JP2002543814A - Genetic modification of male germ cells for transgenic species development and gene therapy - Google Patents

Abstract

Abstract: An in vivo method has been described for integrating exogenous genetic material into the genome of a vertebrate, which comprises administering a gene delivery mixture comprising a viral vector, such as a retroviral vector, to the testes of a vertebrate. , Delivering a polynucleotide encoding the desired trait or product. In addition, in vitro methods of integrating exogenous genetic material into the vertebrate genome have been described, wherein germ cells are obtained from a donor male vertebrate and genetically modified in vitro prior to transfer to a recipient male vertebrate. Add decorations. After transfer, the male vertebrates carrying the genetically modified germ cells are crossed with female vertebrates to produce transgenic offspring carrying the polynucleotide in the genome. Further, non-human transgenic vertebrates, including transgenic progeny, produced according to the method are disclosed. Transgenic cells from transgenic vertebrates are also published as sperm or ova, progenitor cells of any of these, or germ cells such as somatic cells. A method for producing a non-human transgenic vertebrate strain, including native germ cells, carrying at least one heterologous polynucleotide in its genome has been described, including transgenic male germ cells useful in practicing the method. Vertebrate semen is also announced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Throughout this application various publications are referenced in parentheses. The entire texts of these publications are incorporated herein by reference in this application to further elaborate the state of the relevant art. The present invention relates to the medical field, particularly to the field of transgenics and gene therapy. The invention is particularly directed to in vitro and in vivo methods for making genetic modifications to male germ cells and support cells (ie, Leydig and Sertoli cells), which include exogenous genetic material in the vertebrate genome. Includes methods for producing integrated, transgenic vertebrates and transgenic vertebrate strains.

BACKGROUND OF THE INVENTION The field of transgenics was originally developed to understand the action of single genes in relation to animal individuals, as well as gene activation, expression, and interaction phenomena.
This technology has been used to create models of various diseases in humans and other animals. Transgenic technology is the most powerful tool that can be used to study genetics and understand genetic mechanisms and functions. It is also used to study the relationship between genes and disease. About 5,
000 diseases are due to a single gene defect. More generally, other diseases are the result of a complex interaction of one or more genes with environmental factors such as, for example, viruses or carcinogens. Understanding such interactions is particularly important for the development of therapeutics such as gene therapy and drug therapy, as well as treatments such as organ transplantation. Such treatment may compensate for the functional deficits and / or eliminate unwanted functions that develop in vivo.

[0003] Transgenesis has also been utilized for livestock improvement and large-scale production of biologically active drugs. Historically, transgenic animals have been created almost exclusively by microinjection of fertilized eggs. The pronucleus of a fertilized egg is microinjected in vitro with an exogenous, ie, heterologous or allogeneic, DNA or hybrid DNA. The microinjected fertilized eggs are then transferred to the reproductive organs of a pseudopregnant female. (Example: PJA K
rimpenfort et al., Mature T-cell deficient transgenic mice and methods for producing transgenic mice, US Patent Nos. 5,175,384 and 5,434,340; PJA
Krimpenfort et al., Transgenic mice deficient in mature lymphocytic cell types, US Pat. No. 5,591,669).

[0004] This widely used technique requires large numbers of fertilized eggs, equipment for handling embryos, and facilities for microinjecting them in vitro. One reason is the high rate of egg loss due to lysis during microinjection. Moreover, engineered embryos often do not survive implantation in the uterus. These factors contribute to the very low efficiency of the technology. Superovulated mammals (eg, primates, cows, horses, pigs, and mice) also produce only 10-20 or fewer eggs per cycle. Palmiter, RD and
Brinster, RL, mouse germline, Anna. Rev. Genet. 20: 465-99 [1986]),
In addition, only 0.1% of cattle, sheep and pig eggs (Wall, RJ, et al., Transgenic livestock production: large-scale genetic engineering, J. Cell Biochem 49: 113-120
[1992]) develop in transgenic animals. Typically, 300-500 fertilized eggs will need to be microinjected to produce perhaps three transgenic animals. Therefore, the production of large animals by these techniques is extremely expensive. For this reason, mammalian transgenic technology has been almost exclusively restricted to mice due to its high fertility. Little has been done to improve the production of transgenic animals by microinjecting transgenes into fertilized eggs (Gordon, J. and Ruddle, FH; Integrity and stable germline transmission, Science 214: 1244-1246 [1981]).

[0005] Although small animals, such as mice, have proven to be a suitable model for certain diseases, their value in this context is limited. Large transgenic animals are more relevant to most human disease effects and therapeutic studies than mice because of their striking resemblance to humans in various aspects, and their organ systems and behavior More preferred for research. Large mammals are also more suitable as human potential organ donors than mice because their organs are of comparable size. As transgenic animals with potential for human xenotransplantation have begun to develop, more of these large animals will be needed. Transgenic technology allows such donor animals to be immunocompatible with human recipients.

[0006] Even after treatment with superovulatory drugs, most male mammals, including mice and almost all large mammals, usually have at least 10 10 eggs during each ejaculation, in contrast to only 10-20 eggs per female. Produces ^eight sperm (male germ cells). For this reason alone, male germ cells are a more preferred target for introducing foreign DNA into the germline, which, after simple and natural mating, leads to the creation of transgenic animals with greater efficiency. There will be. Nevertheless, attempts to generate transgenic mice that use sperm to transfer DNA into eggs have been described (Lavitr
ano, M., et al., Sperm cells as a vector for introducing DNA into eggs: genetic transformation of mice, Cell 57: 717-723 [1989]; WO-A-90 / 08192), Its effectiveness has not yet been approved. (Brinster, RL, et al., There is no simple solution for making transgenics, Cell 59: 239-241 [1989]). Recently, transgenic mice have been produced after injection of exogenous DNA with sperm heads into oocytes (Perry, AC, et al., Mammalian Transgenesis by Intracytoplasmic Sperm Injection, Science 284: 1180-1183). [1999]). After uterine transfer, 20% of these embryos developed into transgenic offspring.

[0007] Genetic information has been transferred into embryos using retroviral vectors (Ja
enisch, R., Germline integration and Mendelian transmission of exogenous Moloney leukemia virus, Proc. Natl. Acad. Sci. USA 73: 1260-1264 [1976]), but the animals have different gene insertions in different tissues. (Jaenisch, R., Retrovirus and Embryogenesis: Microinjection of Moloney Leukemia Virus into Midgestation Mouse Embryos, Cell 19: 181-188 [1980]). Recently, five transgenic calves have been produced by injection of a pseudo-type replication-defective vector based on Moloney murine leukemia virus. This vector was introduced into the perivitelline space of metaphase II oocytes (Chan, AW, et al., Transgenic Cattle Produced by Introgression Reverse Transcribed into Oocytes, Proc. Natl. Acad. Sci. US
A 95: 14028-14033 [1998]).

[0008] Although not yet fully realized, an alternative is the stable transfection of male germ cells in vitro and its transfer into recipient testes. Transfer of genetically labeled germ cells to the testis produced offspring, but at present no transgenic offspring were produced (Brinster, RL and Avarbok, MR, donor haplotypes after spermatogonia transplantation). Germline transmission of Proc. Natl. Acad. Sci. USA 91:11.
303-11307 [1994]). Spermatogenesis is a self-motile haploid (haploid) in which diploid spermatogonial stem cells supply daughter cells, which, through dramatic and unique morphological changes, can fertilize the ovum when fully matured. ) This is the process of becoming cells (male gametes). Primordial germ cells are first found in the endodermal yolk sac epithelium at stage E8 and are thought to be derived from the ectoderm of the embryo. (A. McLaren and M. Buehr, Cell Diff. Dev
31: 185 [1992]; Y. Matsui et al., Nature 353: 750 [1991]). They migrate from the yolk sac epithelium, through the hindgut endoderm, to the genital ridge, proliferate by mitosis and assemble into the testis.

[0009] Upon reaching sexual maturation, spermatogonia undergo five or six mitosis before entering meiosis. Primordial spermatogonial stem cells (A0 / As) proliferate to intermediate spermatogonia Apr, AaI, A1-4
And then differentiate into B-type spermatogonia. The B-type spermatogonia differentiate to form a primary spermatocyte, which enters a prolonged meiotic prophase, during which homologous chromosomes pair and recombine. The meiotic states that are morphologically distinguishable are: prefilament, fibrosis, mitosis, thickening, secondary spermatocytes, and haploid spermatids occur later. Sperm cells undergo significant morphological changes during spermatogenesis, such as nuclear reshaping, acrosome (acrosome) formation and tail assembly. (AR
Bellve et al., Sperm collection, capacitation, acrosome reaction, and fractionation, Methods E
nzymol. 225: 113-36 [1993]). Spermatocytes and sperm cells establish important contacts with Sertoli cells via a unique hemijunction attachment with Sertoli cell membranes. The final change in sperm maturation (ie, spermatozoa) occurs in the female reproductive tract before fertilization.

[0010] Initially, attempts were made to add transgenic animals to sperm by adding DNA to it and then using it to fertilize mouse ova in vitro. The fertilized eggs were then transferred to pseudopregnant foster females, and 30% of the offspring were reported to be transgenic and express the transgene. Despite repeated efforts by other investigators, however, this experiment was not reproducible and no transgenic pups were obtained. Indeed, there is little doubt that the transgenic animals thought to have been obtained by this method were not at all transgenic and that the reported DNA incorporation was only an experimental artifact. Data collected from laboratories around the world engaged in testing this method showed that no transgenics were obtained from a total of 890 pups generated.

In summary, while it is now possible to produce live transgenic progeny, the methods available are costly and extremely inefficient. Spermatogenic transfection according to the present invention is a simple, economical and minimally invasive method for producing transgenic animals, whether in vitro or in vivo, as well as heterologous genes in animal germ cells. To provide a potentially very effective method of transferring allogeneic genes to E. coli. To perform in vitro transfection of male germ cells and transplantation into the testes of a recipient male vertebrate, the first step is to transfer the transfected male germ cells from the testes of the recipient vertebrate. It is advantageous to reduce the number of untransferred male germ cells. Testicular cytoreduction is usually performed by whole body exposure of vertebrate individuals to gamma irradiation (X-rays) or by local irradiation of the testes. (Example: G. Pinon-L
ataillade et al., Endocrine and histological changes induced by sustained low-dose gamma irradiation of rat testis, Acta Endocrinol. (Copenh) 109 (4): 558-62 [1985]
G. Pinon-Lataillade and J. Maas, Sustained gamma irradiation in rats: dose-rate effects on loss and recovery of spermatogenesis, Strahlentherapie 161 (7): 421-26 [1985]
CR Hopkinson et al., Effect of local irradiation of testes on testicular tissue and plasma hormone levels in male rats, Acta Endocrinol. (Copenh) 87 (2): 413-.
23 [1978]; G. Pinon-Lataillade et al., Effects of germ cells on Sertoli cells during continuous low-dose gamma irradiation of adult rats, Mol. Cell Endocrinol. 58 (l): 51-
63 [1988]; P. Kamtchouing et al., Effects of sustained low-dose gamma irradiation on rat Sertoli cell function, Reprod. Nutr. Dev. 28 (4B): 1009-17 [1988]; C. Pinea
u et al., Evaluation of testis function after acute and chronic irradiation: Additional confirmation of the effect of late sperm cells on Sertoli cell function in adult rats, Endocrinol. 124 (6): 2720-28
[1989]; M. Kangasniemi et al., Cellular regulation of basal and FSH-stimulated cyclic (cyclic) AMP production in irradiated rat testis, Anat. Rec. 227 (l): 32-36 [1990]
G. Pinon-Lataillade et al., Effects of acute exposure of rat testis to gamma radiation on germ cell and Sertoli and Leydig cell function, Reprod. Nutr. Dev. 31 (6).
: 617-29 [1991]).

