CA1316851C - Transgenic animals incorporating exogenous grf genes - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
TRANSGENIC ANIMALS INCORPORATING
EXOGENOUS GROWTH HORMONE RELEASING FACTOR (GRF) GENES

Transgenic animals are produced having an exogenous GRF-encoding gene inserted in their genome. The preferred gene includes a GRF-encoding sequence portion operably linked to a strong promoter, such as the metallothionein promoter, and the preferred GRF-encoding sequence portion is a fusion of natural gene sequences and cDNA sequences. The GRF-encoding gene is microinjected into fertilized eggs which then incorporate the gene into their genome, and the fertilized eggs are implanted into host mothers. The animals that are born as a result are tested to determine whether the exogenous gene is incorporated and expressed. The transgenic animals grow to larger than normal size. Both male and female transgenic animals are fertile, whereby the animals are useful for breeding to establish fast-growing, enlarged animal lines.

Description

- 1316~5 TRANSGENIC ANIMALS INCORPORATING
EXOGENOUS GRF GENES
The present invention is directed to producing faster growing and enlarged animals and to the animals produced thereby.
BACKGROUND OF THE INVENTION
Recombinant DNA technologies have opened up the possibility of producing genetically-altered animalsO
An important field where research has been conducted and where research is ongoing is the possible correction of genetic defects, particularly with respect to eventual correction of genetic defects in humans. For example, it has been proposed that hemoglobinopathies might be corrected by replacing abnormal globin genes with normal globin genes. Preliminary to such effort, E.
Wagner et al. P.N.A.S. 78, 5016-5020 (1981) and T. Wagner et al. P.N.A.S. 78, 6376-6380 (1981) have inserted exogenous globin genes into mammalian embryos and have thereby produced transgenic mammals which carry the gene and in some cases express the gene product.
Although this work may eventually lead to gene therapy in humans, genetic experimentation with humans or human embryos is still considered to be some distance in the future.
Of more immediate potential importance is the production of transgenic, non-human animals which exhibit specific useful attributes. For example, it is desirable to have meat-producing animals which more efficiently metabolize feed and grow both faster and larger. Increased lactation in milk-producing animals and enhanced disease-resistance are other desirable attributes which potentially could be imparted to a transgenic animal.
Palmiter et al., Nature 300, 611-615 (1982) report transgenic mice that developed from embryos which were transformed with a recombinant gene that included a rat growth hormone-encoding DNA sequence. The mice were found to grow to up to double the normal weight of : . . . .

1316~5 similar mice and were found to have up to 1000 times the normal levels of growth hormone (GH) in their peripheral blood serum.
The dramatic increases in growth rate and adult size of transgenic mice incorporating the exogenous growth hormone gene in their genome suggests that transgenic meat-producing animals might similarly be produced which would also grow larger. Reproducing strains of transgenic animals could potentially substantially increase meat supplies and/or reduce the cost of producing meat. To date, the use of transgenic animals for producing meat has not been described.
One important problem with transgenic animals carrying an exogenous GH gene is that the transgenic females tend to be sterile, posing very substantial problems with respect to establishing reproducible strains and particularly in producing strains that are homozygous for the exogenous gene. Why the fertility problem exists has not been determined; however, the highly elevated levels of the powerful growth hormone may upset other endocrine systems, including those which regulate fertility. The exogenous growth hormone as described by Palmiter et al., Nature 300 (1982~ supra., unlike endogenous growth hormone, is expressed in a variety of organs throughout the body and is, therefore, ; not subject to normal regulatory mechanisms which control its level of production and release.
Growth hormone molecules are large, e.g., typically about Y21 kD, and exhibit a good deal of structural differences from species to species~
Although growth hormone molecules frequently exhibit cross-species hormonal activity, the different structures of the various growth hormones may induce undesirable immune responses in foreign species. Thus, a transgenic animal may exhibit a significant autoimmune response to growth hormone expressed by its exogenous genetic material.

