JP2002065261A - How to get germ cells - Google Patents

Abstract

(57) [Summary] [Problem] To enable the detection of germ cell differentiation from ES cells,
Germ cells differentiated in culture are selected and purified to achieve spermatogenesis from germ cells derived from ES cells. SOLUTION: A marker gene is introduced so as to be controlled under the expression of a Vasa gene or its homolog gene.
Culturing the ES cell line in the presence of a germ cell differentiation promoting factor,
A method for obtaining germ cells, which comprises selecting cells having germ cell differentiation ability using the expression of a marker gene as an index.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for obtaining germ cells and a method for using the same. More specifically, the present invention relates to a germ cell comprising culturing an ES cell line into which a marker gene has been introduced so as to be placed under the control of the expression of a Vasa gene or a homolog gene thereof, in the presence of a germ cell differentiation promoting factor. And how to use it.

[0002]

2. Description of the Related Art Conventional methods for producing genetically modified animals are based on genetic manipulation of fertilized eggs and ES cells.
In order to obtain offspring derived from transgenic cells, the efficiency of transplantation to the foster mother and the efficiency of transfer of the transgene to gamete formation are the major rate-limiting factors for the efficiency of genetically modified animals and the work period. . In addition, these methods are mainly used for experimental animals (mouse) in which an ES cell line is established and the generation period is short.
It is difficult to collect fertilized eggs or is not a practical method for many livestock animals with a long generation period. Also, regarding the creation of cloned animals by somatic cell nuclear transfer, which is currently attracting attention, since the embryo manipulation process after fertilized eggs is common, this problem is an unavoidable problem.

[0003] On the other hand, the production of spermatozoa, the male gametes of higher animals, is carried out by the periodic differentiation of spermatopoietic stem cells capable of self-renewal, and the production is continued throughout the life of the individual. Based on this embryological characteristic, such as artificial insemination technology using sperm cells or their nuclei and cryopreservation technology for sperm,
Reproductive engineering techniques for obtaining progeny individuals from living spermatogenic cells are developing rapidly, and these techniques are now existing.

[0004]

As described above, in the production of current genetically modified animals, the operation of living transplantation of a fertilized egg or a blastocyst stage embryo is indispensable. Whether entering the cell lineage, that is, contributing to spermatogenesis, is a key. In this case, if the progress up to the spermatogenesis process is achieved in any form, a genetically modified individual can be obtained by reproductive engineering using sperm cells. Therefore, the present invention solves the problem of advancing the process of differentiation into germ cells in culture, and then establishing a system that can reliably contribute to spermatogenesis by purifying germ cells derived from genetically engineered cells. It should be a task to be done. To achieve this task, there are two issues that need to be overcome.

One is to make it possible to detect the differentiation of germ cells from totipotent cells, specifically, ES cells. The other is to select and purify the differentiated germ cells in culture to obtain ES cells. To achieve spermatogenesis from germ cells of origin. For the former, Oct3 / 4, SSE, which has been used to identify primordial germ cells, which are generally germ cell precursors
Since the detection method based on A-1 antigen and alkaline phosphatase staining positive is a common trait for totipotent cells including ES cells, it may not be a means to distinguish the differentiation of ES cells into primordial germ cells. It was a major factor that hindered the development of a germ cell differentiation induction system in vitro.

[0006]

The embryoid body formed by suspending and culturing ES cells is composed of embryonic somatic cells of endo-, meso- and ectoderm together with extraembryonic ectoderm cells from 3 to 5 days after culture. Differentiation is seen. The germ cell differentiation in the same embryoid body culture is indicated by the germ cell specific gene Mv
h (mouse vasa homolog) expression (Fujuwara et al., PNA
S, 91, 12258-12262, 1994; Toyooka et al., Mech. De
v., 93, 139-149, 2000), which were detectable by visualization by marker gene expression of the lacZ and GFP genes. Marker-positive, ie, Mvh expression-positive, germ cells in embryoid bodies 5 days after culture account for about 0.1 to 0.3% of the total
Appear at a frequency of

[0007] The present inventors' research indicates that growth factor BMP4 (bone morphoge) belonging to the TGFβ superfamily.
By mixing and culturing cells expressing netic protein-4) with ES cells, the frequency of germ cells mentioned above is about 3
55% (approximately 10-20 fold), and its appearance was found to be induced within a short period of one day after culture. Germ cells sorted as marker-positive cells by using a cell sorter (FACS) were Oct3 / 4, SSEA-1, which characterize fetal gonad primordial germ cells (PGC) at day 10.5-13.5.
[Fig. 4] Fig. 4 shows characteristics of positive for GCNA-1 antigen and alkaline phosphatase. In the present experiment of the present inventors, it was possible to purify ES cell-derived germ cells to 90% or more by two FACS sorts.

[0008] The ability of the ES cell-derived germ cells to have a normal spermatogenesis ability was tested by an operation of transplantation into living testes. When cell aggregates mixed with fetal gonad-derived cells were transplanted under the testis capsule, spermatogenesis could be observed in the transplanted mass in the same manner as 12.5 day embryonic primordial germ cells.
ES cell-derived sperm could be prepared.

[0009] These results indicate that the above-mentioned series of operations is an excellent technique for obtaining ES cell-derived spermatozoa in an extremely short time and reliably without going through a process of producing chimeric animals from ES cells subjected to genetic modification. Can be provided.
In addition, the fact that germ cells induced to differentiate in vitro from ES cells showed normal developmental ability means that this differentiation induction system can be used for a totipotency assay of ES cells and a system for assaying the disruption of chemical substances on germ cell establishment. Is shown. The present invention has been completed based on these findings.

That is, according to the first aspect of the present invention, Vasa
An ES cell line into which a marker gene has been introduced so that it is placed under the control of the gene or its homolog gene, is cultured in the presence of a germ cell differentiation promoting factor, and cells having germ cell differentiation ability using the marker gene expression as an index. And a method for obtaining a germ cell.
Preferably, the marker gene to be introduced into the ES cell line is GF
P, BFB, YFP, or lacZ gene. Preferably, the marker gene is introduced into the ES cell line by homologous recombination. Preferably, the ES cell line is a cell line derived from a mammal or bird. Preferably, the differentiation promoting factor is BMP4.

