cell growth and differentiation factor, GDF-11, a polynucleotide sequence encoding the factor, and antibodies immunoreactive with the factor. This factor appears to be associated with various cell proliferative disorders, especially those involving muscle, nerve, bone, kidney and adipose tissue.

In one embodiment, the invention provides a method for detecting a cell proliferative disorder derived from muscle, nerve, bone, kidney or fat and associated with GDF-11. In another embodiment, the present invention provides a method of treating a lean proliferative disease by inhibiting or enhancing GDF-11 activity.

In yet another embodiment, the invention provides a non-human transgenic animal that is useful as a raw material for a food product having a high muscle, bone and protein content and a reduced fat and cholesterol content. . These animals are GDF-11
Germ cells and somatic cells are altered in the chromosome so that the production of GDF-11 is reduced or the production is completely destroyed, so that GDF-11 in the animal body is reduced.
It results in animals with reduced levels and increased bone tissue, such as muscle and ribs, above normal levels (preferably without increasing fat and / or cholesterol levels). Thus, the present invention also includes food products provided by these animals. Such food products have enhanced nutritional value due to the increased muscle tissue and bone tissue associated with the muscle tissue. Non-human transgenic animals of the present invention include, for example, cows, pigs, sheep, and poultry animals.

[0010] The present invention also provides a method of producing an animal food product having an increased muscle content. This method involves altering the genetic makeup of the germ cells of the prokaryotic embryo of the animal, implanting the embryo into the oviduct of a pseudopregnant female, maturing the embryo into a term term offspring, and transfecting the offspring. Testing for the presence of the gene and identifying transgene-positive progeny, cross-breeding the transgene-positive progeny to obtain additional transgene-positive progeny, and processing the progeny to obtain food. Germ cell modification involves altering a genetic construct to disrupt or reduce expression of a naturally encoded gene associated with the production of a GDF-11 protein. In certain embodiments, the transgene comprises an antisense polynucleotide sequence for a GDF-11 protein. Alternatively, the transgene is a natural GDF-11
It may contain non-functional sequences that substitute or intervene in the gene.

[0011] The present invention also provides a method of making an animal food product having an increased bone content. This method involves altering the genetic makeup of the germ cells of the prokaryotic embryo of the animal, implanting the embryo into the oviduct of a pseudopregnant female, maturing the embryo into a term term offspring, and transfecting the offspring. Testing for the presence of the gene and identifying transgene-positive progeny, cross-breeding the transgene-positive progeny to obtain additional transgene-positive progeny, and processing the progeny to obtain food. Germ cell modifications include altering a genetic construct to disrupt or reduce expression of a naturally encoded gene associated with the production of a GDF-11 protein. In certain embodiments, the transgene comprises an antisense polynucleotide sequence for a GDF-11 protein. Alternatively, the transgene may contain non-functional sequences that substitute or intervene in the native GDF-11 gene.

[0012] The present invention also provides a method of producing a poultry food product having improved muscle and / or bone content. This method involves modifying the genetic composition of the germ cells of a prokaryotic embryo of a poultry animal, transplanting the embryo into the oviduct of a pseudopregnant female embryo of the poultry, and culturing the embryo under conditions that allow the offspring to hatch. Testing the progeny for the presence of a genetic alteration and identifying transgene-positive progeny, cross-breeding the transgene-positive progeny, and processing the progeny to obtain food. .

[0013] The invention also provides a method of treating a disease of a muscle, bone, kidney or adipose tissue in a subject. The method comprises administering to a subject a therapeutically effective amount of a GDF-11 agent,
Affecting the growth of kidney or adipose tissue. GDF-11 agents include, for example, antibodies, GDF-11 antisense molecules or dominant negative polypeptides. In one embodiment, contacting an anti-GDF-11 monoclonal antibody, a GDF-11 antisense molecule or a dominant negative polypeptide (or a polynucleotide encoding a dominant negative polypeptide) with a fetal or adult muscle cell or progenitor cell. By GD
Methods for inhibiting the growth regulating action of F-11 are included. These agents can be administered to subjects afflicted with and aging subjects such as, for example, muscle wasting disease, neuromuscular disease, muscular atrophy, obesity or other fat cell diseases. In another aspect of the invention, the agent may be an agonist of GDF-11 activity.

[0014] The present invention also provides methods for identifying compounds that affect GDF-11 activity or gene expression. The method comprises converting the compound to a GDF-11 polypeptide or GDF-11.
And incubating the cells with a recombinant cell expressing under sufficient conditions to allow these compounds to interact, and determining the effect of the compound on GDF-11 activity or expression.

[0015] DETAILED DESCRIPTION OF THE INVENTION The present invention provides a polynucleotide sequence encoding growth and differentiation factors GDF-11, and GDF-11. GDF-11 is expressed at the highest levels in muscle, brain, uterus, spleen and thymus, and at lower levels in other tissues.

[0016] The TGF-β superfamily consists of multifunctional polypeptides that regulate proliferation, differentiation, and other functions in many cell types. Many of these peptides have both positive and negative regulatory effects on other peptide growth factors. GD of the present invention
The structural homology between the F-11 protein and TGF-β family members is
DF-11 has been shown to be a novel member of this family of growth and differentiation factors. Based on the known activities of many other members, GDF-11 can also be expected to have useful biological activities as diagnostic and therapeutic reagents.

Certain members of this superfamily have expression patterns or activities that are associated with nervous system function. For example, GDNF, a member of the family,
It has been shown to be a potent neurotrophic factor that can promote the survival of dopaminergic neurons (Lin et al., Science, 260: 1130). Dorsalin-1, another family member, can promote neural crest cell differentiation (Basle
r et al., Cell, 73: 687, 1993). Inhibin and activin have been shown to be expressed in the brain (Meunier et al., Proc. Natl. Acad. Sci. USA, 85: 247, 1988;
awchenko et al., Nature, 334: 615, 1988). And it has been shown that activin can function as a neuronal survival molecule (Schubert et al., Nature, 344: 868, 1990).
Another family member, GDF-1, is nervous system-specific in its expression pattern (Lee, Proc. Natl. Acad. Sci. USA, 88: 4250, 1991), and some other family members, For example, Vgr-1 (Lyons et al., Proc. Natl. Acad.
Sci. USA, 86: 4554, 1989; Jones et al., Development, 111: 581, 1991), OP.
-1 (Ozkaynak et al., J. Biol. Chem., 267: 25220, 1992), and BMP-4 (Jone
s et al., Development, 111: 531, 1991) are also known to be expressed in the nervous system. Expression of GDF-11 in brain and muscle suggests that GDF-11 may also have activity associated with nervous system function. In particular, for example, skeletal muscle is known to produce one or more factors that promote motor neuron survival (Brown, Trends Neurosci., 7:10, 1984). The known neurotrophic activity of other members of this family and the expression of GDF-11 in muscle suggest that one of the activities of GDF-11 may be as a trophic factor on motor neurons. In fact, GDF-11 is almost exclusively muscle specific in its expression pattern.
Highly related to the F-8. Alternatively, GDF-11 may have neurotrophic activity on other neuronal populations. Thus, GDF-11 may have in vitro and in vivo uses in treating neurodegenerative diseases, such as amyotrophic lateral sclerosis, or in maintaining cells or tissues during pre-transplant culture.

GDF-11 can also be used to repair muscle degenerative disease, osteoporosis, or wounds,
It may also have use in the treatment of disease processes involving the musculoskeletal system, such as. In this regard, many other members of the TGF-β family are also important mediators of tissue repair. TGF-β has a significant effect on collagen formation and has been shown to provoke a striking angiogenic response in neonatal mice (Rob
Natl. Acad. Sci. USA 83: 4167, 1986). TGF-β has also been shown to suppress myoblast differentiation in culture (Massague et al., Proc. Natl. A
cad. Sci. USA 83: 8206, 1986). In addition, because myoblasts can be used as a vehicle to carry genes into muscle in gene therapy, exploiting the properties of GDF-11 to maintain cells prior to transplantation or to increase the efficiency of the fusion process Will be able to. GDF-11 may also have use in the treatment of diseases involving repair of the kidney and wounds in the kidney.

GDF-11 may also have use in treating immune disorders. In particular, TGF
-Β has been shown to have a wide range of immunomodulatory activities, including potent suppressive effects on B and T cell proliferation and function (for review, see Palladino et al., Ann).
NY Acad. Sci., 593: 181, 1990). GDF-11 in spleen and thymus
Expression suggests that GDF-11 may have similar activity and therefore may be used as an anti-inflammatory agent or in the treatment of diseases associated with abnormal lymphocyte proliferation or function .

Animals intended for use in the practice of the present invention are those generally considered to be useful in food processing, ie, poultry, such as chickens and turkeys, raised for meat and laying eggs. Sheep, such as lambs, cattle, such as beef and dairy cows, fish and pigs. For the purposes of the present invention, these animals are referred to as heterologous DNA sequences integrated into the chromosome of germ cells or ones that are normally endogenous to the animal.
These additional DNA sequences (collectively referred to herein as "transgenes")
Is called "transgenic" when such an animal has it. The transgenic animal, including its progeny, will also have the transgene accidentally integrated into the chromosome of the somatic cell.

[0021] The TGF-β superfamily consists of multifunctional polypeptides that regulate proliferation, differentiation and other functions in many cell types. Many of these peptides have both positive and negative regulatory effects on other peptide growth factors. GDF of the present invention
The homology between the -11 protein and TGF-β family members is determined by the GD
F-11 has been shown to be a novel member of this family of growth and differentiation factors. Based on the known activities of many other members, GDF-11 can also be expected to have useful biological activities as diagnostic and therapeutic reagents.

In particular, certain members of this superfamily have activity or expression patterns associated with nervous system function. For example, inhibins and activins are known to be expressed in the brain (Meunier et al., Proc. Natl. Acad. Sci.,
USA, 85 : 247, 1988; Sawchenko et al., Nature, 334 : 615, 1988), and activin has been shown to be able to function as a neuronal survival molecule (Schubert et al., Nature, 344 :
868, 1990). Another family member, GDF-1, is nervous system specific in its expression pattern (Lee, SJ, Proc. Natl. Acad. Sci., USA, 88 :
4250, 1991), specific members of other family members, such as Vgr-1 (Lyons
Natl. Acad. Sci., USA, 86 : 4554, 1989; Jones et al., Development, 11
1 : 531, 1991), OP-1 (Ozkaynak et al., J. Biol. Chem., 267 : 25220, 1992) and BMP-4 (Jones et al., Development, 111 : 531, 1991) are also expressed in the nervous system. Is known to be. Skeletal muscle is known to produce one or more factors that promote the survival of motor neurons (Brown, Trends Neurosci, 7: . 10, 1984) Since the expression of GDF-11 in muscle, the GDF-11 One activity has been suggested to be a trophic factor for neurons. In this regard, GDF-11 may have use in treating neurodegenerative diseases such as amyotrophic lateral sclerosis or muscular dystrophy, or in maintaining cells or tissues during pre-transplant culture.

[0023] Expression of GDF-11 in adipose tissue also raises the possibility of using GDF-11 in the treatment of obesity or diseases associated with adipocyte overgrowth. In this regard, TGF-β is known to be a potent inhibitor of adipocyte differentiation in vitro (Ignotz and Massague, Proc. Natl. Acad. Sci., USA 82 : 8530, 19
85).

Polypeptides, Polynucleotides, Vectors and Host Cells The present invention provides substantially pure GDF-11 polypeptides and isolated polynucleotides encoding GDF-11. The term "substantially pure" as used herein refers to GDF-11 that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. One skilled in the art can purify GDF-11 using standard techniques for protein purification. A substantially pure polypeptide yields a single major band on a non-reducing polyacrylamide gel. GDF-11 polypeptide purity can also be confirmed by amino-terminal amino acid sequence analysis. The GDF-11 polypeptide includes a functional fragment of the polypeptide as long as the activity of GDF-11 remains. Smaller peptides having the biological activity of GDF-11 are also encompassed by the present invention.