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- I 3 1 6 ~ r Growth hormone releasing factor (GRF3 is a hormone which acts on the pituitary to effect the release of GH. However, R. Clark & I. Robinson, Nature 314, 281-283 (1985) reported that continuously administered doses of GRF are ineffective in increasing growth rates, thus suggesting that exogenous GRF, as would be expressed by an exogenous GRF-encoding gene, would not increase either the growth rate or the eventual size of the animals. An exogenous GRF gene, like an exogenous GH gene, would be expected to be expressed in widely distributed organs, not only the hypothalamus, and therefore would be expected to be outside of the control of the normal endocrine regulatory mechanisms, thereby causing the overexpression of GRF, a result which has been shown to be inconsistent with increased growth rate.
The present invention creates transgenic animals which incorporate an exogenous gene encoding growth hormone releasing factor, and surprisingly and unexpectedly, exhibit increased growth rate and an increase in the eventual size of the animal which is produced. The expression of GRF in transgenic animals carrying the GRF gene is widely distributed in various organs, and GRF production in such animals is highly elevated and appears to be continuous; nevertheless, in contrast to experiments in which the exogenous GRF was continuously introduced by infusion into the bloodstream without enhancing growth rate, the exogenous, gene-expressed GRF enhances the growth rate and the eventual size of the animals.
Of significant interest to the background of the invention are numerous publications of prior investigations relating to regulation of mammalian gene expression and to introduction of purified genes into eukaryotic cells.
Specifically indicating the background of the invention and illustrating the state of the prior art . . .
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- 1 3 1 6 ~j r are the following publications: Durnam, et al., "Isolation and Characterization of the Mouse Metallothionein-I Gene", P N.A.S. 77, 6511-6515 (1980);
Durnam, et al., J. Biol. Chem~ 256, 5712-5716 (1981);
Mayo, et al., J. Biol. Chem. 256 2621-2624 (1981);
Hager, et al., Nature, 291, 30-342 (1981);
Glanville, et al., Nature, 292, 267-269 (1981); and Beach, et al., P.N.A.S. 78, 2210-2214 (1981). The foregoing all deal with DNA sequences specifying production of low molecular weight, metal-binding protein found in one or more forms in most vertebrate tissues. More particularly, the publications treat mouse metallothionein genes as well as their promoter/regulator DNA sequences and the responsiveness of such promoter/regulator sequences to metals and steroid hormones.
Additional relevant publications are:
McKnight, et alO, J. Biol. Chem. 255, 148-153 (1980);
and Palmiter, et al., J. Biol. Chem. 256, 7910-7916 (1981). Also relevant is a publication dealing with microinjection of plasmids into germinal vesicles of mouse oocytes or pronuclei of fertilizes mouse ova, Brinster, et al., Science 211, 396-398 (1981).
The following publications of Evans and co-workers dealing with growth hormone releasing factor mRNA sequences and also dealing with the cloning of rat growth hormone genes and their introduction into and expression in mammalian cells are also relevant: (1) M.M. Harpold, P.R. Dobner, R.M. Evans and F.C. Bancroft, Construction and identification by positive hybridization-translation of a bacterial plasmid containing a rat growth hormone structural gene sequence, Nucleic Acids Research 5, 2039-2053 (1978);

(2) M.M. Harpold, P.R. Dobner, R.M. Evans, F.C. Bancroft and J.E. Darnell, Jr., The synthesis and processing of a nuclear RNA precursor to a rat pregrowth hormone messenger RNA, Nucleic Acids Research 6, 3133-3144 .
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135l~85 (1979); (3) H. Soreq, M. Harpold, R.M. Evans, JoEo Darnell, Jr. and F.C. Bancroft, Rat growth hormone gene: Intervening sequences separate the mRNA regions, Nucleic Acids Research 6, 2471-2482 (1979); (4) Doehmer, J., Barinaga, M., Vale, W., Rosenfeld, M.G., Verma, I.M.
and Evans, R.M.I Introduction of rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector:
Expression and regulation, P.N.A.S. 79, 2268-2272 (1982); (5) Evans, R.M., Birnberg, N.C. and Rosenfeld, M.G., Glucocorticoid and thyroid hormone transcriptionally regulate growth hormone gene expression, P.N.A.S. 79, 7659-7663 (1982); (6) Mayo~
K.E., Vale, W., Rivier, J., Rosenfeld, M.G. and Evans, R.M., Expression cloning and sequence of a cDNA encoding human growth hormone releasing factor, Nature 306, 86-88 (1983); (7) Barinaga, M., Yamomoto, G., Rivier, C., and Evans, R.M. Growth ho ~one releasing factor transcriptionally regulates growth hormone expression, Nature 306, 84-85 (1983); (8) Verma, I., Doehmer, J., Barinaga, M., Rosenfeld, M. and Evans, R.M., In:
Eukaryotic Viral Vectors, Y. Gluzman, ed., Cold Spring Harbor (1982).
Also pertinent to the background of the present invention are the publications of Illmensee, et al., Cell 23, 9-18 (1981) and Gordon, et al., P.N.A.S. 77, 7380-7384 (1981) which respectively treat injection of nuclei into nucleated mouse eggs and introduction of plasmids containing the herpes thymidine kinase gene and SV40 (Simian virus) sequences into mice.
SUMMARY OF THE INVENTION
The present invention is directed to producing transgenic animals which incorporate exogenous GRF in their genome and, as a result, grow at a faster than normal rate and achieve a greater than normal adult size. Such transgenic animals are particularly useful as meat-producers and/or milk-producers. To produce the transgenic animals, a gene that encodes a GRF, for .