[0012] The mechanism of spermatogonia degeneration induced by gamma irradiation is considered to be related to apoptotic response. (M. Hasegawa et al., Resistance to irradiation-induced apoptosis and deletion of differentiating spermatogonia in p53-deficient mice, Radiat. Res. 149: 263-70 [1998]). Another method of reducing vertebrate testis cells is to administer a composition comprising an alkylating agent such as busulfan (Myleran). (Eg FX Jiang, spermatogonia behavior after recovery from busulfan treatment in rats, Anat. Embryol. 198
(l): 53-61 [1998]; LD Russell and RL. Brinster, ultrastructural observation of spermatogenesis after transplantation of rat testis cells into mouse seminiferous tubules, J. Androl. 17 (6): 615-27. [19
96]; N. Boujrad et al., Evolution of somatic and germ cell populations after busulfan treatment in uterus or neonatal cryptochidism in rats, Andrologia 27 (4): 223-
28 [1995]; RELinder et al., Endpoints of sperm toxicity in rats after short-term exposure to 14 reproductive toxicants, Reprod. Toxicol. 6 (6): 491-505 [1992];
Kasuga and M. Takahashi, Endocrine function of rat gonads with a reduced number of germ cells after busulfan treatment, Endocrinol. Jpn 33 (1): 105-15 [1986]).

[0013] Cytotoxic alkylating agents such as busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylethanesulfonic acid are often used in cancer chemotherapy to kill malignant cells. (Example: Andersson et
al., Parenteral busulfan for the treatment of malignancies, U.S. Patent Nos. 5,559,148 and 5,430,057; Stratford et al., Stimulation of stem cell growth by bryostatin, U.S. Patent 5,358,711; Luck et al., Cell foci. Using vasoconstrictors in combination with cytotoxic drugs for introduction into cells, US Pat. No. 4,978,332)
. Treatment of mice with busulfan (13 mg-40 mg / kg body weight) has been reported to kill male germ cells in testis; both stem cells and differentiating spermatogonia were killed; 30 mg / kg body weight The above doses resulted in azoospermia up to 56 days after treatment. (LR Bucci and ML Meistrich, effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormal and dominant lethal mutations, Radiation Research 176: 259-68 [1987]).

DISCLOSURE OF THE INVENTION The present invention provides an in vitro or in vivo method for transferring a heterologous gene as well as a heterologous gene into germ cells of an animal to produce a transgenic vertebrate. The need for genetic modification of spermatogenesis in either is illustrated. This technology demonstrates the need for germline and stem cell line gene therapy in humans and other vertebrate species, including the need for superior methods to reduce the number of testicular cells consisting of untransfected male germ cells I do. The technology according to the invention has significant value in producing large breeds of transgenic animals as well as in repairing genetic defects leading to male infertility. Male germ cells that have stably integrated DNA can be selected. These and other benefits and features of the present invention are described herein.

The present invention originates from the existing methods for genetic modification of animal germ cells and the desire by the inventors to improve the methods of producing transgenic animals. Prior art methods involve DN into conjugates produced in vitro or in vivo.
It relies on direct injection of A or generation of chimeric embryos using embryonic stem cells integrated into recipient blastocysts. Following this, embryos treated in this way
Transfer into the primed uterus or fallopian tube. These conventional methods are extremely protracted and costly, and based on several invasive steps, the production of transgenic offspring is sudden and unpredictable. In the search for an economical, rapid and efficient approach to transgenic production, the present inventors have determined that in vivo vertebrate male germ cells can be treated with nucleic acid segments, ie, polynucleotides, that encode desired traits and products. Alternatively, the present method was devised based on in vitro (ex vivo) genetic modification.

The present invention relates to in vivo and in vitro (ex vivo) genetic modification of vertebrate germ cells with desired genetic material, such as transfection or transduction.
In summary, the in vivo method involves injecting the genetic material with the appropriate vector directly into the testis of the animal. In this method, all or some male germ cells in the testis are genetically modified in situ and under effective conditions. The in vitro method employs a novel isolation or selection method to create a suitable donor gonad (
Germ cells obtained from the testis) or the animal's own testis, transfected or genetically modified in vitro, and then substantially reduced in testis cells in the donor or different recipient male vertebrate Including, returning the cells under appropriate conditions such that the cells regrow spontaneously in the reduced testes. This in vitro method screens germ cells transfected by various means before returning to the testis of the same or a different suitable recipient male, and confirms that the transgene is stably integrated into the genome. It has the advantage that it can be confirmed. Furthermore, only high germ cell populations can be returned after screening and cell sorting. This approach provides a high probability of transgenic offspring after mating.

In particular, the in vivo method according to the invention for integrating exogenous genetic material into the genome of a vertebrate comprises a gene encoding the desired trait or product, and optionally
Gene delivery mixtures, including but not limited to retroviral vectors, comprising at least one polynucleotide defining a gene encoding a genetic selectable marker, comprising a viral vector, can be obtained from a male vertebrate. Including administration into the testis. This gene is operatively linked to the promoter sequence (all individual genes used need not be linked to a single promoter sequence), resulting in the formation of a transcriptional unit and in the vertebrate testis It is administered under conditions effective to reach at least one sperm or sperm precursor present. The delivery mixture containing the polynucleotide is such that the polynucleotide encoding the desired trait or product is integrated into the genome of at least one male germ cell, such as a sperm or progenitor cell, thereby resulting in a genetic modification. The added male gametes are administered in amounts and under conditions effective to be produced by the vertebrate. This male vertebrate is then crossed with that species of vertebrate, either naturally or using artificial reproductive technology, to produce transgenic offspring carrying the polynucleotide in its genome.

The invention further includes an in vitro method for integrating at least one polynucleotide encoding a desired trait or product into the genome of a vertebrate. This in vitro method involves obtaining male germ cells, such as sperm cells or progenitor cells, from a donor male vertebrate, and generally in the presence of a gene delivery mixture comprising a viral vector, to the order of the vertebrate body temperature or Below that, the cell is cultured in vitro for a time sufficient for the polynucleotide encoding the desired trait or product to be integrated into the genome of the cell, without at least one encoding for the desired trait or product instead of an immortalizing molecule. Genetic modification with the polynucleotide and the polynucleotide defining the gene encoding the genetic selectable marker. Next, the genetically modified germ cells are isolated or selected using a genetic selectable marker expressed in the genetically modified cells, and are then transferred to the testes of recipient male vertebrates. The cells are transferred to remain in the seminiferous tubules, thus producing a genetically modified male gamete therein.
The male vertebrate is crossed with a female vertebrate of that species to produce transgenic offspring carrying the polynucleotide in its genome.

The present invention further relates to non-human transgenic male vertebrates produced according to either in vivo or in vitro methods for integrating exogenous genetic material into the vertebrate genome. The transgenic vertebrate produced according to the in vivo method is the recipient of the gene delivery mixture. This transgenic vertebrate, produced according to in vitro methods, is a recipient of the genetically modified male germ cells that have been transferred into their testes. This transgenic male vertebrate is mated with a female belonging to that species to contain native male germ cells that carry an exogenous polynucleotide in their genome that defines the gene encoding the desired trait or product. It is possible to However, somatic cells in tissues other than the testis of the transgenic vertebrate lack this polynucleotide. The present invention further relates to non-human transgenic male vertebrates produced according to either in vivo or in vitro methods of incorporating exogenous genetic material into the vertebrate genome. The non-human transgenic vertebrate is a direct or indirect progeny of a male vertebrate that has received the gene delivery mixture according to the in vivo method. In contrast, a non-human transgenic vertebrate is a direct or indirect progeny of a recipient of a genetically modified male germ cell that has been transferred into its testes according to the in vitro method. Thus, the transgenic offspring is a direct offspring of the transgenic male vertebrate, or
Or descendants of more generations. A transgenic vertebrate contains one or more cells in its genome that carry an exogenous polynucleotide that encodes a desired trait or product.

[0020] The invention further includes transgenic cells derived from the transgenic progeny. The cells can be sperm (ie, spermatozoa) or ova, germ cells such as progenitors of any of these, or somatic cells. The invention further relates to vertebrate semen comprising a plurality of transgenic male germ cells according to the invention. The present invention is further directed to a method of producing a non-human transgenic vertebrate line, including native germ cells, carrying at least one heterologous polynucleotide in its genome. The method includes crossing the transgenic progeny with a cognate heterozygous member and selecting progeny that carry the polynucleotide. The technology can be adapted to the production of transgenic animals for use as animal models, and to the modification of animal genomes, including humans, by the addition, modification or subtraction (subtraction) of genetic material, often resulting in phenotypic Create change. The method is also adapted to changes in the carrier status of animals, including humans, where the individual carries a recessive or dominant genetic disorder, or where the individual is prone to passing a multigenic disorder to subsequent offspring. It is possible. These and other advantages and features of the present invention are described in more detail in the detailed description of the preferred embodiments below. In further describing the present invention,
The disclosures of related applications US Serial Nos. 09 / 191,920; 09 / 292,723; and 09 / 311,599 are incorporated by reference.

BEST MODE FOR CARRYING OUT THE INVENTION The method of integrating exogenous genetic material into the genome of a vertebrate is based on at least one of the following strategies. A first method, an in vivo method of incorporating exogenous genetic material into the genome of a vertebrate, is to use a known gene delivery system to generate polynucleotides in situ into male germ cells in male vertebrate testes. (Eg, in vivo transfection or transduction), allowing the genetically modified germ cells to differentiate in the animal's own environment, and then displaying progeny (transgenic animals) that exhibit nucleic acid integration into the germ cells. select. The animals selected in this manner are bred or their sperm are used for insemination or in vitro fertilization to produce further transgenic offspring.

On the other hand, in vitro methods for integrating exogenous genetic material into the genome of vertebrates include:
Male germ cells are obtained from the testis of an appropriate donor or the animal's own testis, and they are genetically modified in vitro, the genetically modified germ cells are isolated or selected, and then spontaneously regenerated. And transferring them to the testes under appropriate conditions to proliferate. By either the in vivo or in vitro route, the method according to the invention comprises:
Suitable for application to a wide variety of vertebrates, all these vertebrates are capable of producing gametes-ie sperm or eggs. Thus, in accordance with the present invention, novel genetic modifications and / or characteristics are described in humans, e.g., non-human primates such as monkeys, marmosets, livestock agricultural animals such as sheep, cows, pigs, horses,
It can be distributed to vertebrates, including marine mammals, macrodermis, rodents such as mice and rats, and mammals such as gerbils, hamsters, rabbits, and the like. Other vertebrates include poultry such as chickens, turkeys, ducks, ostrich, emu, geese, guinea fowl, pigeons, quail, rare and ornamental birds, and the like. Of particular note are endangered species of wildlife, such as rhinoceros, tigers, cheetahs, certain condor species, and the like.

A “gene delivery (or transfection) mixture” in the context of this patent is, for example, a mixture of an effective amount of a lipid transfection factor, eg, a cationic or polycationic lipid such as polybrene. Means the selected genetic material, together with the appropriate vector. (Eg, Clark et al, Polycationic and cationic lipids enhance adenovirus transduction and transgene expression in tumor cells, Cancer Gene Ther. 6 (5): 437-46 [1999]). The efficiency of adenovirus, retrovirus, or lentivirus-mediated transduction is greatly enhanced by including polybrene during infection. The amounts of each component of the mixture are selected to optimize genetic modification of germ cells of a particular species, for example, by transfection or transduction. Such optimization by routine experimentation is sufficient. The ratio of DNA to lipid can vary widely, but is preferably 1: 1; however, other ratios may be used based on the lipid transfection factor used. (Eg, B
anerjee, R. et al. [1999]; Jaaskelainen, I. et al., Lipid carriers with membrane active components and small complex size are required for effective cellular delivery of antisense phosphorothioate oligonucleotides , Eur, J. Pharm.Sci. 10 (3
): 187-193 [2000]; Sakurai, F. et al., Effect of DNA / liposome mixing ratio on physiochemical characteristics of plasmid DNA / cationic liposome complex, cellular uptake, and intracellular trafficking. Resulting gene expression, J. Contro
lled Release 66 (2-3): 255-69 [2000]). "Genetic material," as used herein, means a DNA sequence capable of imparting a novel genetic modification, or biologically functional characteristic, to a recipient animal. Novel genetic modifications or features are defined as 1
Or one or more genes or gene segments, resulting from the exclusion or mutation of one or more genes, and further comprising regulatory sequences such as, for example, enhancers, promoters, or activator / suppressor binding sites. But not limited to. The transfected genetic material is preferably functional, meaning that it expresses the desired trait, i.e., via a product or by suppressing other production. Examples of other mechanisms by which gene function is expressed include genomic imprinting, ie, inactivation of either one of a pair of genes (alleles) very early in embryonic development, or gene sequence. Inactivation of genetic material by mutation or deletion of, or suppression of dominant negative gene products, and others.