The present invention provides a polynucleotide encoding a GDF-11 protein. These polynucleotides include DNA, cDNA and RNA sequences encoding GDF-11. It is understood that all polynucleotides encoding all or part of GDF-11 are also included here, as long as they encode a polypeptide having GDF-11 activity. Such polynucleotides include naturally occurring polynucleotides, synthetic polynucleotides, and intentionally engineered polynucleotides. For example, a GDF-11 polynucleotide can be subjected to site-directed mutagenesis. The polynucleotide sequence of GDF-11 also includes an antisense sequence. The polynucleotides of the present invention also include sequences that have been degenerated as a result of the degeneracy of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Accordingly, all degenerate nucleotide sequences are encompassed by the present invention, unless the amino acid sequence of the GDF-11 polypeptide encoded by the nucleotide sequence is altered functionally.

The present specification specifically discloses a DNA sequence containing the human GDF-11 gene. This sequence contains an open reading frame encoding a polypeptide that is 407 amino acids in length. This sequence has a putative RXXR at amino acids 295-298.
It has a proteolytic cleavage site. Cleavage of the precursor at this site has a length of 10
With 9 amino acids, it would yield an active C-terminal fragment with a predicted molecular weight of about 12,500 kD. The present specification also discloses a partial mouse genomic sequence. Preferably,
The human GDF-11 nucleotide sequence is SEQ ID NO: 1 and the mouse nucleotide sequence is SEQ ID NO: 3.

The polynucleotide encoding GDF-11 includes SEQ ID NOs: 1 and 3, and nucleic acid sequences complementary to SEQ ID NOs: 1 and 3. The complementary sequence can include an antisense nucleotide. When the sequence is RNA, the deoxynucleotides A, G, C and T of SEQ ID NOs: 1 and 3 are replaced by ribonucleotides A, G, C and U, respectively. Fragments of the above nucleic acid sequences having a length of at least 15 bases are also included in the present invention. This length is sufficient to allow the fragment to selectively hybridize under physiological conditions to DNA encoding the protein of SEQ ID NO: 2 or 4 (eg, under stringent conditions). .

[0028] In nucleic acid hybridization reactions, the conditions used to obtain a particular level of stringency will vary depending on the nature of the nucleic acid to be hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (eg, GC vs. AT content) and nucleic acid type (eg, RNA vs. DNA) of the hybridizing region of the nucleic acid
) Can be considered when selecting hybridization conditions. In addition, it is also considered whether one of the nucleic acids is immobilized, for example, on a filter.

One example of increasingly stringent conditions is: 2 × SSC / 0.1% SDS at about room temperature (hybridization conditions); 0.2 × SSC / 0.
1% SDS (low stringency conditions); 0.2 × SSC at about 42 ° C./0.1% SDS (medium stringency conditions); and 0.1 × SSC at about 68 ° C. (high stringency conditions). Washing can be performed using only one of these conditions, e.g., high stringency conditions, or each condition can be performed by repeating any or all of the listed steps, e.g., for 10-15 minutes each in the order described above. It can be used repeatedly. However, as noted above, optimal conditions will vary depending on the particular hybridization reaction involved and can be determined empirically.

[0030] The C-terminal region of GDF-11 following the putative proteolytic processing site is:
Shows significant homology to known members of the TGF-β superfamily. GDF-1
One sequence contains most of the residues that are highly conserved in other family members (see FIG. 1). Like TGF-β and inhibin β, GDF-11
Has an extra pair of cysteine residues in addition to the seven cysteines found in most of the other family members. Among the known family members, GDF-11
Is most homologous to GDF-8 (92% sequence identity) (see FIG. 3).

[0031] Minor modifications of the recombinant GDF-11 primary amino acid sequence may be achieved by modifying the G amino acid sequence described herein.
It can result in a protein having substantially equivalent activity compared to a DF-11 polypeptide. Such modifications may be deliberate, such as by site-directed mutagenesis, or may be spontaneous. All polypeptides resulting from such modifications are included in the invention, as long as the biological activity of GDF-11 is present. In addition, deletion of one or more amino acids can also result in alteration of the structure of the resulting molecule without significantly altering biological activity. This may lead to the development of smaller active molecules with wider utility. For example,
Amino or carboxy terminal amino acids that are not required for GDF-11 biological activity can be removed.

The nucleotide sequence encoding a GDF-11 polypeptide of the present invention encompasses the disclosed sequences and conservative variations thereof. The term "conservativ" used here
The term "e variation" refers to the replacement of an amino acid residue with another, biologically similar residue. Examples of conservative variations include the replacement of a hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another hydrophobic residue, or the replacement of one polar residue with another, such as arginine for lysine. Glutamic acid with aspartic acid or glutamine with asparagine). "Like deformation"
The term also includes using a substituted amino acid in place of the unsubstituted parent amino acid if antibodies raised against the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

[0033] The DNA sequence of the present invention can be obtained by several methods. For example, the above DN
A can be isolated using hybridization techniques well known in the art. These methods include, but are not limited to: That is, 1) genomic DNA or cDNA for detecting a homologous nucleotide sequence
Hybridization to library probes, 2) polymerase chain reaction (PCR) of genomic DNA or cDNA using primers that can anneal to the DNA sequence of interest, and 3) cloning with common structural features This is an antibody screening of an expression library for detecting a DNA fragment.

[0034] Preferably, the GDF-11 polynucleotides of the invention are derived from mammals, most preferably from mice, rats, cows, pigs or humans. GDF-11 polynucleotides from chickens, fish and other species are also included herein. If suitable probes are available, screening methods by nucleic acid hybridization will allow any gene sequence to be isolated from any organism. Oligonucleotide probes corresponding to a part of the sequence encoding the protein in question can be synthesized chemically. This requires that oligopeptide fragments with short amino acid sequences be known. The DNA sequence encoding a protein can be deduced from the genetic code, but the degeneracy of the code must be taken into account. If the sequence is degenerate, a mixed addition reaction is possible. It contains a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in detecting cDNA clones from sources that have extremely low amounts of mRNA associated with the polypeptide of interest. In other words, by using stringent hybridization conditions that are directed to avoid non-specific binding, for example, by hybridizing the target DNA to a single probe that is the perfect complement in the mixture, Visualization of cDNA clones by autoradiography becomes possible (Wallace, et al., Nucl. Acid Res. 2: 879, 1981).

The development of a specific DNA sequence encoding GDF-11 includes: 1) isolation of double-stranded DNA from genomic DNA; 2) chemical modification of the DNA sequence to provide the codons required for the polypeptide of interest. Production; and 3) in vitro synthesis of double-stranded DNA sequences by reverse transcription of mRNA isolated from eukaryotic donor cells. In the latter case, a double-stranded DNA complement of mRNA, commonly called cDNA, is ultimately formed.

Of the above three methods of developing specific DNA sequences for use in recombinant methods, isolation of genomic DNA isolates is less common. This is especially true where it is desirable to express the mammalian polypeptide in a microorganism due to the presence of introns.

The synthesis of DNA sequences is often the best method when the entire sequence of amino acid residues of the desired polypeptide product is known. If the entire sequence of the amino acid residues of the desired polypeptide is not known, direct synthesis of the DNA sequence is not possible and the best method is to use cDN
Synthesis of A sequence. Some of the standard techniques for isolating cDNA sequences of interest are plasmid or phage-borne, where expression of the gene is induced by reverse transcription of mRNA abundantly present in donor cells at high levels. There is the formation of a cDNA library. When used in combination with the polymerase chain reaction technique, even rare expression products can be cloned. In these cases, the knowledge of a significant portion of the amino acid sequence of the polypeptide would result in the generation of labeled single- or double-stranded DNA or RNA probes, possibly overlapping sequences present in the target cDNA. It can be used in DNA / DNA hybridization methods performed on cloned copies of cDNAs that have been denatured into a strand (Jay, et al., Nucl. Acid Res., 11 : 2325).
, 1983).

For example, a cDNA expression library such as lambda gt11 can be indirectly screened for a GDF-11 peptide having at least one epitope using an antibody specific for GDF-11. Such an antibody may be either polyclonal or monoclonal, and can be used to detect an expression product indicating the presence of GDF-11 cDNA.

The DNA sequence encoding GDF-11 can be obtained by introducing the DNA into a suitable host cell.
It can be expressed in vitro. A "host cell" is a cell in which a vector can be amplified and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that it is possible that not all of the progeny will be equal to the parent cell because mutations may occur during replication. However, when the term "host cell" is used, such progeny are included. Stable methods of transfer are known in the art, meaning that the foreign gene is continuously maintained in the host cell.

In the present invention, a GDF-11 polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by inserting or incorporating a GDF-11 gene sequence. Such expression vectors contain a promoter sequence that facilitates efficient transcription of the inserted gene sequence in the host. Expression vectors typically contain an origin of replication, a promoter, as well as specific genes that allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to, T7-based expression vectors for expression in bacteria (Rosenberg, et al., Gene, 56 : 125, 19117), mammalian cells PMSXND expression vector for expression in E. coli (Lee and Nathans, J. Biol. Chem., 263 : 35
21, 1988) and baculovirus-derived vectors for expression in insect cells. The DNA segment contains regulatory elements, such as a promoter (eg, T7
, Metallothionein 1, or polyhedrin promoter) operably linked and present in the vector.

The polynucleotide sequence encoding GDF-11 can be expressed in either prokaryotes or eukaryotes. Hosts include microorganisms, yeast, insects and mammals. Methods for expressing DNA sequences having prokaryotic or viral sequences in prokaryotes are known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a particular host are known in the art. Such vectors are used to incorporate the DNA sequences of the present invention. Preferably, the mature C-terminal region of GDF-11 is expressed from a cDNA clone containing the entire coding sequence of GDF-11. Alternatively, the C-terminal portion of GDF-11 can be expressed as a fusion protein with the pro region of another member of the TGF-β family, or can be co-expressed with another pro region. (See, for example, Hammonds, et al., Molec. Endocrin., 5 : 149, 1991; Gray, A.,
And Mason, A., Science, 247 : 1328, 1990).

Transformation of a host cell with the recombinant DNA can be performed by a conventional technique known to those skilled in the art. If the host is a prokaryote, for example, E. coli, D
Competent cells capable of incorporation of NA can be prepared from cells harvested after the exponential growth phase and subsequently treated by the CaCl ₂ method using procedures known in the art. Alternatively, MgCl ₂ or RbCl can be used. Transformation can be performed after forming the protoplasts of the host cell, if desired.

When the host is a eukaryote, conventional mechanical techniques such as calcium phosphate coprecipitation, microinjection, electroporation, and DNA transfection methods such as insertion of a plasmid or viral vector encapsulated in liposomes Can be used. Eukaryotic cells can also be co-transformed with a DNA sequence encoding a GDF-11 of the invention and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to transiently infect or transform eukaryotic cells with a eukaryotic viral vector, such as Simian Virus 40 (SV40) or bovine papilloma virus, to express the protein (eg, Eukaryotic). Viral Vectors, Cold Spring Harbor Labor
atory, Gluzman ed., 1982).

Isolation and purification of the polypeptides or fragments thereof expressed in the microorganisms provided by the present invention may be performed by conventional means, including preparative chromatography and immunological separation using monoclonal and polyclonal antibodies. Can be.

GDF-11 Antibodies and Methods of Use The present invention includes antibodies that are immunoreactive with GDF-11 polypeptides or functional fragments thereof. In addition to antibodies consisting essentially of pooled monoclonal antibodies with different epitope specificities, separate monoclonal antibody preparations are also provided. Monoclonal antibodies are prepared from antibodies containing fragments of the protein by methods known to those skilled in the art (Kohler, et al., Nature, 256 : 495, 1975). The term antibody, as used in the present invention, refers to naturally occurring molecules as well as fragments thereof capable of binding to the epitope determinants of GDF-11, such as Fab and F (ab ') ₂ , Fv and SCA fragments. It is intended to include (1) Fab fragment is a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digesting a whole antibody molecule with the enzyme papain to obtain a fragment consisting of a complete L chain and a part of an H chain. it can. (2) A Fab ′ fragment of an antibody molecule can be obtained by treating a complete antibody molecule with pepsin and reducing it to produce a molecule consisting of a complete L chain and a part of an H chain. This treatment yields two Fab 'fragments per antibody molecule. (3) The (Fab ') ₂ fragment of the antibody can be obtained without treating the whole antibody molecule with the enzyme pepsin, followed by reduction. (Fab ') ₂ fragments are dimers of two Fab' fragments held together by two disulfide bonds. (4) the Fv fragment comprises a variable portion of an L chain and a variable portion of an H chain expressed as two chains;
Defined as genetically engineered fragments. (5) Single-chain antibodies ("SCAs") are genetically engineered single-chain molecules that include a light chain variable region and a heavy chain variable region linked by a suitable flexible polypeptide linker. .