According to a second aspect of the present invention, a cell into which a gene encoding a germ cell differentiation promoting factor has been introduced,
Co-culture with an ES cell line into which a marker gene has been introduced so as to be under the control of the gene or its homolog gene expression, and selecting cells having germ cell differentiation ability using the marker gene expression as an index And a method for obtaining a germ cell. Preferably, the gene encoding a germ cell differentiation promoting factor is a gene encoding BMP4.

According to a third aspect of the present invention, an ES cell line into which a marker gene has been introduced so as to be under the control of the expression of the Vasa gene or its homolog gene is cultured in the presence of a test substance, and And a method for screening for a germ cell differentiation promoting factor, which comprises selecting a test substance that induces expression of germ cells. According to a fourth aspect of the present invention, there is provided a germ cell differentiation promoting factor obtained by the method for screening a germ cell differentiation promoting factor according to the third aspect of the present invention.

According to a fifth aspect of the present invention, a germ cell obtained by the method for obtaining a germ cell according to the first aspect of the present invention is cultured, and the obtained cell aggregate is subjected to adult male testicular capsule. There is provided a method for obtaining ES cell-derived spermatozoa, which comprises obtaining cells from a cell mass exhibiting a formed seminiferous tubule structure, which is transplanted below. Preferably, the germ cell culture is a method of co-culturing with cells derived from fetal male gonads.

According to a sixth aspect of the present invention, there is provided a sperm obtained by the method for obtaining an ES cell-derived sperm according to the fifth aspect of the present invention. According to a seventh aspect of the present invention, a recombinant cell line obtained by introducing a foreign gene into an ES cell line into which a marker gene has been introduced so as to be placed under the control of the expression of the Vasa gene or its homolog gene is transformed into an ES cell line. A method for obtaining a genetically modified germ cell is provided, wherein the method for obtaining a germ cell according to the first aspect of the present invention is performed using the cell line as a cell line.

According to an eighth aspect of the present invention, there is provided the fifth aspect of the present invention, wherein the recombinant germ cell strain obtained by the method for obtaining a genetically modified germ cell according to the seventh aspect of the present invention is used. A method for obtaining a genetically modified sperm, characterized by performing the method for obtaining a sperm according to the above aspect, is provided. According to a ninth aspect of the present invention, there is provided a genetically modified sperm obtained by the method for obtaining a genetically modified sperm according to the eighth aspect of the present invention.

According to a tenth aspect of the present invention, there is provided a method for producing an ES cell-derived individual by microinsemination using sperm obtained by the method for obtaining sperm according to the fifth or eighth aspect of the present invention. Is provided.

According to an eleventh aspect of the present invention, an ES cell line into which a marker gene has been introduced so as to be under the control of the expression of the Vasa gene or its homolog gene is cultured in the presence of a test substance, and A method for testing toxicity to germ cells is provided, which comprises assaying a substance having toxicity to germ cells using the number of cells in which expression is induced as an index.

According to a twelfth aspect of the present invention, an ES cell line into which a marker gene has been introduced so as to be placed under the control of the expression of the Vasa gene or a homolog gene thereof is transformed into an ES cell line in the presence of a germ cell differentiation promoting factor and a test substance. Germ cell toxicity test method using the number of cells in which expression of the marker gene has been induced as an index.

According to a thirteenth aspect of the present invention, an ES cell line into which a marker gene has been introduced so as to be placed under the control of the expression of the Vasa gene or a homolog gene thereof, together with a BMP4 gene-introduced cell line in the presence of a test substance. Cultured, using the number of cells in which marker gene expression has been induced as an index, characterized by assaying for a substance having toxicity to germ cells,
Methods for testing germ cell toxicity are provided.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a method and an embodiment of the present invention will be described in detail. In order to discriminate differentiation from ES cells into primordial germ cells, it is necessary to use, as an index, a trait that is not expressed in ES cells but is specifically expressed in differentiated germ cells. Germ cell specific gene Mvh (mouse vasa homolo
g) is specific for late primordial germ cells settled in the gonads and is not expressed in undifferentiated early embryonic cells or ES cells before that (Fujuwara et al., 1994; Toyooka e
t al., 2000), the present invention uses the expression of the Vasa gene or its homolog gene as an indicator of germ cell differentiation. However, in embryoid bodies formed by suspension culture of ES cells, differentiation of various cell types including three germ layers occurs similarly to in vivo, and the frequency of germ cells is considered to be extremely low. For this reason, it was expected that conventional molecular genetic detection methods and immunohistological staining methods would not allow accurate identification. In the present invention, as a means to overcome this problem, Vasa
Expression of the gene or its homologue gene to the expression of marker genes such as GFP (green jellyfish green fluorescent protein) and lacZ (bacterial galactosidase enzyme; a chromogenic substrate enables blue coloration or a luminescent substrate to enable fluorescent coloring) genes An ES cell line into which the above marker gene was introduced so as to be placed under the control of the expression of the Vasa gene or its homolog gene was prepared by performing a genetic modification operation for replacement.

More specifically, ES obtained by knock-in (inserting) the GFP and lacZ genes into the Mvh locus by homologous recombination.
Cell clones were established (see FIG. 1) and Mvh gene expression was
A measure was taken to visualize by acZ (GFP) expression. As a result, without using differentiated germ cells as fixed specimens,
As a means of highly sensitive detection while alive, differentiated germ cells can be fractionated and purified from other cell types.

Next, in order to obtain more germ cells, conditions for promoting the differentiation of germ cells were examined by taking advantage of the advantages of the culture differentiation induction system. In vivo, primordial germ cells are presumed to undergo local differentiation-inducing action from extraembryonic tissues (placental progenitor tissues) to differentiate. Furthermore, knockout mouse analysis reports that disruption of the BMP4 gene expressed in extraembryonic tissues results in loss of primordial germ cells (Lawson et al., Genes & Dev., 13, 424-436, 199).
9) It was suggested that BMP4 might be a germ cell differentiation promoting factor. In the present invention, BMP4-expressing cells transfected with a vector forcibly expressing a BMP4 protein are established, and when they are uniformly mixed with ES cells to form embryoid bodies, the appearance frequency increases by 10 times or more. Found that happens.