The desired product is not an immortalizing molecule but a preselected product, such as SV40 large T or polyomavirus large T antigen. Immortalizing molecules can transform cells into "cancer-like" cells. "Immortalization" due to the expression of immortalizing molecules causes male germ cells to undergo meiosis, which is essential for the production of many of its important germ cell features, such as a functioning male gamete Can cause loss of ability. (E.g., Wolkowicz, MJ, Coonrod, SM, Reddi, PP Milla
n, JL, Hofmann, MC, Herr, JC, Subdivision of the differentiation phenotype of the spermatogenic cell line GC-2spd (ts), Biology of Reproduction 55: 923-32 [1996]). Male germ cells that have been genetically modified to express an immortalizing molecule are therefore not useful for producing transgenic vertebrate progeny according to the present invention. Furthermore, the novel genetic modification can be an artificially induced mutation or mutation, or an allelic mutation or mutation of a naturally occurring gene. Mutations and mutations can be artificially induced by a number of techniques, all of which are known in the art, including chemical treatments, gamma irradiation treatments, ultraviolet irradiation treatments, ultraviolet irradiation, certain chimeric DNAs. / Use of RNA oligonucleotides (chimera formation) and their synonyms. Chemicals useful for inducing mutations or mutations include carcinogens such as ethidium bromide and other substances known in the art.

[0025] DNA segments of a particular sequence can also be made by incorporating desired mutations or variants, or by disrupting the gene or altering the genomic DNA. One skilled in the art will readily recognize that genetic material is hereditary and, therefore, is present in almost all cells, including germ cells, of future generations of its progeny. New features include past expression of non-expressed traits, enhancement or reduction of expressed traits, overexpression or underexpression of traits, ectopic expression-expression of traits in tissues that are not normally expressed,
Or there is an attenuation (attenuation) or elimination of a previously expressed trait. Other novel features include qualitative changes in the expression trait, eg, reduction or alleviation, or otherwise preventing the development of a multigenic hereditary disorder. "Transfection factor", as used herein, refers to the presence of genetic material in a eukaryotic cell, preferably a mammalian cell, more preferably a mammalian germ cell, to enhance the uptake of the exogenous DNA segment. It means the composition of the substance to be added. Enhancement is measured relative to uptake in the absence of the transfection agent. An example of a transfection factor is adenovirus-transferrin-polylysine-D
Contains NA complex. These complexes generally enhance the uptake of DNA into the cell and reduce its degradation during passage from the cytoplasm to the nucleus of the cell. These complexes can be targeted to male germ cells using specific ligands recognized by germ cell cell surface receptors, such as c-kit ligands or modifications thereof.

[0026] Other preferred transfectants are lipofectin, lipfectamin (lipfecta
mine), DIMRIEC, Superfect and Effectin (Qiagen), Unifectin, Maxifectin, DOTMA, DOGS (Transfectam; dioctadecylamidoglycylspermine), DOPE (1,2-diol eoyl-sn-glycero-) 3-
Phosphoethanolamine), DOTAP (1,2-diol eoyl-3-trimethylammonium propane), DDAB (dimethyl dioctadecylammonium bromide), DH
DEAB (N, N-di-n-hexadecyl-N, N-dihydroxyethylammonium bromide)
, HDEAB (Nn-hexadecyl-N, N-dihydroxyethylammonium bromide),
Contains polybrene or poly (ethylene imine) (PEI). (Eg, Banerjee, R
et al., a new series of non-glycerol-based cationic transfection lipids for use in liposome gene delivery, J. Med. Chem. 42 (21): 4292-99 [1999]; God.
bey, WT et al., Improved packing of poly (ethyleneimine / DNA complexes increases transfection efficiency, Gene Ther. 6 (8): 1380-88 [1999]; Kichler, A et al., Lipospermine Of DNA complex formation medium on transfection efficiency of DNA / DNA particles, Gene Th
er. 5 (6): 855-60 [1998]; Birchaa, JC et al., Physiochemical characterization and transfection efficiency of lipid-based gene delivery complexes, Int. J. Pharm. 1
83 (2): 195-207 [1999]). These non-viral elements are capable of heterologous integration into the vertebrate genome without being restricted to the size normally associated with viral-derived transfectants.
It has the advantage of promoting stable integration of DNA sequences. "Virus", as used herein, refers to any virus, or transfected fragment thereof, which may facilitate delivery of genetic material into male germ cells. Examples of viruses that are suitable for use herein include adenovirus, adeno-associated virus, retroviruses such as the human immunodeficiency virus, lentivirus, mumps virus (epidemic parotitis), and the Any transfected fragments, and other viral DNA segments that promote uptake of the desired DNA segment and release into the germ cell cytoplasm, and mixtures thereof. Preferred viral vectors are the Moloney murine leukemia virus and a retroviral vector derived from the Moloney virus called vesicular stomatitis virus glycoprotein (VSV-G) -Moloney murine leukemia virus. The most preferred viral vector is the pseudotyped (VSV-G) lentiviral vector from the HIV virus (Naldini et al., [1996]). In addition, mumps virus is particularly suitable for its affinity for immature sperm cells, including spermatogonia. All of the above viruses will require modifications to reduce non-pathogenicity or antigenicity. Other known vector systems, however, are also useful within the scope of the present invention.

In an in vivo method of incorporating exogenous genetic material into the vertebrate genome, administering the gene delivery mixture into the testes of the vertebrate delivers directly to at least one of the testes of the animal, where In vivo transfer of the gene delivery mixture into male germ cells by distributing to the male germ cells at various stages of development. The in vivo method preferably employs the injection of the gene delivery mixture into the seminiferous tubules or testis network, and most preferably into the testicular export tubules or export tracts, for example by micropipette. To ensure a stable infusion of the gene delivery mixture, the infusion is performed under pressure that does not damage the delicate tubules in the testis.
This is done using a micropipette, with a pico pump delivering a precisely metered volume under controlled pressure. Micropipettes are made of a suitable material such as metal or glass and are usually made from a glass tube whose working tip is stretched to a small hole, for example using a pipette making machine.
The tip may be angled in a convenient manner to facilitate entry into the testicular tubule system. In addition, the micropipette can also be supplied at the injection site with a beveled working end to further improve and reduce damage to the delicate tubule upon penetration. This oblique angle can be produced by means of a specially produced polishing device.
The tip diameter of an in vivo injection pipette is typically about 15-45 microns, but other sizes can be used as needed based on the size of the animal. The tip of the pipette should be removed using a binocular microscope with coaxial illumination, using care to avoid damaging the tubule wall opposite the injection point and minimizing trauma. Can be introduced into the system. On average, a magnification of about 25 to 80 times is appropriate, and this procedure can be performed by a single skilled technician without assistance, so that no benches with micromanipulators are required. A small amount of a suitable non-toxic dye can be added to the gene delivery fluid to confirm testicular delivery to the seminiferous tubule and seeding. It can be visualized and tracked under a microscope,
It may include a dilute solution of a non-toxic dye.

In this way, the gene delivery mixture reaches the male germ cells and is placed in intimate contact. Male germ cells contain sperm (ie, male gametes) and their developing precursors. In fetal development, primordial germ cells are thought to arise from the ectoderm of the embryo and are first observed in the endodermal yolk sac during stage E8. From there, they travel through the hindgut endoderm to the genital ridge. In sexually mature male vertebrates, there are several types of cells that are sperm progenitors and are capable of genetic modification, including a primitive spermatogonial stem cell known as A0 / As, Differentiates into type spermatogonia. The latter further differentiates to form primary spermatocytes, which enter into prolonged meiotic prophase, during which homologous chromosomes pair and recombine. Useful progenitor cells at several morphological / developmental stages are also recognizable: prefilament spermatocytes, microfilament spermatocytes, phagocytosis spermatocytes, thick thread spermatocytes Mother cells, secondary spermatocytes, and haploid sperm cells. The latter undergoes large morphological changes during spermatogenesis, including nuclear reshaping, acrosome (acrosome) formation and tail assembly. The final change in sperm (ie, male gamete) maturation occurs in the female reproductive organs before fertilization. The polynucleotide contained in the gene delivery mixture administered into the testis in an in vivo method reaches the germ cell at any one of the stages described above, and the germ cell at the relatively more receptive stage Is preferentially captured.

In vitro (ex vivo) integration of exogenous genetic material into the vertebrate genome
In the method, the male germ cells are preferably, but not limited to, diploid spermatogonia, wherein the diploid spermatogonia are exposed or contacted with the gene delivery mixture. . Whether used in an in vivo or in vitro method, the gene delivery mixture, once contacted with male germ cells, integrates the exogenous genetic material into a suitable cellular location for integration and expression in the genome. Promote uptake and transport. Numerous known gene delivery methods can be used to incorporate nucleic acid sequences into cells. In either in vivo or in vitro methods, the gene delivery mixture typically comprises a polynucleotide encoding the desired trait or product, with an appropriate promoter sequence, and optionally, a liposome, retroviral vector, adenovirus, Includes an agent that increases polynucleotide sequence uptake or comprises the polynucleotide sequence, such as a viral vector, adenovirus enhanced gene delivery system, or a combination thereof. A reporter construct containing a genetic selectable marker, such as the gene encoding green fluorescent protein, may be further added to the gene delivery mixture. A target molecule, such as a c-kit ligand, may be added to the gene delivery mixture to enhance the transfer of genetic material into male germ cells. Immunosuppressants such as cyclosporine or corticosteroid may also be added to the gene delivery mixture, as is known in the art. In in vitro methods for incorporating exogenous genetic material into the genome of a vertebrate, male germ cells are obtained or collected from the donor male vertebrate by means known in the art. The germ cells thus obtained are then exposed to the gene delivery mixture, preferably within a few hours, or cryopreserved for future use.

In one embodiment according to the present in vitro method, obtaining male germ cells from the donor vertebrate can be achieved by testis traversal. Traversal of the isolated testis tissue can be accomplished, for example, by separating the vertebrate testis, desquamating, and severing and chopping the seminiferous tubules. The separated cells are then known to gently cleave the cell substrate to release non-damaged cells, such as pancreatic trypsin, collagenase type I, pancreatic deoxyribonuclease (DNA degrading enzyme) type I, bovine serum Incubation may be in an enzyme mixture containing enzymes such as albumin and modified DMEM medium. The cells are incubated in the enzyme mixture at a temperature between about 5 minutes and about 30 minutes, more preferably between about 15 minutes and about 20 minutes, between about 33 ° C. and about 37 ° C., more preferably between about 36 ° C. and 37 ° C. I can do it. After washing the enzyme mixture from the cells, the cells are placed in a culture medium, such as DMEM or the like, and plated in petri dishes for genetic modification by exposure to the gene delivery mixture. This traversal is inappropriate when the donor and recipient male vertebrate are intended to be the same animal,
In such a case, it is preferable to induce ejaculation by inducing a biopsy method with little destruction or by means known in the art. Any of a number of commercially available gene delivery mixtures can be used, to which polynucleotides encoding the desired trait or product are further mixed. The final gene delivery mixture comprising the polynucleotide is then mixed with the cells for about 2 hours to about 16 hours, preferably for about 3 to 4 hours, about 33 ° C to about 37 ° C, more preferably about 36 ° C to 37 ° C. Interaction at a temperature of ° C., and more preferably at a constant and / or controlled pressure. After this period, the cells are brought to about 33 ° C. to 34 ° C., preferably about 30 ° C.
It is left at a lower temperature of about C to 35C for about 4 to 20 hours, preferably about 16 to 18 hours. Other conditions that do not fundamentally deviate from what has been described may also be used as known to those skilled in the art.