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131~35 example, human GRF (hGRF), is isolated. To enhance expression, a gene portion which encodes GRF is linked to a powerful promoter sequence, such as a mammalian methallothionein gene promoter. A fertilized egg is obtained from an animal of the species into which the exogenous gene is to be introduced, and the exogenous GRF gene is introduced into fertilized eggs, e.g., by microinjection into each egg. The injected fertilized eggs are implanted into host female animals which give birth to transgenic animals. Animals initially selected for further development are those which are shown to incorporate the foreign genetic material in their genome. Development of the animals is followed, and transgenic animals are selected for breeding purposes which exhibit elevated growth rates and achieve greater adult size. Transgenic animals which incorporate an exogenous GRF gene, both male and female, are generally fertile and pass on the exogenous gene to their progeny by normal hereditary mechanisms.
IN THE DRAWINGS
FIGURE 1 Shows structure of a mouse metallothionein-I/human growth hormone-releasing factor fusion gene, MT-GRF. The upper portion of the figure is a restriction map for common 6-bp cutters. BglII/SmaI
indicates the site of the fusion between the mouse MT-I
promoter and a human GRF "minigene". The lower portion of FIGURE 1 is a schematic representation of the 2.5-kb EcoRI-HindIII fragment of DNA used for microinjection.
Open boxes represent 5' and 3' untranslated exon regions, and shaded boxes represent the coding exon regions. The derivations of various parts of the fusion gene are indicated. The fusion occurs in the 5' untranslated regions of both genes. The large exon labelled 3-5 was constructed by replacing part of the human GRF gene with a human GRF cDNA, effectively eliminati ~ intron C (2.4 kb) and intron D (3.0 kb) of the human GRF gene. Consensus sequences involved in : , '~
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- 1 31 6~5 transcription initiation, translation initiation and termination, polyadenylation, and proteolytic processing of the GRF peptide from its precursor are shown.
FIGURES 2A-2E illustrate the stepwise construction of the GRF "minigene" and the fusion gene designated "MT-GRF".
FIGURE 3 is a diagram of pedigree analysis of MT-GRF transgenic mice. Mice that carry the MT-GRF
fusion gene ~determined by DNA blotting of tail snips) are indicated by the solid symbols. Squares represent males and circles represent females. The animal number is given above each symbol; the numbers below the solid symbols indicate increased weight ratios at 9 weeks old compared with age- and sex-matched littermates. FIG. 3a shows the pedigree of MT-GRF mouse 762-5; FIG. 3b shows that of MT-GRF mouse 765-2. Mouse 765-2-6 died but was previously scored as a non-carrier.
Figure 4 is a copy of Northern Blot analysis of messenger RNA for a control mouse depicting the presence or absence of mRNA for MT-I and for GRF in RNAs isolated from the six different tissues obtained from the animal.
DETAILED ~ESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
In accordance with the present invention, transgenic animals incorporating exogenous GRF-encoding genes in their genome express exogenous GRF hormone and exhibit enhanced growth rate and enlarged adult size.
Importantly, both male and female transgenic animals carrying the exogenous GRF gene are fertile and pass on the gene to their progeny. Production of such a transgenic animal begins with isolating a GRF-encoding yene. Preferably the process begins with constructing, through gene splicing techniques, a recombinant GRF-encoding gene with enhanced expression capabilities.
Fertilized eggs are obtained from animals of a desired species, and the constructed GRF genes are microinjected into the eggs, e.g., into the male pronuclei of fertilized eggs. The microinjected eggs, some of which :

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1316~5 -7b-incorporate the injected GRF genes into their genomes, are implanted into host mothers of the species from which the eggs are obtained. Animals which develop from the eggs and are born are initially tested for ., :
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8 1316~i3,, incorporation of the exogenous gene, e.g., with appropriate DNA probes. As the animals which incorporate the exogenous gene develop, they are followed for increased expression of GRF and GRF mRNA, increased secretion of GH, and manifestations of increased growth. Suitable transgenic animals are bred, and those of their progency which similarly express exogenous GRF are used to establish new breeding lines.
A GRF gene used to produce a transgenic animals includes a GRF-encoding DNA sequence and a promoter DNA
sequence to which the GRF-encoding sequence is operably linked. Although the promoter sequence could be the natural GRF promoter sequence, it is preferred to link the GRF-encoding sequence to a stronger promoter, such as a promoter of a mammalian metallothionein gene. This helps to ensure expression of the exogenous G~F in the transgenic animal and desirably promotes expression of the exogenous GRF at significantly elevated levels, relative to normal levels of endogenous GRF. As it is intended that the transgenic animal produce significantly elevated levels of GRF, the promoter preferably helps to ensure that GRF is expressed in widely distributed organs, not only in the hypothalamus, and thus, a promoter is preferably linked to the GRF-encoding sequence which is known to promote expression in widely distributed organs.
Preferably, the promoter sequence includes or is linked to a regulatory sequence by which expression of the gene in the transgenic animal may be controlled by exogenous agents, such as a metal or hormone whi~h is fed to or inoculated into the animalO Promoter/regulator DNA sequences suitable for use in practice of the invention are derived from avian and mammalian cells and include: the iron and steroid hormone-responsive promoter/regulator sequence naturally associated with the transferrin (conalbumin) gene of chickens; the steroid hormone-responsive promoter/regulator sequence .: . . ..
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g associated with ovalbumin gene in chickens; and the metal and steroid hormone-responsive promoter/regulator sequence of the mouse metallothionein-I or metallothionein-II genes.
The invention, however, is not limited to promoters with associated regulator DNA sequences.
Examples of promoter DNA sequences which are not either meta] or steriod- or hormone-responsive which might be employed include: liver promoters, i.e. albumin, glycolytic enzymes, transferrin, caeruloplasmin and alpha-2-microglobulin; histocompatibility gene promoters, immunoglobulin gene promoters, interferon gene promoters, heat shock gene promoters and retroviral gene promoters.
In accordance with a preferred aspect of the invention, the GRF-encoding sequence is a "minigene"
which is a hybrid construct of natural gene sequences and cDNA sequences. The hybrid construct substantially reduces the length of the gene relative to the natural genomic sequence, which is generally so long that it is difficult to work with. The natural human genomic sequence encoding GRF, for example, is 10 kb long and contains 4 introns. Although the entire sequence of the human GRF genomic sequence is known, the long sequence has previously been cloned only in portions, and, therefore, the cloned sequences must be appropriately pieced together to form the entire hGRF genomic sequence. CDNA, on the other hand, is much shorter, by reason of eliminating all introns from the natural genomic sequence. cDNA, however, lacks genomic regulatory elements, such as polyadenylation sequences.
GRF minigenes, according to the invention, are designed to incorporate the desirable features of the natural genomic sequence, including regulatory sequences, and the desirable feature of shortness of the CDNA. The GRF
minigene therefore includes at least one sequence corresponding to the natural genomic sequences and at ; . :