As used herein, the term “epitope” includes, for example, GDF-11
Refers to an antigenic determinant on an antigen, such as a GDF-11 polypeptide, to which an antibody paratope, such as a specific antibody, binds. Antigenic determinants usually consist of chemically active surface molecules, such as amino acids or sugar side chains, and can have unique charge properties in addition to unique three-dimensional structural properties.

As mentioned above, antigens that can be used to produce GDF-11-specific antibodies are GDF-11
Includes a polypeptide or GDF-11 polypeptide fragment. The polypeptide or peptide used for animal immunization can be obtained by standard recombinant, chemical synthesis, or purification methods. As is known in the art, antigens can be conjugated to a carrier protein to increase immunogenicity. Commonly used carriers include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The conjugated peptide is then used for immunization of an animal (eg, a mouse, rat, or rabbit). In addition to such carriers, known adjuvants can be administered with the antigen to promote the induction of a strong immune response.

The term “cell proliferative disorder” refers to both malignant and non-malignant cell populations that appear to differ from surrounding tissue both morphologically and genotypically. Malignant cells (eg, cancer) develop as a result of a multistep process. Polynucleotides that are antisense molecules, or GDF-11 polynucleotides that encode dominant negative GDF-11, are useful for treating cellular malignancies in various organ tissues, particularly, for example, muscle, bone, kidney or adipose tissue. It is useful in. In particular, any disorder in which altered expression of GDF-11 is associated with etiology can be considered susceptible to treatment with a GDF-11 agonist (eg, an inhibitor or activator). One such disorder is, for example, a malignant cell proliferative disorder.

The present invention provides a method for detecting a cell proliferative disorder of muscle, bone, kidney, uterus or nervous tissue. The method includes, for example, a method comprising contacting an anti-GDF-11 antibody with a cell suspected of having a GDF-11-related disorder and detecting binding to the antibody. Antibodies reactive with GDF-11 are labeled with a compound that allows detection of binding to GDF-11. For purposes of the present invention, antibodies that are specific for a GDF-11 polypeptide can be used to detect levels of GDF-11 in biological fluids and tissues. Any sample containing a detectable amount of antigen can be used. Preferred samples of the present invention are muscle, bone, kidney, uterus, spleen, thymus, or nerve tissue. The level of GDF-11 in the suspect cell can be compared to the level in normal cells to determine if the subject has a GDF-11-related cell proliferative disorder. Such a detection method uses nucleic acid hybridization to detect GDF-
11 It is also useful for detecting the level of mRNA or detecting the modified GDF-11 gene. Preferred subjects are humans.

The antibodies of the invention can be used in any subject where it is desirable to perform in vitro or in vivo immunodiagnosis or immunotherapy. The antibodies of the invention are suitable, for example, for use in immunoassays. There, the antibodies can be used in the liquid phase or bound to a solid support. In addition, the antibodies in these immunoassays can be detectably labeled in various ways. Examples of the types of immunoassays in which the antibodies of the invention can be used are direct or indirect competitive and non-competitive immunoassays. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigen using the antibody of the present invention includes an immunohistochemical assay using a physiological sample.
It can be performed using an immunoassay performed in either a forward, reverse, or simultaneous mode. One of skill in the art will know other immunoassay formats or will be able to readily recognize it without undue experimentation.

The antibodies of the present invention can be bound to a number of different carriers and used to detect the presence of an antigen comprising a polypeptide of the present invention. Examples of known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. For the purposes of the present invention, the nature of the carrier may be soluble or insoluble. Those skilled in the art are aware of other suitable carriers for binding the antibody,
Or, ordinary experiments could be used to identify such.

There are many different labels and methods of labeling known to those of skill in the art. Examples of types of labels that can be used in the present invention include enzymes, radioisotopes, fluorescent compounds,
Colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds are included. Those skilled in the art will know of other labels suitable for binding to the antibody, or will be able to recognize such using routine experimentation.

Another technique that may result in higher sensitivity consists of coupling the antibody to a low molecular weight hapten. These haptens can then be specifically detected by performing a second reaction. For example, it is common to use biotin, which reacts with avidin, or haptens, such as dinitrophenyl, pridoxal, and fluorescein, which can react with specific anti-hapten antibodies.

To use the monoclonal antibodies of the invention for in vivo detection of an antigen, the detectably labeled antibody is administered at a diagnostically effective dose. The term "diagnostically effective" refers to a detectably labeled monoclonal antibody in an amount sufficient to permit detection of a site having an antigen comprising a polypeptide of the invention for which the monoclonal antibody has specificity. Administration.

The concentration of the detectably labeled monoclonal antibody administered should be sufficient to allow binding of the polypeptide-containing cells to be detectable relative to background. In addition, to obtain the best target-to-background signal ratio, it is desirable that detectably labeled monoclonal antibody be rapidly cleared from the circulation.

In general, the dosage of a detectably labeled monoclonal antibody for in vivo diagnosis is
It varies depending on factors such as an individual's age, gender, and degree of illness. Such dosages may vary, for example, depending on whether multiple injections are to be taken, antigenic burden, and other factors known to those skilled in the art.

For in vivo diagnostic imaging, the type of detection equipment available is a major factor in radioisotope selection. The radioisotope chosen must have a detectable species attenuation for the given instrument type. Yet another important factor in selecting a radioisotope for in vivo diagnostics is to minimize harmful radiation with respect to the host. Ideally, the radioisotopes used for in vivo imaging lack particle emission, but generate large numbers of photons in the 140-250 keV range,
This can be easily detected by a normal gamma camera.

For in vivo diagnostics, the radioisotope can be conjugated directly to the immunoglobulin, or indirectly by using an intermediate functional group. Intermediate functional groups often used to bind radioisotopes present as metal ions to immunoglobulins are bifunctional chelators, such as diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar Molecule. Typical examples of metal ions that can be bound to the monoclonal antibodies of the present invention are ¹¹¹ In, ⁹⁷ Ru, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ⁸⁹ Zr and ²⁰¹ Tl.

The monoclonal antibodies of the present invention can also be labeled with paramagnetic isotopes for in vivo diagnostics such as magnetic resonance imaging (MRI) or electron spin resonance (ESR).
Generally, any conventional method for visualizing a diagnostic image can be used. Usually, gamma and positron emitting radioisotopes are used for camera image processing, and paramagnetic isotopes are used for MRI. Elements that are particularly useful in such techniques include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr, and ⁵⁶ Fe.

The monoclonal antibodies of the invention can be used to monitor the progress of amelioration of a GDF-11-related disease in a subject in vitro and in vivo. So, for example,
By measuring an increase or decrease in the number of cells expressing an antigen containing the polypeptide of the present invention, or a change in the concentration of such an antigen present in various body fluids, GDF-
11 It will be possible to determine whether a particular treatment regimen aimed at ameliorating the associated disease is effective. The term “improvement” refers to GD in the subject being treated.
Refers to a reduction in the deleterious effects of an F-11-related disorder.

Additional Therapeutic and Diagnostic Methods The present invention identifies nucleotide sequences that may be expressed in a different manner as compared to expression in normal cells, thereby providing an appropriate therapeutic or diagnostic technique for directing this sequence. It is possible to design. Treatment includes the administration of reagents that modulate the activity. The term "modulation" contemplates the suppression or expression of GDF-11 when overexpressed and the enhanced expression of GDF-11 when underexpressed. If the muscle-related disorder is associated with overexpression of GDF-11, a reagent such as an antisense GDF-11 polynucleotide sequence, a dominant negative sequence or a GDF-11 binding antibody can be introduced into the cells. In addition, anti-idiotype antibodies that bind to a monoclonal antibody that binds to GDF-11, or epitopes thereof, can be used in the treatment of the present invention. Alternatively, if the cell proliferative disorder is associated with under-expression or expression of a mutant GDF-11 polypeptide, a sense polynucleotide sequence (DNA coding strand) or GDF-11 polypeptide can be introduced into the cell. Such muscle-related disorders include cancer, muscular dystrophy, spinal cord injury, traumatic injury, congestive obstructive pulmonary disease (COPD), AIDS, or cachexia. It is envisioned that neurodegenerative, musculoskeletal, and renal disorders will also be treated by the methods of the present invention. In addition, the methods of the present invention can be used in the treatment of obesity or disorders associated with adipocyte overgrowth. One of skill in the art will recognize whether a particular course of therapy is successful, using several methods described herein (eg, muscle fiber analysis or biopsy; determination of fat content).
Can be determined by The examples herein show that the methods of the present invention are effective in reducing fat content and, therefore, are useful in treating obesity and related disorders (eg, diabetes).

Thus, where the cell proliferative disorder is associated with GDF-11 expression, nucleic acid sequences that interfere with GDF-11 expression at the translational level can be used. In this approach, for example, antisense nucleic acids and ribozymes can be used to mask their mRNA with an antisense nucleic acid or to cleave it with a ribozyme to produce a specific GDF-11.
Inhibits mRNA translation. The disorders mentioned here include, for example, neurodegenerative diseases. In addition, dominant negative GDF-11 variants are useful for actively disrupting the function of "normal" GDF-11.

An antisense nucleic acid is a DNA or R that is complementary to at least a portion of a particular mRNA molecule.
NA molecule (Weintraub, Scientific American, 262 : 40, 1990). In the cell,
The antisense nucleic acid hybridizes to the corresponding mRNA to form a double-stranded molecule.
Antisense nucleic acids do not translate double-stranded mRNA in cells, and thus interfere with translation of the mRNA.

Antisense oligonucleotides of about 15 nucleotides are preferred because of their ease of synthesis and less likely to cause problems than larger molecules when introduced into target GDF-11 producing cells. The use of antisense methods to inhibit in vitro translation of genes is known in the art (Marcus-Sakura,
Anal. Biochem., 172 : 289, 1988).

A ribozyme is an RNA molecule that has the ability to specifically cleave other single-stranded RNA in a manner similar to DNA restriction endonucleases. By modifying the nucleotide sequences encoding these RNAs, it is possible to create molecules that recognize and cleave specific nucleotide sequences in RNA molecules (Cech, J. Amer. Med. Assn., 2
60 : 3030, 1988). The main advantage of this approach is that only mRNAs with a particular sequence are inactivated because they are sequence-specific.

Ribozymes are of two basic types, the tetrahymena type (Hasselhoff, N
334 : 585, 1988) and the "hammerhead" type. Tetrahymena-type ribozymes recognize sequences that are 4 bases in length, whereas "hammerhead" -type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the higher the probability that the sequence will appear exclusively in the target mRNA species. Therefore, certain
For inactivating mRNA, a hammerhead ribozyme is preferable to a tetrahymena ribozyme, and a recognition sequence of 18 bases is preferable to a shorter recognition sequence.

In another aspect of the invention, there is provided a nucleotide sequence encoding a GDF-11 dominant negative protein. For example, a gene construct comprising a gene encoding such a dominant negative form can be operably linked to a promoter, eg, a tissue-specific promoter. For example, a skeletal muscle-specific promoter (eg, the human skeletal muscle α-actin promoter) or a development-specific promoter (eg, MyHC3 restricted to the embryonic stage of development in skeletal muscle)
), Or an inducible promoter (eg, orphan nuclear receptor TIS1). Such constructs are useful in methods of modulating skeletal mass in a subject. For example, one method involves transforming an organism, tissue, organ, or cell with a genetic construct encoding a dominant negative GDF-11 protein and an operably linked appropriate promoter, and encoding the dominant negative encoding GDF-11. Expressing the gene,
Thereby involving regulating muscle mass, bone content and / or kidney growth by actively interfering with wild-type GDF-11.