For both in vivo and in vitro methods, the most preferred embodiments use a retroviral vector system developed for gene therapy (
Naldini, L., et al., Lentiviral vector in vivo gene delivery of non-dividing cells and transduction with stability, Science 272: 263-267 [1996]),
The system has been used to transduce male germ cells in vivo or in vitro. The present gene delivery system uses retroviral particles generated by the three plasmid expression system. In this system, the packaging construct contains the human cytomegalovirus (hCMV) immediate early promoter, which drives the expression of all viral proteins. The design of this artifact is
Eliminates cis-acting sequences important for viral packaging, reverse transcription and integration of these transcripts. The second plasmid encodes a heterologous envelope (outer membrane) protein (env), a glycoprotein of vesicular stomatitis virus (VSV-G).
The third plasmid, the transduction vector (pHR '), provides the necessary cis-acting sequences for human immunodeficiency virus (HIV), reverse transcription and integration, and cloning of heterologous complementary DNA (cDNAs), which are required for packaging. Unique restriction sites for For example, a genetic selectable marker such as, for example, enhanced green fluorescent protein (EGFP), and
And / or a gene encoding another preselected or desired trait or product is cloned downstream of the HCMV promoter in the HR 'vector and further operatively linked to form a transcription unit. Have been. The VSV-G pseudotype retroviral vector system can infect a wide range of cells, including cells from different species, and can integrate into its genome. Certain retroviruses, ie, lentiviruses such as HIV, have the ability to infect non-dividing cells. They have limited capacity for heterologous DNA sequences and the size limit of this vector is 7-7.5 kb (Verma, IM and Somia, N., Gene Therapy-Perspectives, Problems and Prospects, Nature 389: 239-242 [1997]). In vivo experiments using lentivirus do not block expression like other retroviral vectors and show that in vivo expression in brain, muscle, liver, or pancreatic islet cells is maintained for at least 6 months, This is the longest test time to date (Verma and Somia [1997]; Anderson, WF., Human Gene Therapy, Nature (S
uppl). 392: 25-30 [1998]).

To express the genetic material delivered by transfection, transduction, or other means to obtain expression of the desired trait or product, the promoter sequence encodes the desired trait or product. The polynucleotide sequence is operatively linked. For the purposes of the present invention, "operably linked" means that within the transcription unit, the promoter sequence is located upstream from the coding sequence (
I.e. in the 5 'direction in this regard), the coding sequence is 3' to the promoter.
Or 3 'to the promoter in the gene sequence, meaning that expression is thereby regulated in a consistent manner with each other. Both the promoter and the coding sequence are oriented in a 5 'to 3' manner, so that all essential enzymes, transcription factors, cofactors, activators, and reactants are present and present in desired physical conditions, e.g., At pH and temperature, transcription can occur in vitro. This does not mean that conditions will favor transcription in any particular cell. For example, transcription from a tissue-specific promoter is generally not favored in heterologous cell types from different tissues. The promoter sequence is selected to be the cell type of interest and / or to operate under the physiological or developmental conditions of interest. Useful promoter sequences include, but are not limited to, a cytomegalovirus (CMV) promoter, a constitutive promoter, or a human C-reactive protein (CRP) promoter.
However, this includes but is not limited to inducible promoters (eg, Kanzler, S., et.
al., TGF-betaI in liver fibrosis: an induced transgenic mouse model for studying liver fibrosis, Am. J. Physiol. 276 (4Pt1): G1059-68 [1999]), or insulin-like growth factor (IGF- 1) Promoters (eg, Meton I., et al, growth hormone induces insulin-like growth factor-I gene transcription through the synergistic action of STAT5 and HNF-Ialpha, FEBS Lett. 444 (2-3): 155 -59 [19991]. Useful promoters include promoters that promote transcription in cells of various tissues, such as the insulin receptor (IR) gene promoter (eg, Tewari, DS, et al.).
al., characterization of the promoter region and 3 'end of the human insulin receptor gene, J. Biol. Chem. 264 (27): 16238-45 [1989]); growth hormone receptor (GFIR) P2 or P3 promoter (eg, Jiang, H., et al., Isolation and characterization of a new promoter for the bovine growth hormone receptor gene,
J. Biol. Chem. 274 (12): 7893-900 [1999]); Alternatively, the leptin promoter (
For example, Chen, XL et al., Analysis of the 762-bp proximity leptin promoter that drives and regulates the expression of the transgene of the growth hormone receptor in mice, Biochem. Biophys. Res. Commun. 262 (1): 187- 92 [1999]), but is not limited thereto.

Also useful in a variety of applications are tissue-selective (ie, tissue-specific) promoters, ie, whose expression is preferentially as compared to one or more other tissue types. Appears on certain types of tissue cells. Tissue-specific promoters
It is particularly useful in applications directed at gene therapy or genetic enhancement of non-human vertebrates. For example, a promoter sequence that is active only in a cycling spermatogonial stem cell population can be used for differential expression in male germ cells, for example, c-
kit promoter region, c-raf-1 promoter, ATM (ataxia-telangiectasia) promoter (active in central cells and thymocytes), vascular promoter, cyclin A1 promoter, RBM (
(Ribosome binding motif) promoter, DAZ (deleted in azoospermia)
B-Myb or male germ cell-specific promoters, such as the promoter, XRCC-1 promoter, HSP90 (heat shock gene) promoter, or FRMI (fragile X site) promoter. Promoters useful for hematopoietic stem cell tissue-selective expression in hematopoietic stem cell progenitor cells include the cyclin A1 promoter (eg, Muller, C., et al., Cyclin A
1 Characterization of genome structure and its promoter region, J. Biol. Ch.
em. 276 (16): 11220-28 [1999]); CD34 promoter (eg, Burn, TC, et al.,
Hematopoietic stem cell-specific gene expression, US Pat. No. 5,556,954); c-kit promoter, or integrin alphaIIb promoter (eg, Wilcox, DA, et al.,
Integrin of gene product in megakaryocytes derived from retrovirus-introduced human hematopoietic cells
AlphaIIb promoter target expression, Proc. Natl. Acad, Sci. USA 96 (17): 9654-59 [
1999]).

A cartilage-selective promoter for expression in chondrocytes, such as the osteocalcin (OC) promoter (eg, Newberry, EP, et al., RRM domain of MINT, new M
SX2 binding protein recognizes and regulates the rat osteocalcin promoter, Biochemistry 38 (33): 10678-90 [1999]); SOX9 promoter, aggrecan gene promoter (AGCI), or collagen oligomerization substrate protein (COMP)
Gene promoters (eg, Kanai, Y. and Koopman, P., structural and functional characterization of the mouse Sox9 promoter: inferences about dysplasia,
Hum. Mot. Genet. 8 (4): 691-96 [1999]; Newton et al., Characterization of human and mouse cartilage oligomerization matrix proteins, Genomics 24: 435-39 [19].
94]; alternatively, a promoter derived from a collagen gene, such as, but not limited to, the COL2A1, COL9A1 or COL10A1 promoter (eg, Ganguly,
A., et al. Targeted insertion of two exogenous collagen genes into both alleles of the endogenous locus in cultured human cells: insertion is induced by a relatively short fragment containing the promoter and the 5 'end of the gene. Proc Natl Acad Sci USA 91 (15): 7365-
9 [1994]; Dharmavaram, RM, et al., Detection and characterization of Sp1 binding activity in human chondrocytes and its modification during chondrocyte dedifferentiation, J. Biol Chem.
272 (43): 26918-25 [1997]; Zhou, G., et al., 48 high-mobility glip-like sequences within the Col2al gene 48 enhancer are required for in vivo cartilage-specific expression, J. Biol Chem 273 (24): 14989-97 [1998]; Seghatoleslami, MR, et.
al., Differential regulation of COL2Al expression in developing and mature chondrocytes, Matrix Biol 1
4 (9): 753-64 [1995]; Lefebvre, V., et al., 18 bp sequence in the mouse proalphaI (II) collagen gene is sufficient for expression in cartilage and is selectively expressed in chondrocytes. J. Mol Cell Biol 16 (8): 4512-23
[1996]; Zhou, G., et al., 182 bp fragment of the mouse proalpha I (II) collagen gene is sufficient for direct chondrocyte expression in transgenic mice, J. Cell Sci, 108 (Pt 12): 3677-84 [3677-84]; Mukhopadhyay, K., et al.
Delineates the 119 base pair chondrocyte-specific enhancer element in mouse pro
Use of new rat chondrosarcoma to define an active promoter segment in the alphaI (II) collagen gene, Biol Chem 270 (46): 27711-9 [1995]; Vikkula, M
., et al., Structural analysis of regulatory elements of the tyupe-II procollagen gene. Conversation of promoter and first intron sequence between human and mouse Bioc
hem J 285 (Pt 1): 287-94 [1992]; Beier, F., et al., Localization of silencer and enhancer elements in the human type X collagen gene,
J Cell Biochem 662 (2): 210-8 [1997]; Thomas, JT, three mammalian types X
Sequence comparison of collagen promoters and preliminary and functional analysis of human promoters, Matrix 13 (2): 165-79 [1993]). Cartilage-derived retinoic acid-sensitive protein (C
The D-RAP) gene promoter is also useful for chondrocyte selective expression by chondrocytes. (Eg, Xie, WF, et al., Transactivation of Mouse Cartilage-Derived Retinate-Sensitive Protein Gene by Sox9, J. Bone Miner. Res. 14 (5): 757-63 [1999]).

Useful promoter sequences for liver-selective expression in hepatocytes include albumin gene promoters (eg, Pastore, L., et al; use of a liver-specific promoter is directed to the transgene in an adenovirus vector. Hum. Gen. Ther. 10 (11): 1773-81 [1999]) to reduce the immune response; CYP7A or CYP7A I promoter (eg, Nitta, M., et al., CPF: human cholesterol 7-a-an orphan nuclear receptor that regulates liver-specific expression of the hydroxylase gene, Proc.
Natl. Acad. Sci. USA 96 (12): 6660-65 [1999]; Chen, J., et al., Hepatocyte nuclear factor I binds and transactivates the human but not rat CYP7A1 promoter, human growth. Endo, a liver-specific promoter for hormone receptor genes
crinology 138 (4): 1771-74 [1997]; Jiang, H., et al., Jiang, H., et al., [
1999]; Adams, TE, Differential Expression of Growth Hormone Receptor mRNA from the Second Promoter, Mol. Cell Endocrinol. 108 (1-2): 23-33 [1995]); Alternatively, a thrombin-activatable fibrinolytic inhibitor ( TAFI) promoter (eg, Boffa, MB, e
characterization of the gene encoding human TAFI [thrombin activatable fibrinolysis inhibitor; plasma procarboxypeptidase B], Biochemi
stry 38 (20): 6547-58 [1999]). Nerve-specific promoters are also useful, such as the neurofilament promoter or the neuron-specific enolase promoter. Numerous other tissue-specific promoters are useful for tissue-specific expression of genes preselected for phenotypic expression of a desired trait or product in various tissues and organs of the vertebrate body. Other useful promoters are associated with the expression of cytokine-inducible proteins, such as promoters that regulate the expression of products and modulators of the JaK-STAT signal cascade, such as the SOCS-3 promoter (CJ Auernhammer et al.
al., Autoregulation of pituitary adrenocortical SOCS-3 expression: Characterization of murine SOCS-3 promoter, Proc. Natl. Acad Sci. USA 96: 6964-69 [1999]), STAT-3
Promoter ((C. Bousquet & S. Melmed, J. Biol. Chem. 274; 10723-30 [199
9]), POMC promoter (CJ Auernhammer et al. [1998b]), or Spi 2.1 promo
ter (TE Adams et al. [1995]).