Further, the germ cells differentiated in culture can be easily purified by a cell sorting apparatus (FACS).
We verified that these germ cells have the same developmental potential as primordial germ cells in vivo, and as a means of forming ES cell-derived spermatids, a method of inserting and transplanting a cell mass derived from purified cells into living testis was used. Using. In this method, the transplant is separated from the host tissue to construct a unique seminiferous tubule tissue, so that sperm cells derived from ES cells can be easily separated and purified without mixing with the host sperm. Hereinafter, the present invention will be described in more detail.

1. Vasa gene in embryoid body formation or
Germ cell differentiated ES cells expressing the homolog gene maintain undifferentiation in the presence of LIF (Leukemia Inhibitory Factor) and proliferate with a fast division cycle of about 8 hours, but the growth rate decreases in the absence of LIF Cell differentiation as well as early embryos. In particular, when a cell mass is formed by suspension culture, a spherical tissue structure consisting of a plurality of endo-, meso- and ectodermal cell types called embryoid bodies is formed. For a system that detects whether or not germ lineage differentiation occurs during the in vitro differentiation of ES cells based on the expression of the germ cell-specific Vasa gene or its homolog gene, the ES cell is required to express the Vasa gene or its homolog gene expression. A method is used in which a marker gene is introduced so as to be placed under control, and this is cultured under appropriate conditions.
These can be performed, for example, using the following method. In order to enable highly sensitive visualization and detection of cells expressing the Vasa gene or its homolog gene, a marker gene-introduced ES in which an IRES-lacZ expression cassette, an IRES-GFP expression cassette, and the like have been inserted and replaced (knock-in) by homologous recombination. Establish cell line. By introducing the visualization marker gene, the presence of cells expressing the Vasa gene or a homolog gene thereof can be quantitatively identified by LacZ enzyme activity staining, GFP fluorescence emission, or the like. In fact, the ES cell line in which the Mvh gene was knocked in by the GFP gene was subjected to suspension culture using a culture solution without LIF, and embryoid body formation was performed to examine the appearance of GFP-positive cells. In the embryoid body 3 days later, a small amount of Mvh
The appearance of gene-expressing cells was observed, and the number of GFP-positive cells further increased in the embryoid bodies on day 5 after culture, and the presence of Mvh gene-expressing cells was observed in about 10% of the entire embryoid bodies. Similarly, l
Even when acZ was introduced as a marker gene, the appearance of germ cells expressing Mvh was observed in embryoid body formation using lacZ knock-in ES cells. This emergence of Mvh-expressing germ cells was not detected when suspension culture was performed in the presence of LIF (103 units / ml) (a cell mass similar to the embryoid body was formed).
Even when F is not present, it is not detected when cultured on a normal tissue culture plate (there is no formation of cell clumps), so that its appearance requires the formation of cell clumps in the absence of LIF.

As used herein, the term "Vasa gene" or a homolog gene thereof preferably means a Vasa gene of a mammal (eg, mouse, human, pig, cow, sheep, rat, etc.). In addition, the Vasa gene and its homolog gene are known, and for example, rats (Komiya et al., D
ev. Biol., 162, 354-363, 1994), chicken (Tsuneka)
wa et al., Development, 127, 2740-2750, 2000), zebrafish (Yoon et al., Development, 124, 315).
7-3165, 1999).

The type of the marker gene referred to in the present specification is not particularly limited as long as its expression can be easily detected or measured, and examples thereof include GFP, BFB, YFP and lacZ genes. Expression cassettes for introducing marker genes include IRES (Intern
al ribosome entry site: Jackson, RJ, et al., Tre
(nds Biochem Sci., 15 (12), 477-483, 1990) is advantageous for the functional expression of the marker gene when used so as to be linked to the 5 ′ side of the marker gene.

As a mode of introducing a marker gene under the control of the expression of the Vasa gene or its homolog gene, (1) a marker gene is placed downstream of the promoter of the Vasa gene or its homolog gene under the control of the promoter. A mode to be introduced to be placed, and
(2) There are two embodiments in which a marker gene to which a promoter of a Vasa gene or a homolog gene thereof is linked is introduced into an ES cell line. When the marker gene is introduced in the first embodiment, the gene may be inserted so as to match the reading frame between the DNA sequences of the Vasa gene or its homologue gene, or a part or all thereof. It can be introduced interchangeably. As a method for introduction, homologous recombination can be used. In the case where a marker gene is introduced in the above-described second embodiment, the site of introduction of the marker gene directly linked to the promoter of the Vasa gene or a homolog gene thereof is not particularly limited. In addition, the gene can be introduced using a gene transfer method known per se. Of these, it is preferable to introduce an expression unit consisting of a marker gene and an IRES sequence linked to the 5 'side thereof by homologous recombination to replace the Vasa gene or a part of its homolog gene.

The type of the ES cell line used in the present invention is not particularly limited, but a cell line derived from mammals or birds is preferable.
Specifically, mouse E129 cell line derived from male strain 129, E14TG2a cell line derived therefrom, and human ES cell line H9 (Thomson e
tal., Science, 282, 1145-1147, 1998).

2. Vasa gene or its homolog gene
The cells in which the expression of the preparative preparative marker gene of the present germ cell has been induced can be separated and purified by detecting a signal generated by the marker. As a signal detection method and a cell separation / purification method, an appropriate method known per se can be used. Specifically, for example, it can be performed as described below. La induced to differentiate in the knock-in ES cell embryoid body
cZ / GFP positive germ cells are separated and purified using a cell sorter (FACS). Day 5 embryoid bodies of lacZ knock-in ES cells are dispersed with collagenase, and the dispersed cells are subjected to biofluorescence staining with lacZ enzyme activity, and then analyzed using FACS to collect lacZ positive cells. In the following examples, cells sorted into the lacZ-positive fraction were 0.1 to 0.3% of the whole. The thus purified lacZ-positive cells are positive for Mvh antibody staining, confirming that they are actually endogenous Mvh-expressing cells. At the same time, by confirming the positiveness of Oct3 / 4 antibody staining, AP staining, and Sycp3 protein detection of the fetal germ cell identification index, almost all of the existing primordial germ cell identification indexes can be used to identify the characteristics of germ cells. We can confirm that we have.