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1 3 ~ 6~5 least one sequence corresponding to the cDNA sequence.
Preferably, in order to include both 5' and 3' regulatory sequences of genomic DNA, a minigene includes a 5' sequence corresponding to the natural GRF genomic sequence, a middle cDNA sequence and a 3' sequence corresponding to the natural GRF genomic sequenGe.
Because eukaryotic genes generally include introns, it is assumed that a gene will be best recognized in a eukaryotic host cell as a eukaryotic gene and be appropriately processed if it contains at least one intron, therefore, preferred GRF minigenes have fewer introns than the natural GRF gene, but retain at least one intron.
The invention will now be described in greater detail by way of specific examples.

This example relates to procedures for preparation of a fusion GRF gene and use of the fusion gene to produce transgenic mice. In this example, a DNA
plasmid, pMThGRF, is produced which includes a DNA
sequence encoding the hGRF structural gene which is operatively associated with the promoter/regulator DNA
sequence of the mouse metallothionein-I (M~-I) gene.
The MT portion of the fusion gene was constructed using p d mlpEE3.8, a plasmid in which the 2.8 kb EcoRI-EcoRI fragment of me-lambda 26 is inserted at the EcoRI site of pBR322, as disclosed in Durnam, et al., P N.A.S. 77, 6511-6515 (1980).
-A fragment of a cloned human growth hormone releasing factor gene, from which the 5' regulatoryreyion has been deleted, is fused to the MT-I
promoter/regulator region to form plasmid pMTGRF. More specifically, the unique B~II site of the MT-I genomic clone mlpEE3 8 is destroyed by digesting with BglII, followed by filling in the sticky ends with Klenow fragment of DNA polymerase in the presence of ATP, GTP, CTP and TTP. This plasmid is digested with PvuII and is . . .

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ligated to the blunt ends of a _maI-to-SmaI fragment containing the human GRF structural gene to give MThGRF
(8.3 kb). A 4.3 kb BstEII to _maI fragment is used for subsequent injection studies. The fusion gene is predicted to direct transcription of an mRNA containing 68 bases contributed by MT-I, followed by the entire human GRF sequence.
The 4.3 kb fragment fusion gene extending from the BstEII site of MT-l to the SmaI site of GRF is restricted from MThGRF and separated from other fragments on an agarose gel and used for microinjection into fertilized eggs. The male pronuclei of fertilized eggs are microinjected with 2 picoliters containing about 1,000 copies of this fragment, and 170 eggs are inserted into the reproductive tracts of foster mothers. 20 mice develop from these eggs.
When the mice are weaned, total nucleic acid is extracted from a piece of tail and used for DNA dot hybridization to determine which animals carry MThGRF
sequences. Using a nick-translated probe complementary to the hGRF gene, 7 of the animals give hydridization signals above background, and their DNA is analyzed further by restriction enzyme digestion and Southern blotting. This analysis shows that all 7 animals have a ~ 25 predicted intact hGRF fragment. 7-21 weeks post -; parturition, peripheral blood serum is obtained from each of the hybridization probe-positive mice. The presence of human growth hormone releasing factor in the sera is determined by radioimmunoassay using antibody raised against human GRF. The levels of human GRF in the mice sera are determined by radioimmunoassay to range from about 10 to about 1,000 ng per milliliter.
GRF is normally not present in the peripheral blood of either mice or humans in amounts approaching one nanogram per ml concentrations.
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This example relates to procedures for preparation of another fusion gene of the present invention. In this example, a DNA plasmid, pThGRFr is 5 shown to include a DNA sequence coding for human growth hormone releasing Eactor structural gene which is operatively associated with the promoter/regulator DNA
sequence of the chicken transferrin gene. The fusion gene is constructed using plasmid pl7 disclosed in McKnight, et al., Cell 34, p. 335-341 (1983).
A fragment of a cloned GRF from which the 5' regulatory region has been deleted is fused to a transferrin promoter/regulator region to form plasmid pThGRF. More specifically, the unique EcoRI site in the first intervening sequence of the transferrin genomic clone pl7 is destroyed by digesting with EcoRI, followed by filling in the sticky ends with Klenow fragment of DNA polymerase in the presence of ATP, GTP, CTP and TTP. This plasmid is digested with PvuII and is ligated to the blunt ends of a SmaI to _maI fragment containing the entire human growth hormone releasing factor structural gene to give pThGRF. A 7.3 kb _maI-to~_maI
fragment is used for subsequent injection studies which is isolated from an agarose gel by the NaClO4 method of Chen, et al., Anal. Biochem. 101, 339-341 (1980) .
The fusion gene is predicted to direct transcription of an mRN~ containing 5' untranslated sequences contributed by the transferrin gene, followed by the entire GRF gene.
The 7.3 kb fragment fusion gene extending from the KpnI site of transferrin (-185) to the SmaI site of GRF is restricted from pThGRF, separated from other fragments on an agarose gel and used for injection into eggs. The male pronuclei of fertilized eggs are microinjected with 2 picoliters containing about 1,000 copies of this fragment, and 170 eggs are inserted into the reproductive tracts of foster mothers as in Example I. 20 mice develop from these eggs.