GDF-11 probably forms a dimer, homodimer or heterodimer,
It may even form heterodimers with other GDF family members, such as GDF-11 (see Example 4). Thus, while not wishing to be bound by any particular theory, the dominant negative effects described herein can include the formation of non-functional homodimers or heterodimers of dominant negative and wild type GDF-11 monomers. More specifically, any non-functional homodimers or any heterodimers formed by dimerization of wild-type and dominant negative GDF-11 monomers are 1) synthesized but not processed or secreted; 2) by inhibiting the secretion of wild-type GDF-11; 3) by interfering with the normal protein cleavage of the preprotein, thereby producing a non-functional GDF-11 molecule; 4) non-functional Alters the affinity of the target dimer (eg, homodimeric or heterodimeric GDF-11) for the receptor,
Or by creating an antagonistic form of GDF-11 that binds to the receptor but does not activate the receptor; or 5) inhibits the intracellular processing or secretion of GDF-11-related or TGF-β family proteins Doing so may cause a dominant effect.

[0069] Non-functional GDF-11 may function to inhibit the growth-regulating effects of GDF-11 on muscle cells containing the dominant negative GDF-11 gene. GDF-11 deletion or missense dominant negative endogenous wild type that retains the ability to form a dimer with wild type GDF-11 protein but does not function like wild type GDF-11 protein
It can be used to inhibit the biological activity of GDF-11. For example, in one embodiment, the cleavage site of the GDF-11 protein is altered (eg, deleted), thereby allowing subsequent dimerization with endogenous wild-type GDF-11 but mature GDF. -
GDF-11 molecules can be generated that cannot be subjected to further processing into the 11 form. Alternatively, non-functional GDF-11 may function as a monomeric species to inhibit the growth-regulating effects of GDF-11 on muscle, bone and kidney cells at any point in tissue or organism development.

Any genetic recombination method in the art can be used, for example,
Recombinant viruses can be made to express a dominant negative form of GDF-11 that can be used to inhibit the activity of wild-type GDF-11. Such viruses can be used therapeutically in the treatment of diseases resulting from abnormal overexpression or activity of the GDF-11 protein, for example in denervation hypertrophy, or in treating diseases involving muscle. In such cases, for example, it can be used as a means of regulating GDF-11 expression in tissue repair due to muscular degenerative disease or trauma or regulation of GDF-11 expression in animal management (eg, transgenic animals for agriculture). .

In addition, expression of GDF-11 can be used, for example, to aid kidney development. The method comprises administering to the subject a therapeutically effective amount of a GDF-11 agonist, thereby promoting kidney cell growth and differentiation in kidney tissue. The agent may be a GDF-11 antagonist or agonist. For example, this agonist
GDF-11 antisense molecules or dominant negative polypeptides can be included.

The present invention provides a method for treating a muscle, kidney (chronic or acute) or adipose tissue disorder in a subject. The method comprises administering to the subject a therapeutically effective amount of a GDF-11 agonist, thereby inhibiting abnormal growth of muscle or adipose tissue or stimulating growth in kidney tissue. GDF-11 agonists include, for example, GD
An F-11 antisense molecule or a dominant negative polypeptide can be included. GD
A "therapeutically effective amount" of an F-11 agonist is an amount that ameliorates the symptoms of the disorder or inhibits GDF-11-induced muscle growth, for example, as compared to a normal subject.

The present invention also provides gene therapy for treating a cell proliferative or immunological disorder mediated by the GDF-11 protein. Such treatment achieves its therapeutic effect by introducing a GDF-11 antisense polynucleotide or a dominant negative encoding polynucleotide sequence into cells having a proliferative disorder. Delivery of an antisense GDF-11 polynucleotide can be achieved using a recombinant expression vector, such as a chimeric virus or a colloidal dispersion system. Particularly preferred for therapeutic delivery of antisense or dominant negative sequences is the use of targeted liposomes. In contrast, if it is desired to increase GDF-11 production, a "sense" GDF-11 polynucleotide can be added to the appropriate cells (
One or more).

Various viral vectors that can be used for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors into which one foreign gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine breast cancer virus (MuMTV) And Rous sarcoma virus (RSV). Some additional retroviral vectors can incorporate multiple genes. All of these vectors are capable of transferring or incorporating a gene for a selectable marker, thereby identifying and producing transfected cells. Insertion of the GDF-11 sequence of interest into a viral vector, for example, along with another gene encoding a ligand for a receptor on a particular target cell, renders the vector target specific. Retroviral vectors can be made target-specific, for example, by attaching a sugar, glycolipid, or protein. Preferred targeting is achieved by using an antibody that targets the retroviral vector. One of skill in the art knows certain polynucleotide sequences that can be inserted into the retroviral genome or bind to the viral envelope for target-specific delivery of a retroviral vector containing a GDF-11 antisense polynucleotide. Yes, or could be easily confirmed without undue experimentation.

[0075] Because recombinant retroviruses are defective, they require assistance to produce infectious vector particles. This assistance can be provided, for example, by using a helper cell line that contains a plasmid that encodes all of the structural genes of the retrovirus under the control of regulatory sequences in the LTR. These plasmids have lost the nucleotide sequence that allows the packaging machinery to recognize the RNA transcript for inclusion. Helper cell lines that lack a packaging signal include, but are not limited to, ψ2, PA317 and PA12, for example. These cell lines produce empty virions because the genome is not packaged.
If the retroviral vector is introduced into a cell where the packaging signal is complete but the structural gene has been replaced by another gene of interest, the vector can be packaged and the vector virion is produced I do.

Alternatively, NIH3T3 or other tissue culture cells may contain the retroviral structural genes gag, p
Plasmids encoding ol and env can be directly transfected by conventional calcium phosphate transfection. These cells are then transfected with a vector plasmid containing the gene of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for GDF-11 antisense polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloid system of the present invention is a liposome. Liposomes are artificial membrane vesicles that are useful as in vitro and in vivo delivery vehicles. Unilamellar vesicles (LUVs) ranging in size from 0.2-4.0 μm have been shown to be capable of encapsulating a significant proportion of aqueous buffers containing macromolecules. RNA, DNA and complete virions encapsulated within the aqueous interior can be delivered to cells in the form of a biologically active (Fraley, et al., Trends Biochem Sci, 6:. . 77, 1
9111). In addition to mammalian cells, liposomes have been used to deliver polynucleotides to plant, yeast and bacterial cells. In order to make liposomes an efficient gene delivery vehicle, the following characteristics should be present: (1) high efficiency encapsulation of the genes of interest without impairing their biological activity; (2) preferential and substantial binding to target cells compared to non-target cells; (3) highly efficient delivery of the aqueous contents of the vesicles to the target cell cytoplasm; and (4) genetic information (Manning, et al., Biotechniques, 6 : 682, 1988).

The composition of the liposome is usually a combination of phospholipids, especially those with a high phase transition temperature, generally in combination with steroids, especially cholesterol. Other phospholipids or other lipids can be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids useful for forming liposomes include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are those in which the lipid moiety has 14-18 carbon atoms, especially
Diacylphosphatidylglycerol containing 16-18 carbon atoms and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoyl phosphatidylcholine and distearoyl phosphatidylcholine.

The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based on whether it is passive or active. Passive targeting takes advantage of the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs including sinusoidal capillaries. Active targeting, on the other hand, involves coupling liposomes with specific ligands, such as monoclonal antibodies, sugars, glycolipids, or proteins, to achieve targeting to organs or cell types other than natural localized sites. Or altering the liposomes by changing the composition or size of the liposomes.

The surface of the targeted delivery system can be modified in various ways. In the case of liposome targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain the targeting ligand in stable association with the liposome bilayer. Various linking groups can be used to link the lipid chain to the targeting ligand.

For the expression of GDF-11 in muscle and adipose tissues, there are various uses for the polypeptides, polynucleotides, and antibodies of the invention associated with these tissues. Such uses include treatment of these and other cell proliferative disorders involving other tissues, such as neural tissue. In addition, GDF-11 may be useful in various gene therapy methods. In embodiments where the GDF-11 polypeptide is administered to a subject, the dosage range is about 0.1 μg / kg to 100 mg / kg; more preferably about 1 μg / kg to 75 mg / kg, most preferably about 10 mg / kg to 50 mg. / Kg.

Chromosomal Location of GDF-11 The data in Example 6 indicates that the human GDF-11 gene is located on chromosome 2. GDF-
By comparing the eleven chromosomal locations to the map locations of various human disorders, it should be possible to determine whether mutations in the GDF-11 gene are involved in the pathogenesis of human disease. For example, an autosomal recessive form of juvenile amyotrophic lateral sclerosis has been shown to map to chromosome 2 (Hentati, et al., Neurology, 42
[Suppl.3]: 201, 1992). More accurate mapping of GDF-11 and analysis of DNA from these patients can indeed indicate that GDF-11 is a gene affecting this disease. In addition, GDF-11 is useful for distinguishing chromosome 2 from other chromosomes.

Transgenic Animals and Methods of Making Same Various methods of making the transgenic animals of the subject invention can be used. In general, three such methods can be used. One of such methods
In one, pronuclear stage embryos (“one-cell embryos”) are harvested from females and microinjected with a transgene into the embryo, where the transgene is derived from embryonic and somatic cells of the resulting adult animal. It is integrated into both chromosomes. In another such method, embryonic stem cells are isolated and transgene incorporated therein by electroporation, plasmid transfection or microinjection, and then the stem cells are reintroduced into the embryo, where they form colonies. And contribute to the germ line. Methods for microinjection of mammalian species are described in U.S. Patent No. 4,873,191. In yet another such method, embryonic cells are infected with a retrovirus containing the transgene, thereby integrating the transgene into the chromosomes of the embryonic embryonic cells. If the animal to be transgenic is a bird, the fertilized egg of the bird generally completes cell division in the first 20 hours in the fallopian tube, making it difficult to access the pronucleus and Microinjection into the pronucleus is problematic. Thus, among the usual methods for producing transgenic animals described above, for example, US Pat.
As described in No. 215, retroviral infection is preferred for avian species. However, if one wishes to use microinjection in avian species, a procedure recently published by Love et al. (Biotechnology, 12, Jan 1994) can be used, whereby prior laying eggs from the sacrificed hens Approximately 2 and 1.5 hours after the embryo is obtained, the transgene is microinjected into the cytoplasm of the scutellum and the embryo is cultured in the host shell until maturation.
If the animal to be transgenic is a cow or pig, microinjection can be hindered by the opacity of the ovum, which makes it difficult to identify nuclei by traditional differential interference contrast microscopy. To overcome this problem, the eggs can first be centrifuged to separate the pronuclei for better visualization.

The “non-human animals” of the present invention include bovine, porcine, ovine
) And bird animals (eg, cows, pigs, sheep, chickens). In the present invention,
A "transgenic non-human animal" is produced by introducing a "transgene" into the germline of a non-human animal. Embryonic target cells at various stages of development can be used for transgene introduction. Different methods are used depending on the stage of development of the embryonic target cell. Conjugates are the best target for microinjection. There are greater advantages to using zygotes as targets for gene transfer, in most cases the injected DNA is integrated into the host gene before the first cleavage (Brinster et al., Proc. N
atl. Acad. Sci. USA 82 : 4438-4442, 1985). As a result, all cells of the transgenic non-human animal carry the integrated transgene. This is also reflected in the efficient transmission of the transgene to the offspring of the founder, as generally 50% of the embryonic cells have the transgene.

[0086] The term "transgenic" is used to describe an animal that contains exogenous genetic material in all of its cells. "Transgenic" animals can be produced by cross-breeding two chimeric animals that contain exogenous genetic material in their cells and are used for breeding. Twenty-five percent of the resulting progeny are transgenic, ie, animals that contain exogenous genetic material in both alleles of all of their cells. 50% of the resulting animals contain exogenous genetic material in one allele and 25%
Does not contain exogenous genetic material.