[0036] Useful promoters also include exogenous inducible promoters. There are promoters that are not generally exogenous metabolites or cytokines and can be activated in response to exogenously supplied factors or stimuli. Illustrative are antibiotic-inducible promoters, such as tetracycline-inducible promoters; heat-inducible promoters;
A light-inducible promoter; (Eg, Ha
lloran, MC et al., Laser-induced gene expression in transgenic zebrafish-specific cells, Development. 127 (9): 1953-1960 [2000]; Gerne
r, EW et al., Heat Induced Vectors for Use in Gene Therapy, Int. J. Hyp
erthermia 16 (2): 171-81 [2000]; Rang, A .. and Will, H., tetracycline-responsive promoter contains a functional interferon-inducible response element, Nucl
eic Acids Res. 28 (5): 1120-5 [2000]; Hagihara Y. et al., Evaluation of long-term function of encapsulated cells transfected by Tet-On system, Cell Transplant. 8 (4): 431 -Four
[1999]; Huang, CJ et al. Tetracycline regulation system allows time,
Expression of green fluorescent protein in glial cells in a level-controllable manner, Mol. M
ed. 5 (2): 129-37 [1999]; Forster, K. et al., Nucleic Acids Res. 27 (2), a tetracycline-inducible expression system with reduced basal activity in mammalian cells.
): 708-10 [1999]; Liu, HS et al., Lac1Tet dual induction system function in mammalian cell lines, Biotechniques 24 (4): 624-8, 630-2 [1998]). Other useful promoters include those that are developmentally or transiently regulated. Examples are the myelin PO promoter (P. Thatikunta et al., Reverse Id expression in Schwann cells and myelin gene regulation, Moll. Cell Neurosci, 14 (6):
519-28 [1999]), Gabra3 or GABRA3 promoter (W. Mu and DR Burt
, Mouse GABA (A) receptor a 3 subunit gene and promoter, Brain
Res. Mol. Brain Res. 73 (1-2): 172-80 [1999]), tyrosine hydroxylase (J
.J. Schimmel et al., 4.5 kb of rat tyrosine hydroxylase 5 'flanking sequence
Brain Res. Mol. Brain Res. 74 (1-2): 1-14 [1999]), which induces tissue-specific expression during development and contains a consensus site for multiple transcription factors, a vimentin promoter (A. Benazzouz and P. Duprey, Diffentientiation of the vimentin promoter as a tool for early event analysis of retinoic acid-induced differentiation of cultured embryonic cancer cells (65 (3): 1
71-80 [1999]), GATA-6 promoter (A. Brewer et al., Human and mouse GATA
-6 gene utilizes two promoters and two initiation codons, J. Biol. Chem. 274 (53)
: 38004-16 [2000]), the SHIM or SHW2 promoter (S. Schurmans et al.,
Mouse SHIP2 (InppII) gene: complementary DNA, genomic structure, promoter analysis, and gene expression in embryonic and adult mice, Genomics 62 (2): 260-71 [1999]), or the hGH-N promoter (BM Shewchuk et al. , Somatotropin-specific DNase I of the human growth hormone locus control region, a Pit-I binding site in hypersensitization sites I and II, J. Biol. Chem. 274 (50): 35725-33 [1999]). .

The foregoing examples of useful promoter sequences are not a list of all promoters, but merely an illustration of promoters available to those of skill in the art in practicing the present invention. In vivo and in vitro methods of incorporating exogenous genetic material into the genome of a vertebrate include incorporating a polynucleotide encoding the desired trait or product into the genome of at least one male germ cell, such as a sperm or progenitor cell And that the resulting genetically modified male gametes are produced by male vertebrates.
Thus, vertebrate germ cells with this genetic modification are transgenic and have non-endogenous (exogenous) genetic material integrated into their chromosomes. This is what is often referred to as "stable transfection" or "stable integration". It is applicable to all vertebrates, including humans. Animals that have been shown to harbor properly modified sperm cells are then mated spontaneously, or the sperm is used for insemination or in vitro fertilization. Isolation and / or selection of genetically modified cells, including transgenic germ cells and transgenic somatic cells, and transgenic vertebrates,
Using appropriate means, such as biochemistry, enzymology, immunochemistry, histology, electrophysiology, biostatistics or similar methods, appropriate means such as the physiological and / or morphological phenotype of interest And, for example, hybridization analysis, nucleic acid amplification (
Including polymerase chain reaction [PCR], reverse transcriptase mediated polymerase chain reaction [RT-PCR], transcription mediated amplification [TMA], reverse transcriptase mediated ligase chain reaction [RT-LCR], and / or electroporation techniques, The specific DNA of interest using conventional molecular biology techniques depends on, but is not limited to, the analysis of cellular nucleic acids, such as the presence or absence of RNA.

In a preferred embodiment, the gene delivery mixture comprises a gene encoding a genetic selectable marker such that it is operably linked to a promoter sequence, such that a transcription unit is formed. It contains at least one polynucleotide. The promoter sequence may be the same or different from the promoter that regulates expression from the gene encoding the desired trait or product.
This genetic selection marker, also known as the reporter gene,
98; - Enzymes like galactosidase or green fluorescent protein (GFP),
Genes encoding enhanced green fluorescent protein [EGFP], fluorescent proteins such as yellow fluorescent protein and blue fluorescent protein, phycobiliproteins such as phycoerythrin and phycocyanin, or other proteins that fluoresce at appropriate wavelengths. Another preferred genetic selectable marker or reporter gene suitable for some applications is a gene that encodes a protein that enzymatically induces luminescence from a substrate; for the purposes of the present invention, such a protein is referred to as a "photoprotein". Or luminescent protein. For example, photoproteins include proteins such as luciferase and apoaequorin. Transgenic cells expressing the fluorescent or luminescent protein encoded by the reporter construct can be sorted, for example, by use of a fluorescence activated cell sorter (FACS) set to the appropriate wavelength, or chemically. The method can be selected.

A preferred method for isolating or selecting a male germ cell population is to push the cells out of the seminiferous tubules and to dissociate with a specific male germ cell, such as spermatogonia, from a mixed population of testis cells by mild enzymatic disaggregation. Including obtaining a cell population. The spermatogonia or other male germ cell population to be genetically modified is a reporter construct; e.g., green fluorescent protein (or EGFP), yellow fluorescent protein, blue fluorescent protein, phycobiliproteins such as phycoerythrin or phycocyanin,
Or a promoter that is specifically or selectively active only in the cycling male germline stem cell population, linked to other proteins that fluoresce under light of the appropriate wavelength, or constructs encoding luciferase or apoaequorin Sequences, for example, the B-Myb promoter, or the c-kit promoter region, the c-raf-1 promoter, ATM (Ataxia-Terangie ectasia:
xia-telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter, HSP90 (heat shock gene) promoter, cyclin A1 promoter,
Alternatively, they can be separated from a mixed cell population by methods involving the use of specific promoters, such as the FRMI (fragile X site) promoter. These unique promoter sequences drive the expression of the reporter construct only during certain stages of male germ cell development, as known to those skilled in the art (eg, Muller, C., et al., Cyclin A1 Genome). Characterization of structure and promoter region, J. Biol. Chem. 276 (1
6): 11220-28 [1999]; Schrans-Stassen, B., H. et al., Mouse undifferentiated and differentiated
Differential expression of c-kit in type A spermatogonia, Endocrinology 140: 5894-5900 [1999])
. The population of male germ cells at a particular developmental stage is therefore the only cell in the mixed population that expresses the reporter construct and can therefore be isolated on this basis. In the case of a fluorescent receptor construct, for example, set to an appropriate wavelength
It can be sorted by using FACS or selected by chemical methods. In vitro methods for incorporating exogenous genetic material into the genome of a vertebrate further include obtaining male germ cells from the donor animal and modifying the gene in vitro to distribute genes encoding the desired trait or product. In addition, male germ cells that exhibit evidence that the DNA has been modified in the desired manner are isolated or selected and transfected into the testes of appropriate recipient animals. After transfection, after testis cytology of one or both recipient males, or by examining animal ejaculates amplified by the polymerase chain reaction, the desired nucleic acid sequence is indeed incorporated. Later, further selections can be attempted. As described above, priming gene delivery can be performed using green fluorescent protein, enhanced green fluorescent protein (EGFP), yellow fluorescent protein, blue fluorescent protein, phycobiliproteins such as phycoerythrin and phycocyanin, or fluorescent light at the appropriate wavelength. Or a reporter gene, such as a gene encoding a fermentation protein, or a gene encoding another protein that produces E. coli.

For in vitro methods of incorporating exogenous genetic material into the genome of a vertebrate, the genetically modified germ cells are isolated or selected in this manner, and preferably are recipient male vertebrate-donor It may be, but need not be, identical to the animal-transfected into the testis. Prior to transfecting the genetically modified male germ cells into one or more testes of the recipient male vertebrate, the testes of the recipient animal are preferably depleted of native germ cells. Substantial reduction of endogenous male germ cells promotes colonization of recipient testes by genetically modified germ cells from the donor animal. Cell reduction can be performed by any suitable means, including gamma irradiation, chemical treatment, infectious vehicles such as viruses, or autoimmune deficiencies, or a combination thereof. Whatever means is used for endogenous male germ cell depletion in the testis, the essentially strict architecture of the germ line must not be destroyed or severely damaged. When disruption occurs in the delicate system of tubule formation, it becomes difficult for exogenous spermatogonia to regrow in the testis. Disruption of the tubules further leads to impaired sperm transport in the testes, resulting in infertility. Since Sertoli cells are necessary to provide a base for germ cell development during maturation, testicular damage to this type of testis that can be controlled so that they are not irreversibly damaged should also be minimized. Furthermore, Sertoli cells appear to play a role in preventing the host immune defense system from destroying the transplanted exogenous spermatogonia. However, the vertebrate testis is preferably reduced by a combined treatment of the vertebrate with an alkylating agent and gamma irradiation in accordance with the method according to the invention for substantially reducing vertebrate testes. The method comprises the use of cells such as, but not limited to, busulfan (1,4-butanediol dimethanesulfonate; Myleran, Glaxo Wellcome), chlorambucil, cyclophosphamide, melphalan, or ethylethanesulfonic acid. Toxic alkylating agents, including treatment in combination with gamma irradiation, may be administered in any order. The combination of the alkylating agent and gamma irradiation resulted in unexpectedly superior results in testicular germ cell loss as compared to each individual treatment. The dose of the alkylating agent and the dose of gamma irradiation are sufficient to substantially reduce testis.

A preferred dose of the alkylating agent is about 4-10 mg / kg body weight, with about 6-8 mg / kg body weight being most preferred. Alkylating agents include pharmaceutically acceptable delivery, including, but not limited to, intraperitoneal, intravenous, or intramuscular injection, intravenous infusion, implantation, transdermal or transmucosal delivery systems. It can be administered by the system. Although the recovery period between administration of the alkylating agent and irradiation is not critical, it is most preferred that the two treatments be performed within 0-24 hours of each other. Preferably, 2
The time between treatments should not exceed 2 weeks as optimal results are less likely to be obtained for the purpose of transferring genetically modified or heterologous male germ cells to the recipient testis. The recipient vertebrate may have a dose of about 200-800 Rads, most preferably about 350-
A dose of 450 Rads is directly gamma-irradiated locally to the testis to be reduced. Less than 200 Rads has little effect; above 800 Rads usually causes radiation disease symptoms, especially in the gastrointestinal tract. Within 3 days to 2 months after performing the treatment to reduce the number of cells in the recipient testis according to the method, the male germ cells can be transferred into the testis as described herein. Prior to three days, traces of cytotoxic alkylating agents or endogenous apoptotic signal molecules remain in the recipient testis and can damage male germ cells transferred therein. After two months, the endogenous population of male germ cells typically begins to repopulate and transfected, genetically modified, or xenogeneic male germ cells are introduced into the recipient testes for mating purposes. If populated, it will not produce optimal results.