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~' 1316~5 When the mice are weaned, total nucleic acid is extracted from a piece of tail and used for DNA dot hybridization to determine which animals carry pThGRF
sequencesO Using a nick-translated probe complementary to the GRF gene, 7 of the animals give hybridization signals above background, and their DNA is analyzed further by restriction with restriction enzymes and Southern blotting. This analysis shows that all 7 have a predicted intact hGRF fragment. 7-21 weeks post parturition, peripheral blood serum is obtained from each of the hybridization probe-positive mice. The presence of human growth hormone releasing factor in the sera is determined by radioimmunoassay according to the method of Doehmer, J. et al., P.N.A.S. 79, 2268 (1982), using antibodies raised against GRF as described in Rivier et al., Nature 30_, p. 276 (1982). The level of GRF in the mice sera is determined by radioimmunoassay to range from about 10 to about 1,000 ng per milliliter.

This example represents a preferred embodiment of the present invention in which the promoter sequence is a mammalian metallothionein promoter and the GRF-encoding portion includes segments from -the natural genomic sequence that encodes the human GRF precursor protein as well as sequences from corresponding cDNA, resulting in a "minigene" which is shortened relative to the natural genomic sequence by deleting all introns but the second intron.
The structure of the mouse MT-I/human GRF
fusion gene construct used (referred to as MT-GRF) is shown in Figure 1. A 770-base pair (bp) fragment of the mouse MT-I gene, including sequences responsible for metal-inducibility and transcription initiation, was fused to a human GRF "minigene" which includes the entire coding region of the GRF precursor protein. The human GRF minigene was created by combining cDNA and genomic clones such that the 10-kilobase (kb) human GRF

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- 1316.`j, gene, which normally includes five exons (K.E.
Mayo et al., P.N.A.S. 82, 63-67 (1985)), has been reduced to less than 1 kb and retains a single intron of 230 bp (see Fi~ure 1) . Analysis of human GRF cDNA
clones suggests that the GRF precursor protein can consist of either 107 or 108 amino acids, differing in the presence or absence of serine 103 (U. Gubler et al., P.N.A.S. 80, 4311-4314 (1983)). DNA sequence analysis of the human GRF gene indicates that this difference may be explained by alternative RNA processing. The cDNA
used herein to construct the MT-GRF fusion gene encodes only the 108-amino-acid form of the GRF precursor protein.
More particularly, the GRF "minigene" and the MT-GRF construct were constructed in a multistep process as diag rammed in Figures 2A-2E.
Step A. A vector was constructed that is a derivative of pBR322 but is deleted between the EcoRV and PvuII sites. This was done by digesting with these two enzymes and ligating the resulting blunt-ends together. This vector, therefore, lacks any BamHl and PvuII
sites, a necessity for subsequent steps.
Step B. The EcoRl-to-HindIII insert from human GRF cDNA clone phGRF-54 (see Mayo et al., Nature 306, 86-88 (1983)) was ligated into the EcoRl - HindIII sites of the vector described in Step A. The cDNA essentially provides fused exons 3-5 for the final product.
Step C. The BamHl-to-H_III fragment at the 3'-end of the cDNA (about 200 bp) was removed and replaced with a BamH1 to HindII fragment of about 1 kb from genomic clone hGRF 101 (see - Ma~o et al., P.N .A .S. 82, 63-67 (1985)). This ~ragment provides the polyadenylation site and 3'-flanking sequences for the final product.

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Step D. An EcoRl-to-PvuII fragment at the 5'-end of the cDNA (about 100 bp3 was removed and replaced by~ an EcoRl-to-PvuII fragment of about 1.5 kb f rom genomic clone hGRF cos49 (see Mayo et al., P.N.A.S. 82, 63-~7 (1985)). The 1.5 kb f ragment was removed by partially digesting a plasmid sukclone of hGRF cos49 with PvuII and completely digesting with EcoRl.
This fragment provides exon 2 and intron B for the final product.
Step E. The hGRF "minigene" (the product of Step D) is digested partially with SmaI to cut only at the site within exon 2. This SmaI site is made into a _~II site using synthetic linkers. An EcoRl-to-B~II fragment of about 1 kb is removed and is replaced with a 770 bp EcoRl-to-_glII fragment from the mouse metallothionein-I gene (see Glanville et all., Nature 292, 267-269 (1981)). This fragment provides the promoter/regulatory activity for the final product.
To initially determine whether the MT-GRF
fusion gene could be expressed and regulated correctly, it was introduced into cuItured mouse fibroblast cells using CaPO4-mediated DNA transfection [F.L.
Graham et al., Virology 52, 456-467 (1973)] and co-selection for neomycin resistance conferred by the vector pSV2-neo [P.J. Southern et al., Molec. Appl.
Genet. ul, 327-341 (1982)]. Several stable cell lines that were generated in this manner express a MT-GRF
fusion mRNA of the expected size whose abundance is increased by metal treatment, and accumulate radioassayable hGRF in the cultu re medium.
A 2.5-kb EcoRI-HindIII fragment containing the MT-GRF fusion gene (about 1,600 molecules) was microinjected into the male pronuclei of 350 F2 hybrid eggs (obtained by mating C57BL/6 X SJL hybrid adults), and the eggs were transferred into the oviducts of : ~ :