3. Effect of Inducing Germ Cell Differentiation by Co-Culture In the present invention, in the in vitro differentiation system using ES cells described above, germ cell differentiation is more efficiently promoted using the appearance frequency of Mvh-expressing cells as an index. A method is also provided. In vivo, it is estimated that some germ cell differentiation-inducing action is exerted on epiblast from extraembryonic tissues in embryos at 6.0-6.5 days of embryo. Therefore, as a preliminary experiment, the extra-embryonic ectoderm of the 7.5-day embryo was excised, and an equal amount of primary cultured extra-embryonic ectoderm-derived cells and knock-in ES cells grown in a medium containing FGF4 by long-term culture (about 1 month) As a result of performing a co-culture to form a cell cluster, the appearance of Mvh-expressing cells was observed in almost all cell clusters on the first day after the start of the culture, and the frequency of Mvh-expressing cells was higher than that of ES cell alone culture. A much higher induction enhancement was observed. This indicates that extraembryonic ectoderm cells have a germ cell differentiation-promoting effect, and that the in vitro culture differentiation system correctly reflects the characteristics of germ cell differentiation occurring in vivo.

In addition, BM, which is a candidate factor for a differentiation-inducing signal of the extraembryonic ectoderm by the CMV promoter, is added to the M15 cell line derived from the embryonic (10.5 day embryo) germinating somatic cells.
Gene transfer of a vector expressing the full-length P4 sequence
An M15 cell line (BMP4-M15) that forcedly expressed the protein was created. As a result of examining a mixed cell mass obtained by mixing the BMP4-M15 cells and the GFP knock-in ES cells at a ratio of 1: 1, Mvh
The appearance of expressing cells was observed. In the co-culture of wild-type M15 cells performed as a control experiment, no Mvh-expressing cells were observed on the first day. According to FACS analysis, its appearance frequency was calculated to be about 3%, which is 6 to 10 times that of the embryoid body of ES cells alone (day 5). Further, 80% or more of the cell fraction collected by the first FACS purification was Mvh-expressing cells, and the fraction obtained by subjecting the positive fraction to FACS purification again was 95%.
% Or higher (see FIG. 3). From these results, ES cell lines transfected with a marker gene so that they are placed under the control of the expression of the Vasa gene or its homolog gene, by culturing in the presence of germ cell differentiation promoting factors such as FGF and BMP4, It was found that differentiation induction of marker gene positive cells could be promoted.

The germ cell differentiation promoting factor can be obtained by co-culturing a cell into which a gene encoding the gene has been introduced and an ES cell into which a marker gene has been introduced so as to be controlled under the expression of the Vasa gene or its homolog gene. It has also been found that it is preferred to be supplied. The cells to be introduced may be any cells as long as they can be cultured in the same culture system as the ES cells used in the present invention and express the introduced germ cell differentiation promoting factor gene. M15 (Rassoulzade), which is more preferably a cell derived from a germ progenitor cell, such as a cell of the germ layer system
gan et al., Cell, 75, 997-1006, 1993) are preferably used. The expression cassette containing the gene encoding the germ cell differentiation promoting factor may be any expression cassette as long as it is suitable for expressing the germ cell differentiation promoting factor in an appropriate amount in cells. A gene in which an appropriate promoter sequence is linked to the 5 'side of a gene encoding a differentiation promoting factor can be used.

The promoter to be used may be any promoter as long as it can express the germ cell differentiation promoting factor in the cell into which the expression cassette has been introduced, in a sufficient amount to promote germ cell differentiation. Good. Specifically, for example, a CMV promoter is preferably used.
For the introduction of the expression cassette into the above-described cells and the culture of cells forcibly expressing the germ cell differentiation promoting factor, appropriate methods known per se can be used. The obtained germ cell differentiation-promoting factor-introduced cell is co-cultured with an ES cell into which a marker gene has been introduced so as to be placed under the control of Vasa gene or its homolog gene expression, whereby the ES cell Promotes germ cell differentiation. The ratio of both cells in the case of co-culture is appropriately selected depending on the culture scale and the type of promoter used. Specifically, when the ratio of germ cell differentiation promoting factor-introduced cells to the total number of cells is 1% or more, promotion of germ cell differentiation is induced, but preferably about 10 to 50%.

The conditions for inducing germ cell differentiation in vitro and the conditions for purification described above were obtained using knock-in operated ES cells. Similarly, the behavior of cells expressing the Vasa gene or its homolog gene was used as an index. By doing so, it is considered that the method can be applied to ES cells that have been subjected to another genetic manipulation treatment or untreated ES cells. Also, Mv
h Antibody staining also cross-reacts with other mammalian germ cells, and the Vasa homolog gene is highly conserved across species, so it can be applied to other mammalian ES cells such as humans and pigs. Conceivable.

4. In vitro differentiated Vasa gene or its
Spermatogenesis derived from homologous gene-expressing germ cells As described in 3 above, germ cells purified by FACS after in vitro differentiation induction have the same developmental potential as germ cells generated in vivo, It can be examined by transplantation method under the testicular capsule. Transplantation under the testicular capsule is performed by mixing FACS-purified ES-derived cells with dispersed cells derived from fetal (12.5 day embryo) male gonads and forming a cell aggregate by suspension culture for 1 day. May be implanted under the testis capsule of an adult male nude mouse and cultured in vivo.
One to two months after the start of culture, the transplanted cell mass forms a unique tubule structure in the transplanted testis in a form separated from the host testis. Histological analysis showed that ES-derived germ cells had settled in the seminiferous tubules and spermatogenesis images were observed. From these results, by culturing the germ cells obtained in 3 above, transplanting the obtained cell aggregates under the testicular capsule of an adult male, and obtaining cells from the formed cell mass exhibiting a seminiferous tubule structure It was found that sperm derived from ES cells could be obtained.