Thus, in an in vitro (ex vivo) method, one or more of the gene delivery mixtures may comprise one or more of the testis cells of the recipient male vertebrate, preferably within three days to two months after final treatment to reduce the number of cells. It is administered in vitro to male germ cells of the donor vertebrate in sufficient amounts and under effective conditions as are genetically modified. The genetically modified male germ cells from the donor male vertebrate are then transferred to the testes of the recipient male so as to dwell in the seminiferous tubules of the testes, where they are transformed into genetically modified gametes. To mature. Transferring germ cells, isolated or selected with genetic modification, to the recipient testis can be accomplished by direct injection using a suitable micropipette. Supporting cells, such as Leydig or Sertoli cells, that provide hormonal stimulation for spermatogonia differentiation, can be transferred to the recipient testis along with the modified germ cells. These transfected support cells may be unmodified or, conversely, may be genetically modified themselves, with or without germ cells. These transfection support cells may be allogeneic or heterologous to either the donor or recipient testis. The preferred cell concentration in the transfer fluid can be easily established by simple experimental methods, but will be in the range of about 1 × 10 ⁵ to 10 × 10 ⁵ cells / 10 μl liquid. This micropipette can be placed in an export tube, testis, or tubule,
Optionally, a pico pump can be introduced to regulate the pressure and / or volume, or this delivery can be performed manually. The micropipette used is similar in most respects to that used for in vivo injection, except that the tip diameter is usually about 45-70 microns. The microsurgical method of introduction is similar in all respects to that used for the in vivo method described above. Appropriate dyes or bubbles (less than 1 mm in diameter) can also be incorporated into the carrier liquid to facilitate identification of the transfected germ cells being sufficiently delivered. For both in vivo and in vitro methods of incorporating exogenous genetic material into the genome of a vertebrate, mating a male vertebrate with a homologous female vertebrate requires fertilization due to the association of male and female gametes. Occurs, forms transgenic zygotes, and transgenic offspring or progeny are produced during pregnancy, when the fetus is subsequently developed. The association of male and female gametes can occur by natural mating, ie, coupling by homologous male and female vertebrates, or by in vitro or in vivo artificial means. When artificial means are selected, it is particularly useful to incorporate into the genome a genetic selectable marker that is expressed in male germ cells. Preferably, the expression of the genetic selectable marker is regulated by a constitutive or a male germ cell-specific promoter operably linked to the gene encoding the genetic selectable marker. Artificial means include artificial insemination, in vitro fertilization (IVF), and / or other techniques such as intracytoplasmic sperm injection (ICSI), subzonal insemination (SUZI), partial decapsulation (PZD). Including but not limited to artificial reproductive technology. However, other methods such as cloning and embryo transfer, cloning and embryo cleavage, and synonyms thereof, may also be utilized.

The progeny of the transgenic vertebrate obtained in this manner can be used to obtain natural transgenic, artificial insemination or in vitro fertilization (IVF) and / or cytoplasmic progeny to obtain further transgenic progeny. Intra-sperm injection (ICSI), subzonal insemination (SUZ
I), by other artificial reproductive technologies such as partial delamination (PZD), can be crossed in turn. Those skilled in the art will readily recognize that the desired trait resulting from the change to genetic material of any transgenic animal produced by the method according to the present invention is hereditary. Although the genetic material was initially inserted alone into the germ cells of the parent animal, it will be inherently present in future offspring and subsequent germ cells. Furthermore, this genetic material is also present in non-germ cells of progeny, ie in somatic cells. Broadly, a “transgenic” vertebrate is one that has foreign or exogenous DNA permanently introduced into the cell. Exogenous genes introduced into the cells of an animal are referred to as "transgenes" and are xenogeneic and / or allotransgenic genetic material, including genetic material with biological functionality. The present invention has application to the production of transgenic animals that contain exogenous DNA from a heterologous, ie, different species, in a native, undisturbed form, or an artificially mutated form. In other embodiments, the genetic material is an "allogeneic" genetic material, which is an exogenous transgenic material obtained from a different strain, breed, pedigree, or individual of the same species, e.g., the gene " An animal having a "normal" form,
Alternatively, it is obtained from an animal having the desired allele, mutant, or mutation. Further, the gene may be a hybrid construct consisting of a promoter DNA sequence and a DNA coding sequence operatively linked together. These sequences can be obtained from DNA sequences from different species, or homologs that are not normally aligned. DNA constructs can also be derived from prokaryotes, such as bacteria, or from viruses.
It may include NA sequences. The transgenic germ cells of the transgenic vertebrate preferably have the non-endogenous (exogenous) genetic material integrated into their chromosomes. One skilled in the art will readily recognize that the desired trait resulting from the change to genetic material of any transgenic animal produced according to the present invention is hereditary. The genetic material is inherently present in direct offspring and subsequent generations of germ cells, even though it was originally inserted alone in the germ cells of the parent animal. Genetic material is also present in progeny's differentiated cells, ie, somatic cells.

The present invention includes non-human transgenic male vertebrates produced according to either in vivo or in vitro methods of incorporating exogenous genetic material into the vertebrate genome. The transgenic vertebrate produced according to the in vivo method is the recipient of the gene delivery mixture. Non-human transgenic male vertebrates, on the other hand, are recipients of genetically modified male germ cells that have been transferred into their testes according to in vitro methods. The transgenic male vertebrate breeds with a female of that species to contain native male germ cells that carry an exogenous polynucleotide in their genome that defines the gene encoding the desired trait or product It is possible. However, somatic cells in tissues other than the testis of the transgenic vertebrate lack this polynucleotide. Preferably, the transgenic male vertebrate will, but need not, maintain the production of infinitely genetically modified gametes. However, in some embodiments, the transgenic state is transient and lasts for at least several weeks to several months, after which the unmodified gametes are again produced exclusively or preferentially by the animal. The invention further includes non-human transgenic vertebrates produced according to in vivo or in vitro methods for incorporating exogenous genetic material into the vertebrate genome, the non-human vertebrates described above. It is a direct or indirect progeny of a transgenic male vertebrate. Thus, the transgenic offspring is a direct descendant of the transgenic male vertebrate, male or female, or a descendant of one or more generations. A transgenic vertebrate includes one or more cells that carry an exogenous polynucleotide in their genome that encodes the desired trait or product.

The present invention further includes transgenic cells derived from transgenic progeny. The cell is a germ cell, such as a sperm or an egg, a precursor cell of any of these, or a somatic cell. Male germ cells are obtained from the semen of a male animal, and sperm, spermatogonia, or immature spermatocytes are selected from a total cytology of testis tissue, including male germ cells. On the other hand,
Male germline stem cells can be isolated from embryonic tissue. Female germ cells are obtained by known means, including hormone-induced "maturation" and collection from the fallopian tube or aspiration from the cervix, or laparoscopic incision. Somatic cells include stem cells. Stem cells are undifferentiated mother cells that are capable of self-renewal throughout the life of a living body and have pluripotency. This means that it is possible to generate a variety of defined progenitor cells that are capable of developing into fully mature differentiated cell lineages. (Eg, T. Zigova and PR Sanberg, Rising Star in Neural Stem Cell Research, Nature Biotechnol. 16 (11): 1007-08 [1998]). All vertebrate tissues are derived from stem cells, which Hematopoietic stem cells derived from blood cells of the type; ectodermal stem cells; neural stem cells, such as neural precursors derived from brain and neural tissue. Somatic cells further include all types of progenitor cells or terminally differentiated cells associated with all tissues or organs of the vertebrate body. Somatic cells can be obtained from blood, heart, kidney, ureter, bladder, urethra, brain, thyroid, parotid, pancreas, hypothalamus, pituitary, mandibular gland, hypoglossal gland, lymph by known sampling or cytological means. Gland, bone, bone marrow, cartilage, lung, mediastinum, breast, uterus, ovary,
Testis, prostate, cervix, endometrium, liver, spleen, adrenal gland, esophagus, stomach, intestines, hair roots,
Obtained from any body tissue, organ, or fluid, including, but not limited to, muscle, nerve, urine, amniotic fluid, villous processes, skin, blood vessels or oral epithelium, or cerebrospinal fluid. The transgenic cells according to the present invention can be cultured or stored by known means.

The invention further relates to vertebrate semen comprising a plurality of transgenic male germ cells according to the invention. Vertebrate semen according to the present invention is useful for breeding or other suitable purposes. It is obtained from the ejaculate produced by the transgenic male vertebrate according to the present invention or its transgenic male progeny (direct progeny or progeny separated by one or more generations) and is used for ejaculation induction and semen capture by the vertebrate. The method is known. Semen is washed and / or stored, for example, by means as is known in the art. For example, the storage condition is
A cryopreservation method using a freezing method by a computer program, and / or
For example, the use of cryoprotectants such as dimethyl sulfoxide (DMSO), glycerol, trehalose, or propanediol-sucrose, and the use of storage methods in substances such as liquid nitrogen. Such preservation techniques are particularly beneficial to young adults or children who receive oncological treatment for diseases such as leukemia or Hodgkin's disease. These treatments often irreversibly damage the testes, thus making it impossible to resume spermatogenesis after treatment with, for example, irradiation or chemotherapy. In non-human species, the technique is useful for the transfer of gametes as frozen germ cells. Such transportation is
Promote the establishment of a variety of useful livestock and poultry when donor animals are far away. This approach is also adaptable to the conservation of endangered species on Earth.

Accordingly, the present invention further includes a method of producing a non-human transgenic vertebrate line, wherein such an individual comprises a native germ cell carrying at least one heterologous polynucleotide in its genome. Transgenic vertebrates crossed with other transgenic or non-transgenic animals of the same species include those that produce some transgenic offspring and are fertile. The method comprises crossing a transgenic offspring with fertility with a homologous heterozygous member as described above, and selecting for offspring carrying the polynucleotide. The method according to the invention for producing non-human transgenic vertebrate strains is simple and effective and is more easily achieved in large mammals than in mice due to the large testicular tract. The number of animals required for the production of transgenic offspring by genetic modification of male germ cells is extremely small, and they can be produced continuously by repeated mating without interruption by pregnancy or parturition. It does not require expensive equipment or the training required for microinjection. The technology according to the present invention is adaptable in the field of gene therapy to allow the introduction of genetic material that encodes and regulates certain genetic traits. Thus, in humans, for example, it is possible to correct a number of single genetic disorders that otherwise affect children by treating the parent. Diseases that are completely hereditary,
It is likewise possible to alter the expression of at least partially genetically based diseases that are caused by the interaction of one or more genes or that occur more frequently due to the contribution of multiple genes. The technology may also be applied to correct disorders in animals other than human primates in a similar manner. In some cases, in order to achieve a desired therapeutic effect, one or more "germ cells" are present in the germ cells of an animal, such as when multiple genes are involved in the expression or suppression of a defined trait.
It may be necessary to introduce a "gene". In humans, examples of multigenic disorders are specific forms of diabetes mellitus, inflammatory bowel disease, atheromatous cardiovascular disease and hypertension, schizophrenia and chronic depression caused by a lack of insulin production or response. Includes forms with sexual dysfunction and others. In some cases, as is known in the art, one gene encodes an expressible product, while another gene may encode a regulatory function. Other examples are those in which homologous recombination is applied to repair point mutations or deletions in the genome, inactivation of genes resulting from pathogenicity or disease, or genes expressed in a dominant / negative manner Or the modification of regulatory elements such as the gene promoter, enhancer, untranslated tail region of the gene, or the regulation of repetitive sequence expansion of DNA resulting from Huntington's disease, fragile-X syndrome and similar diseases.

[0048] A particular reproductive application of the present invention is in the treatment of animals, especially humans, with spermatogenic disorders. Incomplete spermatogenesis or sperm perfection often has a genetic cause, and one or more mutations in the genome can result in defective production of normal sperm cells. It appears at various stages of germ cell development and can result from male infertility or sterility. The present invention is adaptable, for example, to insert or incorporate nucleic acid sequences into the genome of the recipient, thereby establishing spermatogenesis in the correction of oligospermia and azoospermia in the treatment of infertility. is there. Similarly, the method is also applicable to men whose infertility or azoospermia results from a motility disorder having a genetic cause. The present invention is further applicable to the generation of transgenic animals that express substances with therapeutic effect for human and veterinary or welfare applications. Illustrative examples include the production in milk of factors that enhance blood coagulation in patients suffering from the hemophilia type, or drugs such as hormonal agents such as insulin and other peptide hormones. The method is further applicable to the generation of transgenic animals having an anatomical and physiological phenotype suitable for transplantation of human xenografts, such as pigs. The transgenic technology according to the invention allows the creation of animals that are immunocompatible with human recipients. Suitable organs can be excised, for example, from such animals, for example, to allow heart, lung, and kidney transplants. Furthermore, male germ cells that have been genetically modified according to the present invention can be obtained from transgenic animals and, as is known in the art, effective conditions for future use. Can be saved below. The present invention will now be described in detail with reference to the following non-limiting examples.
The relevant portions of all reference content, published patent applications cited throughout this patent as necessary for legalization purposes, are hereby incorporated by reference.