1 31 6!~3r, pseudopregnant recipients as described in R.L.
Brinster et al., Cell 27, 223-231 (1981). Fifty-nine animals developed from the microinjected eggs.
At weaning, DNA was isolated from a piece of -tail and analyzed for the fusion gene by dot-blotting using a human GRF cDNA as probe (K.E. Mayo et al., Nature 306, ~6-88 (1983)). Positive animals were re-analyzed by Southern DNA blotting and copy number determined by comparison with a standard curve generated from known amounts of MT-GRF plasmid DNA. Positive animals were maintained on water containing 25 mM
ZnSO4. RNA was isolated following partial hepatectomy and analyzed by Northern blotting using a human GRF cDNA
probe. Autoradiograms were densitometrically scanned to determine relative RNA amounts. Plasma GRF and GH were determined by radioimmunoassay of serum samples from animals at 9 weeks old. The GH radioimmunoassay was able to detect 16 ng/ml serum GH (two control animals had 16 and 46 ng/ml GH), and the GRF radioimmunoassay was able to detect 10 ng/ml serum GRF (two control animals had less than 10 ng/ml GRF). The growth ratio was determined at 9 weeks of age and represents a comparison with age- and sex-matched littermates.
Fourteen animals carried the MT-GRF fusion gene; these animals were maintained on water with 25 mM
ZnSO4 to enhance expression of the fusion gene. Table 1 summarizes the expression of the MT-GRF fusion gene in these 14 transgenic mice.