It has also been found that culturing the obtained germ cells is more preferable by co-culturing with cells derived from fetal male gonads. The germ cell culture method obtained is
Any culture method may be used as long as a cell aggregate known per se is formed. Specifically, a suspension culture method is preferably used. In this culture, it is preferable to co-culture with the cells derived from fetal male gonads as described above. As a cell derived from a fetal male gonad, a cell obtained by dispersing a fetal male gonad of an appropriate animal by an appropriate known method can be used. The mixing ratio is not particularly limited, but the fetal male gonad-derived cells are preferably about 50% of the total cell number. Methods for obtaining the formed cell aggregates and transplanting the adult animal under the testicular capsule are described, for example, in Hashimoto N. et al., J. Exp. Zool., 253, 61-70, 19
The method described in 90 can be used. As the animal to be transplanted, an animal suitable for transplantation of a foreign cell, such as a nude mouse, is preferably used. After breeding the transplanted animal for an appropriate period, the sperm derived from the ES cells can be obtained by obtaining the seminiferous tubule structure formed by the germ cells derived from the ES cells. The seminiferous tubule of the transplanted tissue is a closed system and can be extracted with extremely high purity without the risk of host sperm contamination. It is possible to obtain offspring. For methods of sperm microinjection, artificial insemination, and obtaining offspring, see, for example, KimuraY. Et.
al., Development, 121, 2397-2405, 1995.

5. Screening of germ cell differentiation promoting factor
The present invention also introduces a marker gene to be placed under the control of Vasa gene or its homolog gene expression.
A method for screening a germ cell differentiation promoting factor, comprising culturing an ES cell line in the presence of a test substance and selecting a test substance that induces expression of the marker gene, and germ cells obtained by the screening method It also relates to differentiation promoting factors. Although the type of the test substance is not particularly limited, for example, peptides, polypeptides, synthetic compounds (such as low molecular weight organic compounds and high molecular weight organic compounds), fermented microorganisms, organisms (plant or animal tissues, microorganisms, or cells) And the like, or libraries thereof. Examples of the library include a synthetic compound library (such as a combinatorial library) and a peptide library (such as a combinatorial library). The test substance for screening may be a natural product or a synthetic substance, and a single candidate test substance may be tested independently, or a mixture of several candidate test substances (including libraries) may be tested. You may do. In addition, it is also possible to screen a fraction obtained by fractionating a mixture such as a cell extract, repeat the fractionation, and finally isolate a substance that induces the expression of a marker gene.

6. Genetically modified germ cells and genetic modification
The present invention also introduces a marker gene to be placed under the control of the expression of the Vasa gene or its homolog gene.
Culturing a recombinant ES cell line obtained by introducing a foreign gene into the ES cell line in the presence of a germ cell differentiation promoting factor, and selecting cells having germ cell differentiation ability using the marker gene expression as an index. The present invention also relates to a method for obtaining a transgenic germ cell, which is a feature. The type of foreign gene to be introduced into the ES cell line is not particularly limited, and examples thereof include physiologically active substances such as peptide hormones, neurotransmitters and growth factors, and immunopotentiating substances such as vaccines and drug resistant substances such as neomycin resistance factors. No. In addition, for example, cDNA or the like whose function is unknown can also be introduced. A promoter sequence suitable for expression of such a foreign gene can be introduced by being linked to the 5 'side of the gene.

The method for introducing a foreign gene is not particularly limited.
Conventional gene transfer methods can be used. The introduction position of the foreign gene is not particularly limited and can be appropriately selected depending on the purpose and the like.
The foreign gene is preferably introduced so as not to adversely affect the expression of the marker gene introduced so as to be placed under the control of the expression of the Vasa gene or its homolog gene. Using the transgenic germ cell line obtained as described above, transgenic sperm can also be obtained as described in 4. above.

7. Method for Testing Toxicity to Germ Cells The present invention also introduced a marker gene so as to be placed under the control of Vasa gene or its homolog gene expression.
A germ cell toxicity test comprising culturing an ES cell line in the presence of a test substance and assaying for a substance having germ cell toxicity using the number of cells in which marker gene expression is induced as an index Also about the method. When culturing the ES cell line in the presence of the test substance, a germ cell differentiation promoting factor may be present, and more preferably, the cell line is cultured with the BMP4 gene-introduced cell line. The type of the substance having a toxicity to germ cells (test substance) is not particularly limited. For example, peptides, polypeptides, synthetic compounds (low-molecular organic compounds, high-molecular organic compounds, etc.), fermented microorganisms, organisms ( (Including plant or animal tissues, microorganisms, cells, etc.), or libraries thereof. Examples of the library include a synthetic compound library (such as a combinatorial library) and a peptide library (such as a combinatorial library). The test substance may be a natural product or a synthetic product, and a single candidate test substance may be tested independently, or a mixture of several candidate test substances (including a library) may be tested. You may. In addition, it is also possible to screen a fraction obtained by fractionating a mixture such as a cell extract, and repeat the fractionation to finally isolate a substance that induces the expression of a marker gene. More specifically, an endocrine disrupting substance (environmental hormone) or the like can be evaluated as a substance having toxicity to germ cells by the toxicity test method of the present invention. The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to only these examples.