In a microinjection method useful in the practice of the subject invention, the transgene is digested and purified, eg, by electrophoresis, without any vector DNA. It is preferred that the transgene comprises an operably involved promoter that interacts with cellular proteins involved in transcription and ultimately results in constitutive expression. Useful promoters in this regard include those derived from cytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus,
Metallothionein, skeletal actin, P-enolpyruvate carboxylase (PEP
CK), phosphoglycerate (PGK), DHFR, and those derived from genes encoding thymidine kinase. A viral terminal repeat (LTR) promoter, for example, Rous sarcoma virus, can also be used. If the animal to be transgenic is an avian, a preferred promoter is chicken β
-The globin gene, the chicken lysozyme gene, and those of the avian leukemia virus. Constructs useful in plasmid transfection of embryonic stem cells include additional regulatory elements known in the art, such as enhancer elements that stimulate transcription, splice acceptors, termination and polyadenylation signals, and ribosomes that allow translation. A binding site is used.

[0088] Retroviral infection can also be used to introduce a transgene into a non-human animal, as described above. Non-human embryos that are developing are differentiated in vit
Can be cultured in ro. During this period, blastomeres may be targets for retroviral infection (Jaenich, R., Proc. Natl. Acad. Sci. USA 73: 1260-1264, 19
76). Effective infection of blastomeres is achieved by enzymatic treatment to remove the zona pellucida (Hogan et al. (1986), Manipulating the Mouse Embryo, Co
ld Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus with the transgene (Jahner et al., Proc. Natl. Acad.
Sci. USA 82 : 6927-6931, 1985; Van der Putten et al., Proc. Natl. Acad. Sci. U
SA 82 : 6148-6152, 1985). Transfection is facilitated and efficient by culturing blastomeres on a monolayer of virus-producing cells (Van der Putten,
Supra; Stewart et al., EMBO J. 6: 383-388, 1987). Alternatively, the infection can be performed later. The virus or virus-producing cells can be injected into the blastocoel (D. Jahner et al., Nature 298 : 623-628, 1982). Most of the founders are mosaic about transgenes. Since integration occurs only in the subset of cells that formed the transgenic non-human animal. In addition, the founder may contain various retroviral insertions of the transgene at different locations in the genome, which generally segregate in progeny. Moreover,
Despite the low efficiency, transgenes can also be introduced into the germline by in utero retroviral infection of midgestation embryos (D. Jahne
r et al., supra).

A third type of target cells for transgene introduction is embryonic stem cells (ES). ES cells are obtained from preimplantation embryos cultured in vitro and fused with embryos (M.
J. Evans et al., Nature 292: 154-156, 1981; MO Bradley et al., Nature 309 : 255-2.
58, 1984; Gossler et al., Proc. Natl. Acad. Sci. USA 83 : 9065-9069, 1986; and Robertson et al., Nature 322: 445-448, 1986). Transgenes can be efficiently introduced into ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can then associate with blastocysts from a non-human animal. The ES cells then colonize the embryo and contribute to the germline of the resulting chimeric animal (for a review, see Jaenisch, R., Science 240 : 1468-1474, 1988).

“Transformed” means a cell into which (or to its ancestors) a heterologous nucleic acid molecule has been introduced by recombinant nucleic acid technology. “Heterologous” refers to a nucleic acid sequence derived from another species or modified from its original form or its originally expressed form in a cell.

A “transgene” is an artificially inserted cell that becomes part of the genome of an organism that has arisen from the cell (ie, as a stably integrated or stable extrachromosomal element). Means any piece of DNA. Such a transgene can include a gene that is partially or entirely heterologous (ie, foreign) to the transgenic organism, or exhibits a homologous gene to an endogenous gene of the organism. Can be. Included within this definition are transgenes that are made by providing an RNA sequence that is transcribed into DNA and then integrated into the genome.
The transgene of the present invention includes a DNA sequence encoding GDF-11,
-Sense and antisense polynucleotides and dominant negative coding polynucleotides, which can be expressed in transgenic non-human animals. As used herein, the term “transgenic” is further used by in vitro manipulation of early embryos or fertilized eggs, or by any transgenic technique to induce specific gene knockout.
Includes any organism whose genome has been modified. As used herein, the term `` gene knockout '' refers to the targeted disruption of a gene in vivo with complete loss of function, achieved by any transgenic technique well known to those skilled in the art. Say. In one embodiment, the transgenic animal having the gene knockout is one in which the target gene has been rendered non-functional by an insertion targeted to the gene to be rendered non-functional by homologous recombination. is there. As used herein, the term "transgenic" refers to an organism having an introduced transgene, or that an endogenous gene has been rendered non-functional or "
It includes any transgenic technique well known to those skilled in the art, which can produce a "knocked out" organism. Examples of transgenes used to “knock out” GDF-11 functions in embodiments of the present invention are described in Example 6 and FIG. Thus, in another embodiment, the present invention provides a transgene in which the entire mature C-terminal region of GDF-11 has been deleted.

[0092] The transgene used in the practice of the present invention is a DNA sequence that includes a modified GDF-11 coding sequence. In a preferred embodiment, the GDF-11 gene is disrupted by homologous targeting in embryonic stem cells. For example, the maturation of the GDF-11 gene
The entire C-terminal region can be deleted, as described in the examples below. Optionally, the disruption or deletion of GDF-11 can be caused by other DNA sequences, such as non-functional GDF
-11 sequence insertion or substitution therewith. In other embodiments, the transgene comprises antisense DNA to the coding sequence for GDF-11. In another embodiment, the transgene comprises DNA encoding an antibody or receptor peptide sequence capable of binding GDF-11. The DNA and peptide sequences of GDF-11 are known in the art, and the sequence, localization and activity are described in WO95 / 08543 and pending US patent application Ser. No. 08 / 706,958 (1996). (Filed September 3, 2009), all of which are incorporated herein by reference. The disclosures of both of these applications are incorporated herein by reference. Where appropriate, DNA sequences which encode proteins having GDF-11 activity, but which differ in nucleic acid sequence due to the degeneracy of the genetic code, may also be referred to as truncated, allelic and interspecies homologs. It can be used in the invention.

Accordingly, the present invention also includes animals having a heterozygous mutation in GDF-11. Heterozygotes appear to have a modest increase in muscle mass compared to homozygotes.

Embryos can be microinjected with transfected embryonic stem cells to form colonies or infected with a retrovirus containing a transgene (but not with birds treated elsewhere herein). (Except for the practice of the invention in species), embryos are transferred into the fallopian tubes of pseudopregnant females. The resulting offspring are
Blood samples are tested for transgene incorporation by Southern blot analysis using a transgene-specific probe. In this regard, PCR is particularly useful. Positive progeny (G0) are crossed to produce progeny (G1), which is analyzed for transgene expression by Northern blot analysis of tissue samples. A marker gene fragment was included in the construct in the 3 'untranslated region of the transgene so that expression of the transgene of a similar species could be distinguished from expression of the endogenous GDF-11 gene in the animal. Northern probes can be designed to catch marker gene fragments. The serum concentration of GDF-11 can be measured in transgenic animals to establish appropriate expression. Expression of the GDF-11 transgene, and thereby GDF-11 reduction in tissue and serum concentrations of the transgenic animal, and consequent increase in muscle tissue content is preferably achieved without increasing fat and cholesterol concentrations. , More preferably reduced,
Food from these animals with significantly increased muscle content (ie, eggs, beef, pork, chicken, milk, etc.) results. By the practice of the present invention, a statistically significant increase in muscle content, preferably at least a 2% increase in muscle content as a percentage of body weight (eg in chicken), more preferably a 25% increase in muscle content,
More preferably, more than a 40% increase in muscle content in these foods can be obtained.

In addition, the reduction of GDF-11 in tissue and serum concentrations of transgenic animals can be used to increase the bone content of animals used as food, for example, transgenic animals with reduced GDF-11. A transgenic animal can be provided having a greater number of ribs.

Further Uses Accordingly , the present invention relates to a method of increasing the muscle mass and / or rib content of a domestic animal, comprising the inactivation or deletion of the gene encoding growth and differentiation factor-11 (GDF-11). Includes methods characterized by loss. Domestic animals are preferably sheep, cows, pigs,
It is selected from the group consisting of fish and birds. The animal can be treated with an isolated polynucleotide sequence encoding a growth and differentiation factor-11, wherein the polynucleotide sequence is also selected from the group consisting of sheep, cows, pigs, fish and birds. From livestock animals. The invention includes a method of increasing the muscle mass or rib content of a domestic animal, comprising administering to the domestic animal a monoclonal antibody directed against a GDF-11 polynucleotide. This antibody is anti-GD
It can be F-11 and can be a monoclonal or polyclonal antibody.

The present invention encompasses a method comprising using an anti-GDF-11 monoclonal antibody, an antisense or a dominant negative mutant as a therapeutic agent for inhibiting the growth regulating effect of GDF-11 in muscle cells. . Muscle cells are defined to include fetal or adult muscle cells, as well as progenitor cells that can differentiate into muscle. The monoclonal antibody can be a humanized (eg, whole or chimeric) monoclonal antibody of any source, eg, mouse, sheep, cow, pig, or bird. Methods for producing antibody molecules using various combinations of "humanized" antibodies are well known in the art and combine mouse variable regions with human constant regions (Cabily et al., Proc. Natl. Acad. Sci. USA, 81 : 3273, 1984) or grafting mouse antibody complementarity determining regions (CDRs) to human skeletal regions (Ric.
hmann et al., Nature 332 : 323, 1988). Other general references that teach how to make humanized antibodies include Morrison et al., Science, 229 : 1202, 1985; Jon.
es et al., Nature, 321 : 522, 1986; Monroe et al., Nature, 312 : 779, 1985.
Oi et al., BioTechniques, 4 : 214, 1986; European Patent Application No. 302,620; and U.S. Patent No. 5,024,834. Thus, humanizing a monoclonal antibody of the invention for use in vivo greatly reduces the immune response to the antibody.

The present invention encompasses a method comprising using an anti-GDF-11 monoclonal antibody, an antisense or a dominant negative mutant as a therapeutic agent for inhibiting the growth regulating effect of GDF-11 on bone cells. . Osteocytes are defined to include fetal or adult bone cells, as well as progenitor cells that can differentiate into bone. The monoclonal antibody can be a humanized (eg, whole or chimeric) monoclonal antibody of any source, eg, mouse, sheep, cow, pig, or bird. Methods for producing antibody molecules using various combinations of "humanized" antibodies are well known in the art and combine mouse variable regions with human constant regions (Cabily et al., Proc. Natl. Acad. Sci. USA, 81 : 3273, 1984) or grafting mouse antibody complementarity determining regions (CDRs) to human skeletal regions (Richmann et al., Nature 332 : 323, 1988). Other general references that teach how to make humanized antibodies include Morrison et al., Science, 229 : 1202, 1985; Jones et al., Nature, 321 : 522, 1986; Monroe et al., Nature, 312 : 779, 1985; Oi et al., BioTechniques, 4 : 214, 1986; European Patent Application No. 302,620; and US Patent No. 5,024,834. Thus, humanizing a monoclonal antibody of the invention for use in vivo greatly reduces the immune response to the antibody.

A monoclonal antibody, GDF-11 polypeptide or GDF-11 polynucleotide (all “GDF-11 agents”) can have the effect of increasing skeletal muscle or skeletal bone development. In a preferred embodiment of the claimed method, the GDF-11 monoclonal antibody, polypeptide or polynucleotide is afflicted with a disease selected from the group consisting of muscle wasting disease, neuromuscular disease, muscular atrophy or aging. To be administered. In another preferred embodiment, the present invention provides a method for treating GD in a patient suffering from a disease.
Provided is a method of treating an osteodegenerative disease, eg, osteoporosis, by administering an antibody, polypeptide or polynucleotide that elicits F-11 activity. GDF-11
The drug is also used for muscular dystrophy, spinal cord injury, trauma injury, congestive obstructive pulmonary disease (congest
ive obstructive pulmonary disease (COPD), AIDS or cachechia (cachechia
) Can be administered to patients suffering from a disease selected from the group consisting of: In a preferred embodiment, the GDF-11 agent is administered to a patient with a muscle or bone wasting disease or disorder by intravenous, intramuscular or subcutaneous injection,
Preferably, the monoclonal antibody is administered at a dosage ranging from about 0.1 mg / kg to about 100 mg / kg, more preferably from about 1 mg / kg to 75 mg / kg, most preferably from about 10 mg / kg to 50 mg / kg. You. Antibodies can be administered, for example, by bolus injection or by slow infusion. Slow infusion over a period of 30 minutes to 2 hours is preferred. The GDF-11 agent can be a formulation suitable for administration to a patient. Such formulations are known in the art.