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Table 1 Expression of the MT-GRF fusion gene in transgenic mice Liver No. of GRF
gene mRNA Plasma Plasma 5copies (relative hGRF mGH Growth Mouse ~ amount) (ng/ml) (ng/ml) (ratio) M760-5 1 3 24 1,051 1.35 M762-3 2 0 10 21 0.93 F762-5 ~ 63 26 141 1.33 F765-2 2 102 207 415 1.24 M800-1 1 0 10 149 1.12 F800-8 1 3 14 402 1.05 M801-3 3 0 10 38 0.97 F801-5 8 38 99 809 1.35 M801-9 1 4 20 541 1.42 F802-3 5 1 45 913 1.51 M803-4 10 118 263 1,095 1.41 F803-5 1 5 24 166 1.45 M803-6 10 16 50 302 1.24 F803-7 20 * * * 1.36 * F803-7 died prematurely, Although liver GRF mRNA has measured, the amount could not be quantitated because of RNA degradation in the postmortem liver. Plasma GRF and GH
levels could not be determined accurately for the same ; 25 reason.
Eleven of the 14 animals express the MT-GRF
messenger RNA in the liver, a major site of metallothionein expression. The amount of the fusion mRNA in the livers of these mice varies by as much as 100-fold and does not seem to be strongly correlated with the number of copies of the fusion gene in each animal. Animals expressing the fusion gene have measurable levels of hGRF in their serum, and they have increased levels of serum growth hormone. Ten of the mice showed significant increases in growth at 9 weeks old and were 25-50% larger than control littermates.
Examination of the data in Table 1 reveals that there is no strong correlation between GRF levels, GH levels and growth.
To determine whether the MT-GRF fusion gene and the large size are heritable~ two the MT-GRF founder animals were bred with control animals; offspring from , - ' ,~
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-`` 1 3 1 6 1~ 5 these matings were analyzed for the presence of the MT-GRF fusion gene and for their rate of growth.
Figure 3 shows two pedigrees demonstrating that both the fusion gene and the "large" phenotype are inherited by about 50% of the total off spring. DNA analysis showed that all positive offspring carried the same number of copies of the fusion gene as the corresponding parent.
Four other females were also mated and produced litters. Thus, all six of the females tested were fertile (two were killed before breeding) . In addition, male MT-GRF mice 760-5 and 801-9 (see Table 1) sired litters that included offspring carrying the fusion gene and displaying the large phenotype, indicating both males and females carrying the MT-GRF fusion gene are fertile and transmit the gene. It is now known that both the MT-GRF fusion gene and the large phenotype are inherited by about 50% of the offspring examined in the F2 generation of animals.
To determine whether expression of the MT-GRF
fusion gene in transgenic mice demonstrates a tissue specificity similar to that of the endogenous MT-I gene, both MT-I and MT-GRF RNAs we re analy zed in six tissues f rom either a control mouse or an MT-GRE' mouse (female 765-2-3) (see Fl, in Figure 3b). Both animals were maintained on water with 76 mM ZnSO4 for several weeks before killing. The indicated organs were removed and frozen at -70C until use. Total RNA was prepared by homogenization in guanidine isothiocyanate and centrifugation through cesium chloride, as described in J.M. Chirgwin et al., Biochemistry 18, 5294-5299 (1979);
5 ug of each RNA was denatured and electrophoresed on formaldehyde/l.5% agarose gels (J. Meinkoth et al., Analyt. Biochem. 138, 267-284 (1984)) and the RNA
transferred to nitro-cellulose filters as described in P. Thomas, P.N.A.S. 77, 5201-5205 (1980). Filters were probed with either a mouse MT-I genomic clone, pSH (D.M.
Durnam et al., P.N.A.S. 77, 6511-6515 (1980)), or a .:
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.~
: ' ' 1 31 6~5 human GRF cDNA clone, phGRF-54 (K.E. Mayo et al., Nature 306, 86-88 (1983))~ Probes were labelled by nick-translation using [alpha-32P]dCTP. Exposure time of the autoradiograms was 16 hours.
The control animal expressed MT-I mRNA at high levels in liver, kidney, gut and pancreas, and at lower levels in brain and spleen, as can be seen in Figure 4 obtained using standard Northern Blot analysis of RNAs obtained from six different tissues. This agrees well with the known tissue distribution of MT-I mRNA (D.M.
Durnam et al., ~. Biol. Chem. 256, 5712-5716 (1981)).
The control animal did not express detectable hGRF mRNA
in any tissue. Although the hGRF probe might detect endogenous mouse GRF mRNA in the brain, the low abundance of GRF mRNA even in the hypothalamus (less than 0.01%) makes detection unlikely. The transgenic mouse F765-2-3 showed a tissue distribution of MT-I mRNA
much like that of the control mouse with one exception;
the amount of liver MT-I mRNA was substantially reduced.
In the transgenic mouse, the MT-GRF fusion mRNA
is expressed at high levels in the liver, gut and pancreas, at low levels in kidney and spleen, and is not detected in the brain. Although the expression of the fusion gene in this animal demonstrates a tissue specificity similar to that of the mouse MT-I gene, there are clear and reproducible differences, most noticeably the low level of expression of MT-GRF mRNA in the kidney. The pattern of MT-GRF gene expression has recently been examined in a second pedigree (N801-5-9), and it is found that, although the general pattern of tissue specificity parallels that of mouse MT-I, there are subtle differences particular to this animal and distinct from the MT-GRF animal F765-2-3. Similar results were obtained on examination of the tissue specificity of metallothionein/growth hormone fusion genes (R. D. Palmiter et al., Science 222, 809-814 (1983)). In this case, an extensive survey of multiple animals showed that expression varied among different :
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1 31 6~5 tissues and different animals, suggesting that factors, such as the site of integration, probably influence expression of the exogenous gene.
Although many tissues apparently express the MT-GRF mRNA and presumably synthesize the GRF precursor protein, it is not known which tissues are capable of proteolytically processing this precursor to generate the mature GRF hormoneO The antibody used to detect plasma GRF would detect both the precursor and the mature peptide and cannot be used to distinguish the mature peptide from the precursor. Such processing must occur to some extent, as the free N-terminus of the GRF
peptide is known to be required for biological activity (J. Rivier, et al., Nature 300, 276-278 (1982) R.
Guillemin et al., Science 218, 585-587 (1982)).
The generation of transgenic mice which develop from microinjected eggs and carry the MT-GRF fusion gene have elevated levels of plasma growth hormone and grow at a rate of 25-50% greater than that of control littermates; this is significantly less than the increased growth observed in transgenic mice which develop from microinjected eggs and express growth hormone, many of which grow to twice the normal size (R.
D. Palmiter et al., Nature 300, 611-615 (1982); R. D.
Palmiter et al., Science 222, 809-814 (1983)). However, in the transgenic animals expressing growth hormone, most body tissues express the gene and have the potential to make growth hormone. In the experiments in which animals carry exogenous GRF gene, growth hormone is only made in the anterior pituitary and thus may be rate-limiting.
The first generation of progeny of the transgenic mice which develop from the microin~ected eggs tend to grow even larger than their parents, which may be a artifact of general weakening of the parent mice caused by the microinjection process, which weakening is overcome in their progeny. The increased growth rates of the second and subsequent generations of . .