[0041]

EXAMPLES Example 1 Establishment of Mvh Knock-in ES Cell Line 129 / svj M isolated from mouse genomic library of line
Based on the vh genomic gene clone, a 2.2 kb XbaI-SpeI genomic fragment cut out from the phage clone and
The 1.2 kb XbaI-XhoI fragment was used as the 3′-side and 5′-side homologous flanking regions, respectively.
IRES-LacZ + pGK-
neo (neomycin resistance gene) or IRES-GFP + pGK-ne
o A knock-in vector to replace the cassette was prepared (Fig.
1). Note that an MC1DT-A (diphtheria toxin) expression cassette for negative selection was added to the 3 ′ region of the vector. The linearized knock-in vector was transferred to an ES cell line (E14TG2
a) was introduced by electroporation, and selection by drug resistance was performed in the presence of G418 (175 μg / ml) for 5 to 7 days. From a G418-resistant ES cell clone (about 400 strains), 0.7 kb HindIII-XbaI and 2.6 kb EcoRI-PstI
The fragment was subjected to Southern blot analysis using a probe for detecting homologous recombination on the 3 ′ side and 5 ′ side, respectively, and IRES-LacZ
A recombinant ES cell clone was selected for each of the IRES-GFP knock-in vector and the IRES-GFP knock-in vector. Using the obtained ES cell clone, the following in vitro differentiation induction system was constructed.

Example 2: Germ cell formation during embryoid body formation
Promotion of differentiation induction by co-culture with differentiation and BMP4-expressing supporting cells
For the culture of advanced ES cell lines, use the E14TG2 cell line medium [Glasgow
(Glasgow) Eagle's medium (GMEM; Gibco), 10% fetal bovine serum, 100 μM non-essential amino acid (Gibco), 1 mM sodium pyruvate (Gibco), 107 units / ml Leukemia Inhi
Using a bitory factor (LIF; Gibco), 10 ^-4 M 2-mercaptoethanol], the cells were subcultured into a 6 cm-diameter culture dish which was usually coated with gelatin. Cultured cell lines used as feeder cells, e.g., mouse somatic cells 10.5
DMEM (Gibco), 10% FCS, 100 μM non-essential amino acids (Gibco), for the maintenance of the M15 cell line, which is a cell line derived from the gonad somatic cells of the day embryo, and for embryoid body formation of ES cells 1 mM
Sodium pyruvate (Gibco) was used as the culture solution.

For embryoid body formation, ES cells were suspended at a density of 1 × 10 ⁵ cells / ml, and the suspension was cultured at 37 ° C., 5 ° C. on a tissue culture plate for suspension culture.
After culturing under a concentration of% carbon dioxide, the appearance of Mvh-expressing cells was examined over time. For GFP knock-in ES cells, observe under a fluorescence microscope, and for lacZ knock-in ES cells,
The appearance of Mvh-expressing cells was detected by gal staining (see FIG. 2). X-gal staining was performed using ice-cold fixative (1% paraformaldehyde, 0.2%
Fix with glutaraldehyde / PBS for 5 minutes, wash 3 times with ice-cold PBS, then add X-gal staining solution (50mg / ml X
-gal (Takara Shuzo), 5 mM K ₄ Fe (CN) ₆ 3H ₂ O, 5 mM K ₃ Fe (CN) ₆ ,
(2 mM MgCl ₂ / PBS) to carry out a color reaction at 37 ° C. for 12 to 24 hours, and lacZ positive cells are colored blue.

In order to establish mouse cells for forcibly expressing BMP4, BMP4 cDNA was inserted into an expression vector for mammalian cells (pTarget, Promega) having a CMV promoter and a neomycin resistance gene and capable of inserting any foreign gene.
A forced expression vector was made. Gene transfer was performed by electroporation and selection was performed using neomycin resistance, and a resistant cell line incorporating the BMP4 forced expression vector was established. The expression of the introduced BMP4 gene can be confirmed by performing BMP4-specific RT-PCR detection on the mRNA of the established strain. Although the above-described M15 cells are used in FIG. 3, it has been found that the differentiation-inducing effect and the host cell type are not specific. Mix ES cells and equal amounts of BMP4 forced expression cells,
As a result of performing the embryoid body formation culture in the same manner as described above, not only the appearance of Mvh-expressing cells was detected under the culture conditions of one day, but also the appearance frequency reached about 10 times that of the case of ES alone.

Example 3: Analysis and analysis of cells by FACS apparatus
The image embryoid bodies were collected by centrifugation, suspended in PBS containing 0.01% Koragenesu (Sigma), an enzymatic reaction was carried out for 30 minutes at 37 ° C.. After the cells of the embryoid body were dispersed by a pipetting operation, a centrifugation operation was performed again to remove collagenase. After washing the cells with PBS, the cells are dispersed in phenol red-free DMEM + 10% FCS to a cell density of 10 ⁵ / ml or less, and the cells are purified using FACS (FACStarPLUS, Becton Dickinson Japan). And analysis was performed. Consort-30 was used as software for setting and analyzing cell sorting conditions. When analyzing lacZ expressing cells
Using a lacZ vital staining substrate (Imagene green C12FDG lacZ gene expression kit; Molecular Probes), the cells were stained without fixing cells (30 minutes at 37 ° C.) and sorted. Incidentally by one of the sorting operation, approximately 10 ⁵ cells were collected in positive fraction. The positive fraction obtained here was used for the following assays of germ cell traits and assays for developmental potential in living body transplantation.

Example 4: Reproduction by immunohistochemical staining
Assay of cytoplasm The cells selected by FACS were fixed with 4% paraformaldehyde, washed with PBS, adhered to a slide glass by centrifugation using cytospin, and blocked with a blocking solution [0.1% BSA (Bovin Serum Albumen, Sigma). / PBS-
T] (at room temperature for 60 minutes)
It was allowed to react with the next antibody solution overnight (4 ° C.). Primary antibody dilution
The reaction solution was 1: 1000 for the Mvh antibody, and 1: 100 for the Oct3 / 4 antibody and Sycp3 antibody. After the reaction, 3 times with PBS (15
) And washed with an AP (alkaline phosphatase) -labeled or HRP (peroxidase) -labeled anti-rabbit IgG (Bio-Rad) as a secondary antibody for 30 minutes. After washing three times with PBS, the antibody reaction was performed using a BCIP / NBT solution (AP-labeled antibody) and a DAB coloring solution [0.05% 3,3'-diamino-benzidine, 0.0
[1% H2O2 / PBS] solution (HRP-labeled antibody) was detected by an enzyme antibody method, and used as a microscopic sample using an encapsulant Eukitt (O. Kindler). All of the sorted cells showed a positive reaction with respect to the antibody staining for the antigen protein and the AP staining, which are characteristics of the primordial germ cells (see FIG. 4).
It was confirmed that the cells that differentiated as Mvh-expressing cells were germ cells.