The dosage regimen will depend on the various factors that modify the action of the GDF-11 protein, such as the amount of tissue desired to be formed, the site of tissue damage, the damaged tissue, Condition, wound size, damaged tissue type, patient age, gender and diet, severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and the type of agent (eg, anti-GDF-11 antibody) to be used in the composition. Generally, it is a systemic or injectable administration, for example, intravenous (IV), intramuscular (IM) or subcutaneous (Sub-Q) injection. Dosing is generally begun with a dose with the least effect and increasing the dose over a preselected period of time until a positive effect is observed. A dose escalation is then made, taking into account any adverse effects that may appear, with such escalation limited to an amount that produces a corresponding increase in effect. The addition of other known growth factors, such as IGF I (insulin-like growth factor I), human, bovine or avian growth hormone, which can help increase muscle mass, to the final composition is also a matter of dosage. Can be affected. In embodiments where an anti-GDF-11 antibody is administered, the anti-GDF-11 antibody will generally have a dose of about 0.1 μg / kg to about 100 mg / kg; more preferably about 10 mg / kg to about 50 mg / kg. It is administered within a range.

Progress can be monitored by periodic assessment of tissue growth and / or repair. Progress can be monitored by, for example, X-rays, histomorphometric measurements, and tetracycline labeling.

Screening for GDF-11 Modulating Compounds In another embodiment, the invention provides a method for identifying a compound or molecule that modulates the activity or gene expression of a GDF-11 protein. This method measures the effect of a compound on a component, including a compound, a GDF-11 polypeptide (or with a recombinant cell that expresses a GDF-11 polypeptide), and the GDF-11 activity or expression. And incubation under conditions sufficient to allow the The effect of a compound on GDF-11 activity can be measured by a number of assays, and can include measurements before and after incubation in the presence of the compound. Compounds that affect GDF-11 activity or gene expression include peptides, peptidomimetics, polypeptides, compounds and biological agents (bio
logic agent). Assays include Northern blot analysis (for gene expression), Western blot analysis (for protein concentration) and muscle fiber analysis (for protein activity) of GDF-11 mRNA.

[0103] The screening assay described above involves the isolation of a molecule that binds to the GDF-11 gene in order to detect a compound or molecule that binds to the GDF-11 receptor or GDF-11 polypeptide. It can be used to determine the amount of 11 (polypeptide or RNA (mRNA)), to identify molecules that can act as agonists or antagonists, and the like. For example, a GDF-11 antagonist
Useful for the treatment of muscle and adipose tissue disorders such as obesity.

Incubation includes conditions that allow contact between a test compound and a GDF-11 polypeptide (or with a recombinant cell that expresses a GDF-11 polypeptide).
Contacting includes in solution and in solid phase or in cells. The test compound can optionally be a combinatorial library for screening a plurality of compounds. Compounds identified in the methods of the invention can be used in solution or after binding to a solid support, by any of the methods commonly applied for the detection of specific DNA sequences, such as PCR, oligomer restriction (Saiki et al., Bio / Technology, 3 : 1008-1012, 1985),
Allele-specific oligonucleotide (ASO) probe analysis (Conner et al., Proc. Nat.
l. Acad. Sci. USA, 80 : 278, 1983), oligonucleotides (Landegren et al., Scie
nce, 241 : 1077, 1988) can be further evaluated, detected, cloned, sequenced, and the like. Review of molecular techniques for DNA analysis (Lande
gren et al., Science, 242 : 229-237, 1988).

[0105] All references cited herein are hereby incorporated by reference in their entirety.

The following examples are intended to illustrate, but not limit, the invention. They are typical examples that can be used, but other methods known to those skilled in the art can be substituted.

Example 1 Identification and Isolation of Novel TGF-β Family Members To identify new members of the TGF-β superfamily, GDF-β
Murine GDF-8 spanning the region encoding the C-terminal portion of precursor protein 8
A murine genomic library was screened at moderate stringency using a probe (FIG. 8: nucleotides 865-1234). Literature (Lee, Mol
Endocrinol., 4: 1034, 1990). Hybridization was performed at 65 ° C., and final washing was performed at the same temperature in a buffer containing 0.5 M NaCl. Some hybridized phage were distinguished from GDF-8 containing phage by a weaker hybridization intensity with the GDF-8 probe. Analysis of the partial nucleotide sequence of the genomic insert present in the weakly hybridizing phage showed that this clone was strongly related to murine GDF-8 but contained a different sequence.

The partial nucleotide sequence of the genomic insert present in this phage is shown in FIG. This sequence contained an open reading frame spanning nucleotides 198 to 575 showing significant homology to known members of the TGF-β superfamily (see below). This sequence was preceded by a 3 'splice consensus sequence at exactly the same position as the GDF-8 gene. This new TGF-β family member was named GDF-11 (growth / differentiation factor-11).

Example 2 Expression of GDF-11 To examine the expression pattern of GDF-11, RNA samples prepared from various tissues were screened by Northern analysis. RNA isolation and Northern analysis were performed by the method described in the literature (Lee, Mol. Endocrinol., 4: 1034, 1990), except that hybridization was performed using 5 × SSPE, 10% dextran sulfate, 50% formamide, 1% SDS, 200 μg / Performed in ml salmon DNA and 0.1% each of bovine serum albumin, ficoll and polyvinylpyrrolidone. 5 μg of RNA selected twice with poly A prepared from each tissue (3.
(Using 3 μg of RNA) on a formaldehyde gel, blotted,
GDF-11 was used as a probe. As shown in FIG. 2, the GDF-11 probe is
Approximately 4.2 kb in adult thymus, brain, spleen, uterus and muscle, and whole embryos isolated at 12.5 or 18.5 days, and brain samples taken at each stage of development
And two RNA species of 3.2 kb length were detected. With longer exposure of these blots, low levels of GDF-11 RNA were detected in many other tissues.

Example 3 Isolation of a cDNA clone encoding GDF-11 To isolate a cDNA clone encoding GDF-11, RNA prepared from human adult spleen was used to isolate a cDNA clone into a λZAP II vector (Stratagene). cDN
An A library was prepared. RNA selected twice with poly A prepared from human spleen
From 5 μg, a cDNA library consisting of 21 million recombinant phages was constructed according to the instructions of Stratagene. This library was screened without amplification. Library screening and characterization of the cDNA insert were performed by methods described in the literature (Lee, Mol. Endocrinol., 4: 1034, 1990). 23 hybridizing phages were obtained from the library.

The complete nucleotide sequence of the clone extending farthest from the 5 'end of the gene was determined. The 1258 base pair sequence contained one long open reading frame starting at the 5 'end of the clone and ending at the TAA stop codon. GDF-8 extending to the 5 'end of the clone with this open reading frame
(See below), this clone appeared to lack the coding sequence corresponding to the N-terminal portion of the GDF-11 precursor protein. GDF-1
To obtain the remainder of one sequence, a human genomic library was screened using a human GDF-11 cDNA probe to isolate some genomic clones. By analyzing a partial sequence of one of these genomic clones, this clone was identified as GDF-
It was found to contain 11 genes. From this clone the remaining GDF-11
The coding sequence was obtained. FIG. 1b shows G assembled from genomic and cDNA sequences.
3 shows the predicted sequence of DF-11. Nucleotides 136 to 1393 represent the sequence obtained from the cDNA clone. Nucleotides 1-135 were obtained from genomic clones. The sequence was arbitrarily numbered starting from the Sac II site present in the genomic clone, but the start site of the mRNA is not known. The sequence contains nucleotide 5
4 contains the putative starting methionine. The sequence upstream of this methionine codon is mRN
It is not known whether all are present in A. Starting from the methionine codon, the open reading frame is 407 amino acids long. The sequence contains one putative N-linked glycosylation site at asparagine 94. Amino acid 2 in the sequence
95-298 contains a predicted RXXR proteolytic cleavage site where cleavage of the precursor results in a predicted amino acid length of 109 amino acids and a predicted molecular weight of approximately 12,50.
Generates an active C-terminal fragment of 0 kD. In this region, the predicted GD of rats and humans
The F-11 amino acid sequences are 100% identical. The high degree of sequence conservation across species suggests that GDF-11 plays an important role in vivo.

The C-terminal region following the predicted cleavage site contains all the features present in other TGF-β family members. GDF-11 contains most of the residues that are highly conserved among other family members, including seven cysteine residues and their characteristic spatial arrangement. Like TGF-β, inhibin β and GDF-8, GDF-11 also contains two cysteine residues. In the case of TGF-β2, these additional cysteine residues are known to form intramolecular disulfide bonds (Daopin, et al., Science, 257: 369, 1992; Schlunegger and Grutter,
Nature, 358: 430, 1992). Amino acid sequence homology between GDF-11 and other TGF-β family members is tabulated in FIG. The numbers represent the amino acid identity between each pair calculated using the first conserved cysteine to the C-terminus.
Boxes represent homology between highly related members within a particular subgroup. In this region, GDF-11 is most closely associated with GDF-8 (92% sequence identity).

FIG. 4a shows the alignment of the amino acids of GDF-8 (SEQ ID NO: 5) and GDF-11 (SEQ ID NO: 6). The two sequences contain a similar site at the potential N-linked glycosylation signal (NIS) and a putative proteolytic processing site (RSR).
R). The two sequences are related not only in the C-terminal region (90% amino acid sequence identity) following the putative cleavage site, but also in the pro-region of the molecule (45% amino acid sequence identity).

Example 4 Construction of Hybrid GDF-8 / GDF-11 Gene In order to express GDF-11 protein, the N-terminal region of GDF-11 was
It was replaced with a similar region of DF-8. Such hybrid constructs are biologically active BMP-4 (Hammonds, et al., Mol. Endocrinol., 5: 149, 1991) and Vg-1 (Thomsen and Melton, Cell, 74: 433, 1993). ) Has been used to produce. To confirm that the GDF-11 protein produced from the hybrid construct was authentic GDF-11, a hybrid gene was constructed so that the fusion of the two gene fragments occurred exactly at the predicted cleavage site. In particular, at positions corresponding to the predicted proteolytic cleavage site, both sequences have AvaI
There is an I restriction site. The N-terminal pro-region of GDF-8 up to this AvaII site was obtained by partial digestion of the clone with AvaII,
Fused with the C-terminal region of GDF-11 beginning at the I site. The resulting hybrid construct is then transformed into a pMSXND mammalian expression vector (Lee and Nathans, J. Biol. Ch.
em., 263: 3521) to transform Chinese hamster ovary cells. As shown in FIG. 5, the conditioned medium derived from G418-resistant cells was subjected to Western analysis using an antibody against the C-terminal part of GDF-8.
Eleven proteins were secreted into the medium, indicating that at least some of the hybrid proteins were processed and proteolytically digested. Furthermore, this study indicated that antibodies against the C-terminal portion of GDF-8 also reacted with GDF-11 protein.