[0049] Inbiboa adenovirus of Green Lantern reporter gene delivery system to the genetic modification testis cells of male germ cells in Example vivo and in vitro - enhanced transferrin - polylysine - delivery Adenovirus enhancement mediated - transferrin - polylysine - gene delivery mediated The system is described and patented by Curiel et al. (Curiel DT, et al., Adenovirus Enhancement of Transferrin-Polylysine-Mediated Gene Delivery, PNAS USA 88: 8850-8854 (1991). Delivery of DNA is dependent on transferrin receptor-mediated endocytosis (Wagner et al.,
Transferrin-polycation complex, PN as a carrier for DNA uptake into cells
AS (USA) 87: 3410-3414 (1990). In addition, the method relies on the ability of adenovirus to disrupt cell vesicles, such as endosomes, and release the contents trapped therein. . The system can enhance gene delivery to mammalian cells by a factor of 2,000 over other methods. The gene delivery system used for in vivo experiments was prepared as shown in the examples below.

Example 1 Preparation of Transferrin -Poly-L-Lysine Conjugate Human transferrin was purified from Gabarek and Gergely using EDC (1-ethyl-3 (3-dimethylaminopropylcarbodiimide hydrochloride) (Pierce). (Gabarek and Gergely, Zero-length cross-linking method using active ester, Analyt. Biochem 185: 131 (1990)). The activated protein is reacted with the poly (L-lysine) component for 2 hours at room temperature, reacting with the carboxyl group of transferrin to form an amine-reactive intermediate, and the reaction is brought to a final concentration of 10 mM. The conjugate was stopped by the addition of hydroxylamine and the conjugate was purified by gel filtration.
Stored at 0 ° C.

Example 2: Preparation of DNA for in vivo transfection [0051] Green Lantern-I vector (Life Technologies, Gibco BRL, Gaithersberg,
MD) is a reporter construct used to monitor gene transfer in mammalian cells. It consists of a gene encoding green fluorescent protein (GFP) driven by the cytomegalovirus (CMV) immediate early promoter. Downstream of this gene is the SV40 polyadenylation signal. Green Lantern-1
Transfected cells emit bright green fluorescence when illuminated with blue light. The excitation peak is at 490 nm.

Example 3 Preparation of Adenovirus Particles The adenovirus dI312, a replication-incompetent strain lacking the E1a region, was isolated from Jones and Shen.
g was propagated in the E1a transcomplementing cell line 293 as described by
Jones and Shenk, PNAS USA (1979) 79: 3665-3669).
It was performed using the method of ittereder and Trapnell (Mittereder et al.,
“Evaluation of Adenovirus Vector Concentration and Bioactivity for Gene Therapy”, J. Urolo
gy, 70: 7498-7509 (1996)). Virion concentration was measured by ultraviolet spectroscopy, with one extinction unit being equivalent to 10 virus particles / ml. Purified virus was stored at -70 ° C.

Example 4: Formation of transferrin-poly-L-lysine-DNA-virus complex 6 μg of the transferrin-polylysine complex of Example 1 was combined with 7.3 × 10 ⁷ adenovirus d1312 particles prepared in Example 3. Mix, then use Green L of Example 2
The mixture was mixed with 5 μg of the anternDNA product and left at room temperature for 1 hour. About 100 μl of this mixture
Was stretched with a pipette making machine and slightly bent on a microforge.
Aspirated into a micropipette. Fill the micropipette with the pico pump (
Eppendorf), and delivered the DNA complex under sustained pressure to the mice in vivo as described in Example 6. Controls were performed according to the same procedure, but omitting the transferrin-poly-lysine-DNA-virus complex from the dosing mixture.

[0054] Example 5: Adenovirus enhancement transferrin - polylysine and lipofectin medium conjugated adenovirus particles complexed with comparative DNA transfection efficiency by intervention and in vitro studies in CHO cells before in vivo testing. In these experiments, the luciferase reporter gene was used because luciferase activity was easy to quantify. An expression construct consisting of a reporter gene encoding luciferase was obtained from the CMV promoter (Invitrogen, C
arlsbad, CA 92008). CHO cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. For gene transfer experiments, CHO cells were seeded in a 6 cm tissue culture plate and approximately 50% confluent (
5 × 10 ⁵ cells). Prior to transfection, media was aspirated and replaced with serum-free DMEM. Cells were transfected with either transferrin-polylysine-DNA complexes or lipofectin DNA aggregates. For transferrin-polylysine-mediated DNA transfer, the DNA-adenovirus complex was added to the cells at a concentration of 0.05-3.2 × 10 ⁴ adenovirus particles per cell. Plates were returned to a 5% CO ₂ incubator at 37 ° C. for 1 hour. One hour later, 3 ml of complete medium was added to the wells and the cells were incubated for 48 hours before harvesting. Cells were removed from the plate, counted and then lysed for luciferase activity measurement. For cells transfected with lipofectin, CMV-luciferase DNA
1 μg was incubated with 17 μl of Lipofectin (Life Technologies). Add DNA-Lipofectin aggregates to CHO cells and add 4% at 37 ° C with 5% CO ₂
Incubated for hours. 3 ml of complete medium was then added to the cells and incubated for 48 hours. Cells were collected, counted, and lysed for luciferase activity measurement. Luciferase activity was measured with a luminometer. The results obtained are shown in Table 1. The data contained in Table 1 below shows that the adenovirus-enhanced transferrin-polylysine gene delivery system is 1,808 times more efficient than lipofectin for transfecting CHO cells.

[0055]

[Table 1]

Example 6: Animal via transferrin-L-lysine-DNA-virus complex
In Vivo Delivery of DNA to Germ Cells The GFP DNA-transferrin-polylysine virus conjugate, prepared as described in Example 4 above, was delivered into the seminiferous tubules of 3 week old B6D2F1 male mice. DNA delivery by transferrin receptor-mediated endocytosis has been described by Schmidt et al. And Wagner et al. (Schmidt et al., Cell 4: 41-51 (1986);
Wagner, E., et al. PNAS (1990), (USA) 81: 3410-3414 [1990]). Furthermore, the delivery system relies on the ability of the adenovirus to disrupt cell vesicles, such as endosomes, and release the contents trapped therein. The transfection efficiency of this system is approximately 2000 times higher than lipofection. Male mice are treated with 2% Avertin (100% Avertin contains 10 g of 2,2,2-tribromoethanol (Aldrich) and 10 ml of t-amyl alcohol (Sigma))
Anesthesia was made and a small incision was made in the skin and body wall on the ventral side of the body at the level of the hind legs. The testicular fat pad was grasped with tweezers, the testis of the animal was pulled out through the opening, and the testicular efferent tubules were exposed and supported with a glass syringe. GFP DNA-transferrin-
The polylysine virus complex is injected into a single export tube using a glass micropipette attached to a hand-held glass syringe or a pressure-operated automatic pipettor (Eppendorf), and the mixture is introduced into the seminiferous tube. Trypan blue was added for visualization. The testes were then returned to the body cavity, the body wall was sutured, the skin was closed with wound clips, and the animals were allowed to recover on a warm pad.

Example 7: Detection of DNA and transcribed messages 9 days after delivery of genetic material to animal testes, two animals are sacrificed and their testes excised
Cut in half and frozen in liquid nitrogen. DNA was extracted and analyzed from one half of the tissue and RNA from the other half.

(A) Detection of DNA The presence of GFP DNA in the extracts was tested 9 days after administration of the transfection mixture using the polymerase chain reaction method and GFP-specific oligonucleotides. GFP DNA
Was present in the testes of animals that received the DNA complex, but not in sham control (sham operated) animals.

(B) Detection of RNA The presence of GFP mRNA in testes of experimental animals was assayed as follows. RNA extracted from injected and non-injected testes, and the presence of GFP message, was detected using reverse transcription PCR (RT PCR) with GFP specific primers. The GFP message was present in the injected testis, but not in the control testis. The DNA detected by PCR analysis above is episomal GFP DNA. The transfected gene was transiently expressed.

Example 8: Expression of non-endogenous DNA Two males-one that received an injection of the GFP transfection mixture and one control that performed surgery alone-were sacrificed four days after injection. The testes were excised and fixed in 4% paraformaldehyde at 4 ° C. for 18 hours. 30% in fixed testes were then 2 mM MgCl ₂ was added to PBS
The sections were placed in sucrose at 4 ° C. for 18 hours, embedded in OCT frozen on dry ice, and sectioned. When testes from both animals were examined on a confocal microscope using fluorescence at a wavelength of 488 nM, bright fluorescence was detected in the tubules of GFP-injected mice but not in control testes. Many cells within the tubules of GFP-injected mice displayed bright fluorescence, confirming that green fluorescent protein was expressed.

Example 9 Development of Progeny by Normal Mating GFP-transfected males were crossed with normal females. The female completed the gestation period and the offspring were born. Offspring (F1 descendants or progeny) were screened for the presence of new genetic material.

Example 10 In Vitro Transfection of Testis Cells Cells were isolated from the testes of three 10-day-old mice. The testes were decapsulated and the tubules were split and minced using a sterile needle. Cells were incubated in the enzyme mixture at 37 ° C. for 20 minutes. Enzyme mixture contains 10 mg bovine serum albumin (embryo tested), 50 mg
mg bovine pancreatic trypsin type III, clostridial collagenase type I, 1 mg
Bovine pancreatic desoxyribonuclease type I was added to 10 ml of modified HTF medium (Irvine Scient
ific, Irvine, CA). Enzyme is from Sigma Company (St. Louis,
Missouri 63178). After digestion, cells were washed twice by centrifugation at 500 × g in HTF medium and resuspended in 250 μl HTF medium. Count cells 0.5 x
10 ⁶ cells were transferred to 5 ml DMEM (Gibco-BRL, Life Technologies, Gaithesburg
, MD 20884) in a 60 mm Petri dish. Transfection mixture is 5 μg Green
n Lantern DNA (Gibco-BRL, Life Technologies, Gaithesburg, MD 20884)
20μl Superfect (Qiagen, Santa Clarita, CA 91355) and 150μl DME
Prepared by mixing with M. Add transfection mix to cells, 37 ° C, 5
Incubated for 3 hours under the condition of% CO ₂ . Cells were transferred to a 33 ° C. incubator and incubated overnight. The next morning, the transfection efficiency of the cells was evaluated by counting the number of fluorescent cells.
In this experiment, the transfection efficiency was 90% (not shown in the figure). Nomaski opt
Testis cells transfected with Green Lantern observed on ics 20X showed the same cells observed on FITC. Almost all cells were fluorescent, confirming successful transfection.

Example 11 Preparation of Cell Suspension Derived from Testis Tissue for Cryopreservation Cell suspensions were prepared from mice of different ages as described below. Group I: 7-10 days of age Group II: 15-17 days of age Group III: 24-26 days of age Testes of mice were dissected, placed in phosphate buffered saline (PBS), and decapsulated. The tubules were torn. The seminiferous tubules from groups I and II contain 1 mg / ml bovine serum albumin (BSA) (Sigma, St. Louis, MO 63178) and collagenase type I (Sigma) (Sigma) to remove stromal cells HEPES buffer medium (D-MEM) (Gibco-BRL, Life
Technologies, Gaithersberg, MD 20884). After incubation at 33 ° C. for 10 minutes, the tubules were transferred to fresh medium. This test was not performed because the testes of Group I were fragile. Tubules from Group II and III mice or whole tissue from Group I were transferred to Petri dishes with medium and cut into 0.1-1 mm pieces using a sterile scalpel and needle.
The minced tissue was centrifuged at 500 xg for 5 minutes and the resulting pellet was resuspended in 1 ml of enzyme mix. Enzyme mix is D-DME with HEPES (Gibco-BRL)
M, 1 mg / ml bovine serum albumin (BSA) (Sigma, embryo tested), 1 mg / ml
Collagenase I (Sigma) and 5 mg / ml bovine pancreatic trypsin (Sigma) and 0.1 mg
/ ml Deoxyribonuclease I (DN-EP, Sigma). The tubules were incubated in the enzyme mix for 30 minutes at 33 ° C. After the incubation, 1 ml of medium was added to the mix and the cells were centrifuged at 500 xg for 5 minutes. Cells were washed twice in medium, centrifuged and resuspended. After final wash, 250 μl of cell pellet
And re-suspended in the culture medium.