Example 5: Evaluation of sperm formation ability by aggregate transfer
Assay 12.5 days Fetal male gonad cells are dispersed and allowed to form reagglomerates, and when transplanted under the adult testis capsule, spermatogenesis is known to occur in the graft approximately 1-2 months later. ing. This indicates that fetal male germ cells from day 12.5 onward acquire the fertility under an appropriate culture environment. Therefore,
Similar transplantation experiments were performed on FACS-purified Mvh-expressing cells, and their spermatogenesis ability was assayed. FACS purified lacZ
Knock-in ES-derived cells (approximately 10 ⁵ cells) and dispersed cells (approximately 10 ⁵ cells) derived from fetal male gonads at day 12.5 are mixed, and cell aggregates are formed by suspension culture for 1 day. The cells were transplanted under the testicular capsule of male nude mice and cultured in vivo. In this aggregate culture, the viability of primordial germ cells derived from host gonads is extremely low, and even if contaminated, lacZ knock-in ES-derived cells can easily express lacZ (the Mvh expression continues to sperm cells). Can be distinguished. Two months after the transplantation, the transplanted cell mass formed a unique seminiferous tubule structure in the transplanted testis, and an image of sperm formation by ES-derived cells was observed inside the seminiferous tubule (see FIG. 5). No induction of differentiation as control experiment
When similar transplantation experiments were performed using ES cells, tumors derived from the characteristics of ES cells were formed in the host testis. Therefore, it was found that germ cells that differentiate as Mvh-expressing cells in in vitro culture of ES cells can be differentiated into spermatozoa by using at least the biological culture method.

[0048]

At present, the analysis of gene function and the creation of applied animals using reproductive engineering mainly involve DNA injection into fertilized eggs or genetic recombination using totipotent ES cells. It is based on the fact that engineered cells differentiate into germ cells and the transgene is passed on to the next generation. The present invention provides a technique for inducing germ cells in vitro from totipotent cells such as fertilized eggs and ES cells to obtain sperm derived from genetically engineered cells more quickly and directly,
It greatly contributes to the efficiency and simplification of genetic modification technology. In addition, through its simplicity and high efficiency, the range of application of genetic modification technology will be expanded, and it will contribute to the development of a wide range of basic research, including regenerative medicine and animal husbandry.

That is, according to the present invention, a mouse not specifically expressed in undifferentiated stem cells but specifically expressed in differentiated germ cells
A cultured cell line that can detect the expression of the Vasa homolog (Mvh) gene with high sensitivity has been established. The present invention has succeeded for the first time in inducing germ cell differentiation in in vitro embryoid body formation of embryonic stem (ES) cells and in isolating and purifying germ cells derived from in vitro differentiation induction of ES cells. Things. Therefore, the present invention includes the following application aspects.

1. Genetic modification of ES cells is performed to analyze gene function because ES cells have the property of becoming the next generation through germ cells (sperm). And the most easily missing property is also its ability to contribute to the germline. The present invention provides an excellent experimental system for assaying the ability of the ES cell system to differentiate into germ cells.

2. Conventionally, the production of a genetically modified animal via an ES cell is performed through the production of a chimeric animal in which the isolated ES cell is transplanted into a blastocyst stage embryo, followed by the production of a hetero animal derived from the ES cell. Here, the degree to which ES cells participate in spermatogenesis in chimeric animals is an important step to determine the success or failure of creating modified animals. In contrast, the present invention provides a technique for selecting germ cells from ES cells by an in virto differentiation system, and for directly producing ES cell-derived sperm by a transplantation method. This has the advantage that a heterozygous animal can be obtained within one generation shorter than the conventional method, and that ES cells can be reliably introduced into the reproductive lineage. Specifically, the present invention provides a means for surely obtaining spermatozoa and their offspring from ES cell clones whose contribution to the germ lineage is extremely low (see FIG. 6).

3. The above indicates that when ES cells are animals other than mice, for example, large livestock animals and rare animals, the generation time to sexual maturation and the supply of host early embryos and mothers,
It becomes a factor that causes further difficulties. Therefore, the application of the present invention to animal species other than mice has even greater significance.

4. In vitro which is the main element of the present invention
The establishment of germ cell differentiation in culture can be used as an experimental model system for the establishment and differentiation of the reproductive lineage that is the basis of life phenomena. For example, the present invention can be applied to an evaluation system for observing the effects of environmental chemicals on germ cell differentiation, including humans.

[Brief description of the drawings]

FIG. 1. ES in which lacZ and GFP genes were knocked in by homologous recombination to visualize Mvh gene expression.
Cell clones were generated. The upper part of FIG. 1 schematically shows the design diagram, and the lower part is a photograph showing the test results of two types of knock-in ES cells determined by genomic Southern analysis. Germ cell-specific Mvh gene expression was shown in these clones to be lacZ (clone # 417) and GFP (clone # 3).
7) Identified as gene expression.

FIG. 2 is a micrograph showing the appearance of Mvh-expressing cells due to embryoid body formation. Embryoid body formation was performed using GFP knock-in ES cells, and the morphology on days 0, 2, 3, and 5 after culture
The appearance of GFP-expressing (Mvh-expressing) cells was observed. After culture, 3
GFP expression (Mvh expression) in some embryoid bodies after day
The appearance of cells was detected. The right shows a bright-field image and the left shows a dark-field fluorescent image.

FIG. 3 shows the effect of co-culture with BMP4 forced expression cells.
It is a chart and micrograph which show the refinement | purification by FACS sorting. A cell line transfected with a BMP4 forced expression vector was established for the mesodermal cell line M15 cells derived from the embryonic genital ridge. GFP (Mvh gene expression) -positive cells were not observed when the ES cell clone was mixed with the parent strain M15 for coagulation culture, whereas when mixed with BMP4-expressing M15 cells, the cells were already cultured for only one day. The appearance of GFP-positive cells was observed, and the frequency of appearance also increased to about 3%. In the case of ES cells alone, no GFP (Mvh gene expression) -positive cells appeared (upper row), whereas in the case of co-culture with BMP4 forced expression cells, Mvh-expressing cells were observed on day 1 of culture (Middle). Further, the positive cell fraction was again subjected to FACS sorting, whereby high-purity Mvh-expressing cells were recovered. The stained images on the right show the Mvh expression of each fraction assayed by the anti-Mvh antibody. The upper part shows the antibody staining, and the lower part shows the bright field image.