Example 5 Chromosomal Location of GDF-11 To determine the chromosomal location of GDF-11, a DNA sample from a human / rodent somatic cell hybrid (Drwings, et al., Genomics, 16 : 311-313, 1993;
Dubois and Naylor, Genomics, 16: 315-319, 1993) were analyzed by polymerase chain reaction followed by Southern blotting. Polymerase chain reaction was performed using primer # 101, 5'-GAGTCCCGCTGC
TGCCGATATCC-3 '(SEQ ID NO: 7) and primers # 102, 5'
Using -TAGAGCATGTTGATTGGGGGACAT-3 '(SEQ ID NO: 8), 35 cycles were performed at 94 ° C for 2 minutes, 58 ° C for 1 minute, and 72 ° C for 1 minute. These primers correspond to the reverse complement of nucleotides 981-1003 and 1182-1204, respectively, of the human GDF-11 sequence. The PCR product was electrophoresed on an agarose gel, blotted, and oligonucleotide # 104 corresponding to the internal sequence of the region adjacent to primers # 101 and # 102, 5'-AAATA
TCCGCATACCCATTT-3 ′ (SEQ ID NO: 9) was used as a probe. The filters were hybridized in 6xSSC, 1xDenhard's solution, 100 g / ml yeast transfer RNA, and 0.05% sodium pyrophosphate at 50C.

As shown in FIG. 6, the human-specific probe was a positive control sample (whole human genome D
A band of the expected size (about 224 base pairs) was detected in NA) and in one DNA sample from a human / rodent hybrid panel. This positive signal corresponds to human chromosome 12. The human chromosome contained in each of the hybrid cell lines was identified at the top of each of the first 24 lanes (1-22, X, and Y). In the lanes designated CHO, M, and H, the starting DNA template was total genomic DNA from hamster, mouse and human, respectively. In the lane marked B1, no template DNA was used. The numbers on the left indicate the mobility of the DNA standard. These data indicate that the human GDF-11 gene is located on chromosome 12.

To determine the more precise location of GDF-11 on chromosome 12, the GDF-11 gene was located by fluorescence in situ hybridization (FISH). FISH localization research was conducted by BIOS Laboratories (New Haven, Con
necticut). Purified DNA from the human GDF-11 genomic clone was labeled with digoxigenin dUTP by nick translation.
The labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes from PHA-stimulated peripheral blood leukocytes in a solution containing 50% formamide, 10% dextran sulfate and 2 × SSC. Specific hybridization signals were detected by incubating the hybridized slides in a fluorescently conjugated sheep anti-digoxigenin antibody. The slides were then counterstained with propidium iodide and analyzed. As shown in FIG. 7a, the results of this experiment resulted in specific labeling of the proximal long arm of the group C chromosome (its size and morphology correspond to chromosome 12). To confirm the identity of the specifically labeled chromosome, a second experiment was performed in which a chromosome 12-specific centromere probe was co-hybridized with GDF-11. As shown in FIG. 7b, this experiment clearly shows that GDF-11 is located at 23% of the centromeric to telomere distance of the long arm of chromosome 12, and this region corresponds to band 12q13 (FIG. 7c). A total of 85 metaphase cells were analyzed, of which 80 showed specific labeling.

Example 6 GDF-11 Homology in Mammalian Species Like most other TGF-β family members, GDF-11 also appears to be highly conserved among species. By Southern analysis of the genome, homologous sequences were detected in all mammalian species examined as in birds and frogs (FIG. 4b). In most species, the GDF-11 probe also detected a second, more subtlely hybridizing fragment corresponding to the myostatin gene (McPherron et al., 1997).

Example 7 To determine the biological function of GDF-11 transgenic knockout mouse GDF-11, we disrupted the GDF-11 gene in embryonic stem cells by homologous targeting. The murine 129 SV / J genomic library was prepared using the λFIXI
Prepared in I. The structure of the GDF-11 gene was derived from restriction mapping and partial sequencing of phage clones isolated from the library. Vectors for preparing the targeting constructs were kindly provided by Philip Soriano and Kirk Thomas. To ensure that the resulting mice do not express GDF-11 function, the entire mature C-terminal region was deleted and replaced with the neo cassette (Fig.
9a, b). Rl ES cells were transfected with the targeting construct, selected with ganciclovir (2 μM) and G418 (250 μg / ml), and analyzed by Southern analysis. Homologous targeting of the GDF-11 gene was found in the 8/155 ganciclovia / G418 double resistant ES cell clone. After injecting some targeted clones into C57BL / 6J blastocysts, we produced heterozygous offspring when crossed with both C57BL / 6J females and 129 / SvJ females Chimeras were obtained from one ES clone. Hybridization of C57BL / 6J / 129 / SvJ hybrid F1 heterozygotes produced mature offspring of 49 wild-type (34%) and 94 heterozygous mutants (66%), but suddenly homozygous Mutant mature offspring were not produced. Similarly, 129 / Sv
Even in the J background, no mature homozygous null animals were found (32 wild-type animals (36%) and 56 heterozygous mutant animals (64%)).

To determine the age at which a homozygous mutant dies, we used the litter genotype of embryos isolated at various gestational years from heterozygous females mated with heterozygous males. Were determined. When all embryonic stages were tested, homozygous mutant embryos were present at an estimated frequency of approximately 25%. Some hybrid neonatal mice have the expected Mendelian ratio
Different genotypes were also shown at 1: 2: 1 (34 + / + (28%), 61 +/- (50%), and 28-/-(23%)). Homozygous mutant mice were born alive, able to breathe and lactate. However, all homozygous mutants died within the first 24 hours after birth. The exact cause of the death is unknown, but its lethality may be related to the fact that the kidney of the homozygous mutant was severely hypoplastic or completely defective. The kidney abnormalities in these mice are summarized in FIG.

Example 8 Anatomical Differences in Knockout Mice Homozygous mutant animals could be easily identified by their very short or missing tails (FIG. 11a). To further characterize the tail defect in these homozygous mutant animals, we examined the animal's skeleton to determine the degree of tail vertebral destruction. However, comparing wild-type and mutant skeletal specimens of late embryo and neonatal mice revealed differences not only in the tail region of the animal, but also in many other regions. In almost all cases where differences were noted, the abnormalities indicated homeotic transformation of the vertebral somites, and in certain segments appeared to have a morphology typical of the anterior segment. These transformations, summarized in FIG. 12, were evident throughout the axial skeleton extending from the cervical region to the tail region. Other than the defects found in the axial skeleton, other skeletons, such as the skull and limb bones, appeared normal.

[0122] Anterior transformation of the vertebrae in the mutant neonatal animal was most easily identified in the thoracic region, where the number of thoracic (T) somites was dramatically increased. All wild-type mice tested exhibited a typical pattern of 13 thoracic vertebrae, each with attached rib pairs (FIG. 11 (b, e)). In contrast, homozygous mutant mice showed a marked increase in thoracic vertebrae number. All homozygous mutants tested were 4-5 pairs extra, totaling 17-
It had 18 pairs of ribs (Fig. 11 (d, g)), but more than one third of these
The ribs appeared stunted. Thus, it appeared that segments normally corresponding to the lumbar (L) segments L1-L4 or L5 were transformed into thoracic segments in the mutant animals.

In addition, transformation in the thoracic region where one thoracic vertebra had a characteristic morphology to the other thoracic vertebra was also evident. For example, in wild-type mice, the first seven pairs of ribs attach to the sternum,
The remaining 6 pairs are not attached, that is, free (FIG. 11 (e, h)). In homozygous mutants, the number of attached and free rib pairs was 10-11 and 7 respectively.
88 (FIG. 11 (g, j)). Thus, thoracic segments T8, T9, T10, and in some cases even T11, which have free ribs in wild-type animals, are also transformed in mutant animals and are typical of the anterior thoracic segment Characteristic (ie, the presence of ribs attached to the sternum). Consistent with this finding, migratory spinous processes and migratory articular processes normally found in T10 of wild-type animals were instead found in the homozygous mutant T13 (data not shown). Also, additional transformation in the thoracic region was observed in some mutant animals. For example, in wild-type mice, the ribs from T1 usually touch the upper part of the sternum. However tested 2 /
In 23 hybrids and 2/3 129 / SvJ homozygous mutant mice, T2 was T1
It was thought that it was transformed to have a morphology similar to In other words, in these animals, the ribs derived from T2 extended to touch the upper part of the sternum. In these cases,
Ribs from T1 appeared to fuse to the second rib pair. Finally, in 82% of the homozygous mutants, the long spinous processes normally present in T2 were shifted to T3. In some other homozygous mutants, asymmetric fusion of the true cost pair was observed at other breast levels.

[0124] Forward transformation was not limited to the thoracic region. The most anterior transformation we observed was at the level of the sixth cervical vertebra (C6).
In wild-type mice, C6 can be easily identified by the presence of two ventral anterior nodules. In some homozygous mutant mice, one of these two prenodes has C
At 6, but the other was instead at the C7 position. Therefore, in these mice, C7 appeared to have been partially transformed to have a morphology similar to C6. One other homozygous mutant had two prenodes at C7, but due to complete transformation of C6 to C7, but partial transformation of C6 to C5, one of them at C6 Was held.

Transformation of the axial skeleton also extended into the lumbar region. Wild-type animals usually have only six lumbar vertebrae. On the other hand, there were 8 to 9 homozygous mutants. The above data is 4-5
At least six of the lumbar vertebrae in the mutant are derived from the segment that normally gives rise to the sacral and caudal vertebrae, suggesting that lumbar segments have been transformed into thoracic segments It must be. Therefore, homozygous mutant mice had a total of 33-34 presacral vertebrae compared to the 26 presacral vertebrae normally present in wild type mice. The most common presacral vertebral patterns were C7 / T18 / L8 and C7 / T18 / L9 in mutant mice compared to C7 / T13 / L6 in wild-type mice. The presence of an additional anterior sacral vertebrae in the mutant animals was evident without detailed skeletal examination as the hind limb position had moved 7-8 somites posterior to the forelimb.

The sacral and caudal vertebrae are also affected in homozygous mutant mice, but the exact identity of each transformation was not readily identifiable. In wild-type mice,
Sacral segments S1 and S2 typically have wider transverse processes as compared to S3 and S4. The mutant did not appear to have any identifiable S1 or S2 vertebrae. Instead, the mutant animals had some vertebrae that appeared to have a morphology similar to S3. In addition, the transverse processes of all four sacral vertebrae are usually fused together, but in neonates often only the fusion of the first three vertebrae is observed. However, in homozygous mutants, the transverse processes of the sacral vertebra were not usually fused. In the caudalmost region, all mutant animals had severe malformed vertebrae with over-fused cartilage. Although the severity of the fusion makes it difficult to count the total number of vertebrae in the tail region, we were able to count up to 15 transverse processes in some animals. We could not establish morphological criteria to distinguish S4 from the caudal vertebra even in wild-type newborn animals, so these vertebrae represent the sacral or caudal vertebrae in the mutant Could not be determined. Regardless of their identity, the total number of vertebrae in this area has been significantly reduced from a normal number of about 30. Thus, the mutant had significantly more thoracic and lumbar vertebrae than wild-type mice, but in the mutant the total number of somites was reduced due to tail amputation.

Heterozygous mice also showed abnormalities in the axial skeleton, but the phenotype was much lighter than in homozygous mice. The most obvious abnormality in the heterozygous mice was the presence of an additional thoracic segment with attached rib pairs. (FIG. 11 (c, f)). This transformation was present in every heterozygous animal tested in each case, with additional rib pairs attached to the sternum (FIG. 11 (I)). Thus, T8, in which the attached ribs did not normally touch the sternum, appeared to have transformed into a morphology characteristic of the more anterior thoracic vertebra. Ll also appeared to have transformed into a characteristic morphology in the posterior thoracic vertebra. Furthermore, in heterozygous mice, many abnormalities indicating forward transformation were found to different degrees. These forward transformations include a one-segment shift to T3 of the long spinous process characteristic of T2, T
Shift of articular and spinous processes from 10 to T11, shift of the anterior nodule at C6 to C7, and transformation of T2 to T1 (the ribs attached to T2 touched the top of the sternum)
Is included.

To understand the basis of axis patterning abnormalities found in GDF-11 mutant mice, we examined mutant embryos isolated at various developmental stages and And compared. Overall morphological studies showed that homozygous mutant embryos isolated by 9.5 days of gestation were not easily distinguishable from their corresponding wild-type embryos. In particular, the number of somites present at a given age of development was the same between mutant and wild-type embryos, suggesting that the rate of segment formation was not altered in mutants. I have. By 10.5-11.5 days after conception, mutant embryos could be easily distinguished from wild-type embryos by the hind limb moving backward by 7-8 somites. Tail development abnormalities were also easily identified at this stage. Taken together, these data indicate that the abnormalities observed in the mutant scaffold were not due to additional segment insertion but to true transformation of the segment itself (eg, due to an increased rate of somatogenesis). Abnormal).