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progeny tend to be reflective of the increased growth rate exhibited in the first generation of progeny.
Transgenic animals containing exogenous GRF
genes tend to vary in size ranging from somewhat larger than the normal mice to up to about twice as large.
This contrasts with transgenic mice incorporating exogenous GH gene which tend to generally grow to about double normal size. Developing transgenic animals incorporating exogenous GRF gene rather than exogenous GH gene may therefore be advantageous in allowing a desired size to be selected for breeding lines Although it is an object of the invention to produce faster growing and larger animals, too high growth rate and/or too large eventual size may prove to be incompatible with animal health, e.g., placing too large a burden on the skeletal structure of larger animals.
In such cases, animals exhibiting enhanced growth rate, but not greatly exaggerated growth rate, may be selected. Where exaggerated growth proves to be consistent with animal health and viability, such faster growing animals may be bred.
An unexpected finding was that females expressing the MT-GRF fusion gene are generally fertile, although transgenic female mice expressing the growth hormone gene are generally infertile (R. E. Hammer et al., Nature 311, 65-67 (1984)). This suggests that the enhancement of growth in the MT-GRF females is more physiological, perhaps because the effect is mediated through endogenous somatotropes.
The fertility of both sexes of animals incorporating an exogenous GRF gene is considered highly important for producing breeds of rapidly growing transgenic animals. Any economic advantage derived by elevated growth of an animal overproducing GH may be negated by the infertility of a substantial portion of the progeny, particularly a substantial portion of the female progeny. Transgenic animals incorporating exogenous GRF genes do not suffer this disadvantage.

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While the invention has been described in terms of certain preferred embodiments, modifications obvious to one with ordinary skill in the art may be made without departing from the scope of the present inventionO For example, although the invention has been described in terms of fusion genes that encode human GRF
precursor, there is a substantial degree of homology between GRF's of different vertebrate species, particularly different mammalian species and also a substantial degree of cross-species reactivity. This is particularly true among mammals, but GRF's can be cross-reactive between species of different genuses, e.g., between mammalian and avian species. Similarity in GRF protein structures between species is considered important when producing transgenic animals that express an exogenous protein from a different species, because it is less likely that autoimmune responses will be induced in transgenic animals expressing closely similar exogenous proteins. Accordingly, GRF-encoding sequences may be obtained from a wide variety of animals.
Similarly, although the invention has been described in terms of linking the GRF-encoding sequence to certain strong promoters, such as a promoter of a metallothionein protein, a variety of promoters may be used, including weaker promoters. The gene used to transform the fertilized egg may very well include the - natural promoter of the GRF precursor protein. Weaker promoters may be advantageous in some instances, particularly if transgenic animals having GRF-encoding sequences linked to strong promoters should grow at excessively elevated rates or to excessively elevated size.
; Various features of the invention are emphasized in the following claims.

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Claims (14)

1. A method of producing a transgenic, non-human, economically useful, vertebrate animal which exhibits an increased growth rate and/or achieves a greater adult size relative to normal members of its species, the method comprising providing a growth hormone releasing factor (GRF) gene including a promoter sequence and a GRF-encoding sequence operably linked to and promoted by said promoter sequence, obtaining a fertilized mammalian egg, introducing said GRF gene into said fertilized egg, and implanting said fertilized egg into a host female animal of the species from which fertilized egg was obtained so that said host female animal gives birth to a transgenic animal incorporating said GRF gene in its genome.

2. A method according to claim 1 wherein said GRF-encoding sequence encodes human GRF.

3. A method according to claim 1 including isolating a promoter sequence, isolating a GRF-encoding sequence, and linking said promoter sequence to said GRF-encoding sequence to form said GRF gene.

4. A method according to claim 3 wherein said promoter sequence is a promoter of a metallothionein gene.

5. A method according to claim 4 wherein said promoter sequence is a promoter of murine metallothionein gene.

6. A method according to claim 3 wherein said promoter sequence is a promoter of a transferrin gene.

7. A method according to claim 1 wherein said GRF-encoding sequence is formed by isolating a natural eukaryotic GRF-encoding gene sequence, isolating a corresponding cDNA
sequence, removing a portion of said GRF-encoding gene sequence, splicing a portion of said cDNA sequence into a corresponding portion of said natural eukaryotic GRF-encoding gene sequence, whereby at least one intron in the natural eukaryotic GRF-encoding gene sequence is deleted while at least one intron is retained.

8. A method according to Claim 1 wherein said GRF-encoding sequence encodes the entire GRF precursor protein.

9. A combination of a promoter sequence operably linked to a GRF-encoding fusion DNA sequence comprising at least one DNA sequence portion obtained from a natural eukaryotic genomic sequence encoding a mammalian GRF and at least one DNA sequence portion obtained from the corresponding cDNA encoding a mammalian GRF, said fusion DNA
sequence encoding at least the entire GRF amino acid sequence and said fusion sequence omitting one or more introns from said natural eukaryotic genomic sequence while retaining one or more introns therefrom, and said promoter sequence being that of a promoter of a mammalian metallothionein or transferrin gene.

10. A GRF-encoding sequence according to Claim 9 wherein said GRF-encoding fusion sequence encodes human GRF.

11. A GRF-encoding sequence according to Claim 10 wherein said GRF-encoding fusion sequence encodes the entire human GRF precursor protein.

12. A GRF-encoding sequence according to either Claim 9 or 10 comprising a 5' sequence portion obtained from the natural human GRF genomic sequence, a middle sequence portion obtained from human GRF-encoding cDNA and a 3' sequence portion obtained from the natural human GRF genomic sequence.

13. A GRF-encoding sequence according to either Claim 9 or 10 containing the first intron from the natural human GRF genomic sequence and no other introns.

14. A sequence in accordance with any one of Claims 9, 10 or 11 wherein said promoter is that of a murine metallothionein or transferrin gene.