FIG. 4 is a micrograph showing characteristic analysis of FACS-purified Mvh-expressing cells. That is, FIG. 4 shows the results of analysis of GFP (Mvh) -expressing cells appearing in the embryoid body on day 5 of culture by FACS. Perform PI staining on the dispersed embryonic somatic cells,
The stained cells were classified as dead cells from the object of analysis. The ratio of Mvh expressing cells was 0.3-0.5%. The upper six cells show purified cells as anti-Mvh antibody, anti-TRA98 antibody, anti-Oct3
4 is a micrograph showing the result of staining with a / 4 antibody, an anti-Sycp3 antibody, and AP staining. Purified Mvh expressing cells were positive for these stains. The control image shows the result of staining the dispersed embryoid body cells on day 1 of culture with an anti-Mvh antibody as a control experiment. In this case, no cells showing staining were observed. The lower two panels are micrographs showing the results of dispersing embryoid bodies on day 5 of culture and double staining with an anti-Mvh antibody (left) and an anti-Sycp3 antibody (right). Mvh-expressing cells and Sycp3-expressing cells were found to be identical.

FIG. 5 is a micrograph showing an image of sperm formation generated by living body transplantation of Mvh-expressing cells. FACS purified la
As a result of transplanting a cell aggregate obtained by mixing cZ knock-in ES-derived cells and dispersed cells derived from fetal male gonads at day 12.5 under the adult testis capsule, the transplanted cell mass within the host testis at 2 months after transplantation had its own It formed a seminiferous tubule structure (upper). The seminiferous tubules were filled with strongly positively stained cells by X-gal staining, indicating that they were derived from lacZ knock-in ES cells. Inside the seminiferous tubules, an image of sperm formation by lacZ knock-in ES-derived cells was observed (lower row).

FIG. 6 is a schematic diagram showing a comparison between the present invention and a conventional method.

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Claims (19)

[Claims] 1. A marker gene is introduced so as to be under the control of the expression of the Vasa gene or its homolog gene.
Culturing the ES cell line in the presence of a germ cell differentiation promoting factor,
A method for obtaining germ cells, which comprises selecting cells having germ cell differentiation ability using the expression of a marker gene as an index. 2. A marker gene to be introduced into an ES cell line,
2. The method for obtaining a germ cell according to claim 1, which is a GFP, BFB, YFP, or lacZ gene. 3. The method for obtaining a germ cell according to claim 1, wherein the marker gene is introduced into the ES cell line by homologous recombination. 4. The method for obtaining a germ cell according to claim 1, wherein the ES cell line is a cell line derived from a mammal or a bird. 5. The method for obtaining a germ cell according to claim 1, wherein the differentiation promoting factor is BMP4. 6. A co-culture of cells into which a gene encoding a germ cell differentiation-enhancing factor has been introduced and an ES cell line into which a marker gene has been introduced so that the gene is placed under the control of the expression of the Vasa gene or its homolog gene. A method for obtaining germ cells, comprising selecting cells having germ cell differentiation ability using gene expression as an index. 7. The method for obtaining a germ cell according to claim 6, wherein the gene encoding a germ cell differentiation promoting factor is a gene encoding BMP4. 8. A marker gene is introduced so as to be under the control of the expression of the Vasa gene or its homolog gene.
A method for screening a germ cell differentiation promoting factor, comprising culturing an ES cell line in the presence of a test substance and selecting a test substance that induces expression of the marker gene. 9. A germ cell differentiation promoting factor obtained by the method according to claim 8. 10. A germ cell obtained by the method according to any one of claims 1 to 7, and the obtained cell aggregate is transplanted under a testis capsule of an adult male to form a formed fine cell. A method for obtaining an ES cell-derived sperm, which comprises obtaining cells from a cell mass having a tubular structure. 11. The method for obtaining sperm according to claim 10, wherein the germ cell culture is a method of co-culturing with cells derived from fetal male gonads. A sperm obtained by the method according to claim 10 or 11. 13. A recombinant cell line obtained by introducing a foreign gene into an ES cell line into which a marker gene has been introduced so as to be placed under the control of the expression of the Vasa gene or a homolog gene thereof, is used as the ES cell line. Item 8. A method for obtaining a genetically modified germ cell, comprising performing the method according to any one of Items 1 to 7. 14. A method for obtaining spermatozoa according to claim 10 or 11, using the recombinant germ cell strain obtained by the method for obtaining germ cells according to claim 13. The method for obtaining transgenic sperm. 15. A transgenic sperm obtained by the method according to claim 14. 16. A method for producing an ES cell-derived individual by microinsemination using sperm obtained by the method according to claim 10, 11, or 14. 17. An ES cell line into which a marker gene has been introduced so as to be under the control of Vasa gene or its homolog gene expression, is cultured in the presence of a test substance, and the number of cells in which marker gene expression is induced is determined. A test method for toxicity to germ cells, which comprises examining a substance having toxicity to germ cells as an indicator. 18. An ES cell line into which a marker gene has been introduced so as to be controlled under the expression of the Vasa gene or its homolog gene, is cultured in the presence of a germ cell differentiation promoting factor and a test substance to induce expression of the marker gene. A test method for toxicity to germ cells, comprising testing a substance having toxicity to germ cells using the number of cells as an index. 19. An ES cell line into which a marker gene has been introduced so as to be under the control of the expression of the Vasa gene or a homolog gene thereof, is cultured together with a BMP4 gene-introduced cell line in the presence of a test substance to induce expression of the marker gene. A method for testing toxicity to germ cells, comprising assaying a substance having toxicity to germ cells using the number of cells as an index.