By altering the expression of the gene-containing homeobox, Drosophila (Drosop
hila) and vertebrates are known to cause transformation.
To determine whether the expression pattern of the Hox gene (vertebrate homeobox-containing gene) has been altered in the GDF-11 null mutant, we
Three representative H in wild-type, heterozygous and homozygous mutant embryos at 12.5 days post conception by whole mount in situ hybridization
The expression patterns of the ox gene, Hoxc-6, Hoxc-8 and Hoxc-11 were examined. The expression pattern of Hoxc-6 in wild-type embryos extended to the anterior vertebrae 8-15 corresponding to thoracic somites T1-T8. However, in the homozygous mutant, the Hoxc-6 expression pattern shifted backwards and extended to the anterior vertebrae 9-18 (T2-T11). A similar shift is Hoxc-8
It was also found using a probe. In wild-type embryos, Hoxc-8 is anterior vertebrae 13-18 (
Hoxc-8 was expressed in the anterior vertebrae 14-2
2 (T7-T15). Finally, Hoxc-11 expression also changed in that the anterior border of expression changed from anterior vertebra 28 in wild-type embryos to anterior vertebra 36 in mutant embryos.
Shifted backwards. (Note that the Hoxc-11 expression patterns of wild-type and mutant appear to be similar to hind limbs, as hind limb positions also shift backwards in mutant embryos.) Provides further evidence that skeletal abnormalities found in mutant animals indicate homeotic transformation.

The phenotype of the GDF-11 mouse suggested that GDF-11 acts as a global regulator of axis patterning early in embryogenesis. To begin investigating the mechanism by which GDF-11 exerts its effects, we started whole mount in situ.
The expression pattern of GDF-11 in early mouse embryos was examined by hybridization. At these stages, major sites of GDF-11 expression correlated exactly with known sites where mesoderm cells were produced. GDF-11 expression is initially at 8.25-8.5 days after conception (8-10
Somites) were detected in the primordial streak region where the transferred cells formed the mesoderm of the developing embryo. At day 8.75, but by day 9.5 after conception, GDF-11 expression is maintained when expression is maintained in the primordial streak and the tail bud replaces the primordial streak as a source of new mesoderm cells. Migrated to tail bud. Thus, at these early stages, GDF-11 appears to be synthesized in areas of the developing embryo, where new mesoderm cells arise and perhaps acquire their positional identity.

In some respects, the phenotype of the GDF-11 knockout mouse is that the receptor for some members of the TGF-β superfamily, the activin type IIB receptor (ActRI
IB) mimics the phenotype of mice with deletions. As in GDF-11 knockout mice, ActRIIB knockout mice have extra rib pairs and kidney defects ranging from renal hypoplasia to complete loss of kidney. The phenotypic similarity of these mice raises the possibility that ActRIIB may be a receptor for GDF-11. However, ActRIIB cannot be the only receptor for GDF-11. Because G
This is because the phenotype of DF-11 knockout mice is even more severe than that of ActRIIB mice. For example, GDF-11 knockout animals have 4-5 extra rib pairs and exhibit homeotic transformation throughout the axial skeleton, whereas ActRIIB knockout animals have only 3 extra rib pairs, Does not show transformation at other axis levels. Other data indicate that kidney defects in GDF-11 knockout mice are also more severe than those in ActRIIB knockout mice. ActRIIB
Knockout mice show defects in left / right axis formation, such as pulmonary isomerism and a range of cardiac defects that we have not yet observed in GDF-11 knockout mice. ActRIIB can bind to activin and certain BMPs, but none of the knockout mice generated for these ligands show defects in left / right axis formation.

If GDF-11 acts directly on mesodermal cells to establish positional identity, the data presented here are consistent with either short-range or morphogen models for GDF-11 action right. That is, GDF-11 may act on mesoderm progenitors to establish Hox gene expression patterns as if these cells were produced at the GDF-11 expression site, or to be produced at the posterior end of the embryo GDF-11
Can diffuse to form a morphogen gradient. Regardless of the mechanism of action of GDF-11, the fact that rough pre / post pattern formation still occurs in GDF-11 knockout animals suggests that GDF-11 may not be the only regulator of pre / post specification. It suggests. Nevertheless, it is clear that GDF-11 plays an important role as a global regulator of axis patterning, and further studies on this molecule will show how positional identity along the anterior / posterior axis can New important insights about what is established in animal embryos will be gained.

Although the present invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention.
Accordingly, the invention is limited only by the claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the nucleotide and deduced amino acid sequences of mouse (FIG. 1a) and human (FIG. 1b) GDF-11. Putative proteolytic processing sites are indicated by shaded boxes. In the human sequence, potential N-linked glycosylation signals are indicated by open squares, and the common polyadenylation signal is underlined. The poly A tail is not shown.

FIG. 2 shows Northern blots of RNA prepared from adult (FIG. 2a) or fetal and neonatal (FIG. 2b) tissues, caught using a mouse GDF-11 probe.

FIG. 3 shows amino acid homology with different members of the TGF-β superfamily. Numbers indicate percent amino acid identity between each pair calculated from the first conserved cysteine to the C-terminus. Boxes indicate homology between highly related members within a particular subgroup.

FIG. 4a shows the putative amino acid sequences of human GDF-11 (top) and human GDF-8 (bottom) side by side. Vertical lines indicate that they are the same. The dots represent gaps introduced to maximize alignment. Numbers represent amino acid positions from the N-terminus. Putative proteolytic processing sites are indicated by open squares. Conserved cysteine residues in the C-terminal region are indicated by shaded squares. FIG. 4b shows mouse (McPherron et al., 1997) and human (McPherron and Lee, 1997).
The deduced amino acid sequences of myostatin (MSTN) and mouse and human GDF-11 are shown side by side. Shaded boxes indicate amino acid homology with mouse and human GDF-11 sequences. Amino acids are numbered relative to the human GDF-11 sequence. The putative proteolytic processing site is located at amino acids 295-298.

FIG. 5 shows GDF-11 expression in mammalian cells. If prepared from Chinese hamster ovary cells transfected with the hybrid GDF-8 / GDF-11 gene (see text) cloned in the antisense (lane 1) or sense (lane 2) direction into the MSXND expression vector Dialysed medium
Lyophilized and subjected to Western analysis using antibodies raised against the C-terminal part of the GDF-8 protein. Arrows on the right indicate putative unprocessed (pre-GDF-8 / GDF-11) or processed GDF-11 protein. The numbers on the left indicate the mobility of the molecular weight standard.

FIG. 6 shows chromosome mapping of human GDF-11. DNA samples prepared from human / rodent somatic cell lines were subjected to PCR, electrophoresed on agarose gels, blotted and probed. The human chromosome contained in each of the hybrid cell lines is identified at the top of each of the first 24 lanes (1-22, X and Y). In the lanes marked CHO, M and H, the first DNA template was whole genomic DNA from hamster, mouse and human, respectively. In the lane marked B1, no template DNA was used. The numbers on the left indicate the mobility of the DNA standard.

FIG. 7 shows FISH localization of GDF-11. A digoxigenin-labeled human GDF-11 probe (a) is used for metaphase chromosomes derived from peripheral blood lymphocytes.
), Or a mixture of a human GDF-11 genomic probe and a chromosome 12-specific centromere probe (b), and analyzed as described herein. A diagram showing that GDF-11 exists at position 12q13 is listed in panel (c).

FIG. 8 shows Southern blot analysis of genomic DNA isolated from different species.

FIG. 9 shows the construction of GDF-11 null mice by homology targeting. a) Map of the GDF-11 locus (top line) and targeting construct (second line). Solid and shaded boxes represent coding sequences for the pro- and C-terminal regions, respectively. The targeting construct is GDF-1
It has 11 kb homology with one gene in total. Probes derived from the region upstream of the 3 'homology fragment and downstream of the first EcoR1 site shown are GDF-
Hybridizes to a 6.5 kb EcoR1 fragment in 11 genes and a 4.8 kb fragment in a homologously targeted gene. Abbreviations: X is Xba1; E is EcoR1. b
3.) Southern blot of genomic DNA prepared from F1 heterozygous mutant mice (lanes 1 and 2) and progeny derived by crossing these mice (lanes 3-12).

FIG. 10 shows kidney abnormalities in GDF-11 knockout mice. The kidneys of the newborn animals were examined and classified according to the number of normal or small kidneys, as indicated above. The numbers in the table are the number of animals that fall into each category according to genotype.

FIG. 11 shows homeotic transformation in GDF-11 mutant mice. a
) Newborn dog without tail (first and second from left) and normal tail. b
) -J) Skeletal preparations of newborn wild type (b, e, h), heterozygous (c, f, i) and homozygous (d, g, j) mutant mice. Whole skeletal preparations (b-d), spine (eg), showing transformation and deletion in homozygous and heterozygous mutant mice,
True ribs (h to j). The numbers indicate thoracic segments.

FIG. 12. Ventral (an) profiles in wild-type, heterozygous and homozygous GDF-11 mice.
terior) Table summarizing the transformations.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 13/12 A61P 13/12 19/00 19/00 21/00 21/00 C12N 15/09 ZNA A23L 1 / 31 A // A23L 1/31 C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE) , LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, B A, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor McFarron, Alexandra, C. United States 21201 Platte Street, Baltimore, MD 218 No. 519 F-term (reference) 4B024 AA05 BA21 BA43 CA03 CA04 DA02 EA04 GA11 HA12 HA15 4B042 AC04 AC05 AG01 AH01 AP30 4C084 AA13 BA35 DB52 NA14 ZA812 ZA85 AA1322C DDB EE01 4C086 AA01 AA02 AA04 EA16 MA04 MA24 NA14 ZA81 ZA94 ZA96 ZB21 ZC78

Claims (12)

[Claims] 1. A method for producing an animal food having an increased number of ribs, the method comprising: a) transgene disrupting or preventing the expression of growth differentiation factor-11 (GDF-11); B) maturing the embryo into a term term offspring by implanting the embryo into a fallopian tube of a pseudopregnant female animal; c) transducing the offspring for the presence of the transgene. Testing and identifying transgene-positive progeny; d) performing a cross-breeding of the transgene-positive progeny to obtain further transgene-positive progeny; and e) processing said progeny to obtain food. The above method, comprising: 2. The method of claim 1, wherein said transgene comprises a GDF-11 antisense polynucleotide. 3. The method of claim 1, wherein said transgene comprises a gene encoding a dominant negative GDF-11 polypeptide. 4. A method for producing an avian, porcine or bovine food with an increased number of ribs, comprising: a) a transgene that disrupts or prevents the expression of growth differentiation factor-11 (GDF-11). Introducing into an avian, porcine, or bovine embryo; b) culturing said embryo under conditions that will produce offspring; c) testing said offspring for the presence of a transgene, transgene positive Identifying said progeny, d) performing a cross-breeding of transgene-positive progeny, and e) processing said progeny to obtain food. 5. The method of claim 4, wherein said transgene comprises a GDF-11 antisense polynucleotide. 6. The method of claim 4, wherein said transgene comprises a gene encoding a dominant negative GDF-11 polypeptide. 7. The transgenic animal of claim 4, wherein said transgene comprises a polynucleotide encoding a truncated GDF-11 polypeptide. 8. A method for treating chronic or acute kidney disease in a subject, comprising administering to a subject suffering from chronic or acute kidney disease a reagent that affects GDF-11 activity or expression. 9. The method of claim 8, wherein said reagent is an agonist of GDF-11. 10. The method of claim 8, wherein said reagent is an antagonist of GDF-11. 11. The method of claim 10, wherein said antagonist is an antibody against GDF-11. 12. The method of claim 10, wherein said antagonist is an antisense polynucleotide to GDF-11.