Example 12:Transfer transfected male germ cells into recipient testes Cells were injected into the testes via an export tube using a micropipette. 3 x 10 ^Five A total volume of 50 μl containing cells was used for injection. Prior to injection, cells should be mixed with trypan blue.
Mixed. Recipient mice were anesthetized with 0.017 mL / g body weight Avertin. under
Make an incision across the abdominal wall and pull on the fat pad connected to the testis.
The testes of the animals were gently pulled out through the incision. Exposing testicular export tubules
Glass holder held by a steel micropipette holder (Leitz).
Using a clopipet, 20 μl of the cell suspension was injected into the testicular efferent tubules. Fine
The vesicles are ejected from the pipette using pneumatic pressure from a 20 ml glass syringe.
Was. Prior to transferring the transfected germ cells to the recipient animal, the recipient
The testes underwent endogenous male germ cell reduction.

Example 13: Comparison of reduction treatments to reduce male germ cells from recipient testes . Eight week old C57BL / 6J mice were acclimated for several days before being assigned to one of the following three treatment groups: Mice received the following treatments: (1) 400 Rad gamma irradiation; (2) 4 μg / g body weight busulfan (1,4-butanediol dimethanesulfonate; Myleran, Glaxo Wellcom); or (3) busulfan ( 4 μg / g body weight) One week after treatment, combination treatment with 400 Rad gamma irradiation (“Busulfan / 400 Rad
"Treatment". A fourth group of untreated C57BL / 6J of the same age was used as a control. Each treatment group consisted of 24 mice, of which 3 mice were each sacrificed at the following time intervals after treatment: : 5 hours, 24 hours, 48 hours, 72 hours, 1 week, 2 weeks,
1 month and 2 months. In addition, other C57BL / 6J mice that received the busulfan / 400 Rad combination treatment were histologically examined at each time point up to 5 months post-treatment (testis in these other mice was 4% in PBS). They were fixed in paraformaldehyde at pH 7.4 at 4 ° C, dehydrated, embedded in paraffin, sectioned, and H & E stained).

Delivery of the alkylating agent to the recipient vertebrate. Male mice that received busulfan received 4 μg busulfan / g body weight. Busulfan was first dissolved at 8 mg / mL in 100% dimethyl sulfoxide (DMSO) and diluted 1: 1 with phosphate buffered saline, pH 7.4, immediately prior to injection. Mice were injected intraperitoneally with the diluted busulfan solution.

Radiation treatment of recipient vertebrates. Mice were anesthetized with 0.017 mL / g body weight 2.5% avertin for gamma irradiation treatment. Gamma rays were irradiated by specifying the testes by the following method. Each mouse was placed in a lead chamber so that only the testis and the lower abdomen were exposed to the irradiation source ( ¹³⁷ Cs Gammacell 40 irradiator [Nordion]) through the oblong hole. The chamber floor and roof had six aligned holes through which gamma rays passed without interruption. After the irradiation, the animals
Recovered from anesthesia on a warm pad or warm water bed.

Histology. At specific selected time points, mice from each treatment group were sacrificed and testicular tissues examined were fixed with 10% formalin in PBS at pH 7.4, 4 ° C for 24 hours. A small slit was created in the testis capsule to facilitate penetration of the fixative. The fixed samples were washed four times with PBS and embedded in paraffin using a Tissue Tek-II tissue processor (MET). Sections 8 μm thick were made, stained with hematoxylin and eosin (H & E), and mounted on glass slides with coverslips with Aquamount (Lerner Laboratories). Sections were viewed on a Zeiss or Olympic light microscope using a 40X objective (400X total magnification).

Quantitative tissue analysis. Two months after treatment, quantitative data was collected from testes of two animals for each treatment group. (Table 2). Only one mouse was used as a control group. The seminiferous tubules in one section were counted with a Zeiss optical microscope with a 5X objective lens (50X total magnification). Individual tubules were examined at a total magnification of 400X. The seminiferous tubules appear to be severely damaged, with few cells remaining in the tubules, and the tubules are composed of a basement membrane with a monolayer of cells, most of which are spermatogonia, It was located along the membrane. In the moderately damaged tubules, some of the spermatogonia near the lumen was partially detached. Sperm heads in the tubules were counted and averaged for the total number of tubules counted.

Results of tissue analysis. No clear histological changes were observed in testes up to 2 weeks after treatment. (No data displayed). After two weeks, the changes included a severe disruption of spermatogenesis; all mature sperm were deleted and no sperm cells or spermatocytes were present. 2-3 Sertoli cell nuclei and spermatogonia were detectable around the basement membrane. Six weeks after busulfan / 400 Rad treatment, reestablishment of spermatogenesis was confirmed. Some sperm cells and sperm were observed as well as 2-3 spermatocytes. By about three months, most seminiferous tubules had at least partially recovered, presenting all stages of spermatogenesis. (No data displayed). By 5 months after busulfan / 400 Rad treatment spermatogenesis had returned to normal at this stage. A comparison was also made for the three treatment groups described above. The largest difference between the groups was observed at 2 months after treatment. At two months, mice that received the busulfan / 400 Rad gamma irradiation combination treatment showed virtually a very large number of seminiferous tubule reductions. The seminiferous tubules from this group had a lower average number of sperm heads per seminiferous tubule and had the highest number of severely and moderately damaged seminiferous tubules compared to the other treatment groups and control mice. (Table 2). Treatment of mice using 400 Rad gamma irradiation or busulfan alone resulted in damage to the spermatogenesis process, detachment of cells into the tubule lumen, and substantially less mature sperm heads compared to controls However, to a lesser extent than was demonstrated by the busulfan / 400 Rad treatment group. These results clearly demonstrate that the combined treatment with alkylating agents and gamma irradiation is a more effective method for reducing male germ cells from vertebrate testes than using the two treatments individually.

[0071]

[Table 2]

Example 14 In Vivo Transduction Using Viral Vectors Transduction (genetic alteration or modification) of mouse male germ cells in vivo was performed using a retroviral vector. In particular, in order to express the green fluorescent protein (GFP) instead of the LacZ reporter gene under the transcriptional control of the CMV promoter (HR'GFP), a pseudotyped HIV-derived virus vector (L. Naldini) was modified according to the modified method by Carlos Lois. et al., Lentiviral Vector In Vivo Gene Delivery and Viability of Nondividing Cells Transduced, Science 272: 263-67 [
1996]). Recipient C57BL / 6J mice received busulfan treatment for 44 days prior to viral infection. C57BL / 6J male mice were injected intraperitoneally with 0.1 ml busulfan at a concentration of 1 mg / ml. The dose was 4 μg busulfan / g body weight. One pretreated mouse was anesthetized with Avertin (0.017 ml / g body weight), a ventral midline incision was made, and the right testis was exposed. The export tubes were dissected from the fat and 10 μl of the HIV-derived GFP vector, HR'GFP, at a titer of 1 × 10 ⁹ particles / ml, were delivered via testicular export tubules of busulfan-treated C57 BL / 6J mice. Injected into the seminiferous tubules of the right testis. Injections were made with a quartz glass micropipette attached to a Picospritzer II. The Picospritzer was set at 80 psi and allowed a one second burst when the foot pedal was manually depressed. It is possible to reach all the seminiferous tubules in the testis with a single injection, because the testicular efferent tubules communicate with the common chamber, the testis network, from which all the tubules are radially spread. The left testis was not injected and was used as a control. Transduction of testicular cells in tubules was widespread. Twenty-one days after infection, the mice were sacrificed and their testes fixed in 4% paraformaldehyde in PBS, pH 7.4, 4 ° C. overnight. The testes were washed three times with PBS and left overnight at 4 ° C. in 20% sucrose. The testes were frozen in OCT and sectioned at 8 μm in a cryostat. The sections were immersed in phosphate buffered saline, thawed at room temperature, and Zeiss 310
Observed with a confocal microscope. The laser was set at a wavelength of 488 nm. Green fluorescence was observed in all tubules examined, but its intensity was highest in the tubules on the testis surface. Transduction was observed in Sertoli support cells as well as in spermatogonia along the basement membrane, but was rarely observed in spermatocytes and sperm cells. There were few mature spermatozoa due to busulfan treatment. No fluorescence was observed in the left testis used as a control. This indicates that the male germ cells can be transduced by the viral vector and that the transducing gene is being expressed.

[0073] Example 15: microinjection click optimization Deployment of gene delivery mixture via testicular export tubules. Single Testicular Export Tubules of Viral Particles and Methods for Delivery Therefrom to Seminiferous Tumors (Winston,
RML, re-arteriovenous anastomosis by microsurgical operation of rabbit oviducts and its functional and pathological sequelae, Brit. J. Obstet. Gynaecol. 82: 513-522 ([1975]) (Figures 1a-1d). Mice were treated with busulfan 14 days prior to microsurgery to maximize the opportunity for viral particles to gain access to spermatogonia located in the basal lamellar tubules. Busulfan, an alkylating cytotoxic agent, reduces testicular cells (Bucci, L. and Meistrich, M., Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations, Mut. Res. 176: 259-268 [1987]). Administered intraperitoneal (IP) dose (4 μ
g / g body weight), a large number of spermatocytes, sperm cells and sperm were excluded from the tubules, but after 3-4 months the testes recovered and fertility was restored. This means that the stem cells are alive and regrow in the testes. Stem cell spermatogonia are known to be resistant to injury and often survive the destruction of other germ cell types (Huckins, C. & Oakberg, WF; Morphological and Quantitative Analysis of Spermatogonia Using Total Mounting, 11. Irradiated Testes, Anat. Rec. 1
92: 529-42 [1978]).

[0074] Example 16: transgenic offspring of production microsurgery by in vivo transduction of male germ cells following the natural mating. After testicular cell depletion as described in Example 15, virus particles were delivered to seminiferous tubules as follows: Mice were anesthetized with isofluorane (0.5-2% in oxygen). Each testis was exposed through a midline abdominal incision. Using Microsurgery (Wins
ton [1975]; Zeiss microscope, magnification 4-50x), tissue bundles containing testicular efferent tubules were made visible (Figures la-lb). Disconnect from surrounding fat with the flow pressure of phosphate buffered saline through a fine needle. 10 μl of quartz glass micropipette mixed with 1 μl of polybrene (80 mg / ml)
He was charged with the viral particle (10 ⁹ pfu / ml). This is a micropipette (Eppendorf
) And the particles were introduced into the testicular efferent tubules, controlled by a foot pedal at 2.2 bar pressure and 1.5 second pulses. Initial studies using 1% bromophenol dye showed that most tubules were full (Figure 1c), but during the actual procedure, no dye was used and small bubbles were introduced to remove virus particles. It was introduced into the contained liquid and its diffusion into the seminiferous tubules was confirmed (Fig. 1d). To preserve reproductive function, only one export tube was injected, reducing damage to the remaining tubes.

Preparation of viral vector . Plasmid, pHR'-CMVLacZ (L. Naldni et al [1996]
), Were modified by replacing the BamHI-XhoI fragment containing the LacZ gene with a fragment containing the EGFP gene ('humanized GFP', Clontech).
. For the production of virus particles, 40 μg plasmid DNA was used to transfect 10-cm plates of 293T cells. This 40 μg of plasmid DNA contains 10 μg
PCMV R9, 20 μg of modified pHR ′ and 10 μg of envelope plasmid. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotype vector
Vector plasmid was produced by co-transfection of Moloney murine leukemia virus (MLV) gag-pol packaging plasmids pCMV-GAGPOL and VSV-G plasmid. The supernatant was collected 48-60 hours after transfection, centrifuged at high speed,
The solution was filtered through a 0.45 μm filter and evaluated and analyzed. The transduced virus particles
Instead of the broad spectrum env protein from vesicular stomatitis virus glycoprotein, it had the MLV-limited envelope protein, env.

[0076]In vivo transduction of male germ cells. Six mice were transformed with the transduction vector pHR '(10 ⁹ Particles / ml). 10 μL per testis export tubule
Retrovirus concentrate was injected with 1 μL (80 mg / mL) polybrene. 24 days later
The mice are sacrificed and testes are removed and fixed for frozen sectioning and histology
did. Testes are fixed in 4% paraformaldehyde pH 7.4 for 48 hours and phosphate buffered
It was left for 24 hours at 4 ° C. in 20% sucrose in saline, pH 7.4. Embedded in OCT
And stored at -70 ° C. These were cryosectioned at 8 μm and Zeiss 410 confocal microscopy.
Inspected under a microscope (FIG. 2). Almost all tubules sectioned contained cells expressing GFP. Expression is
It was highest in Rutoli cells and spermatogonia (Fig. 2a-b).

Natural mating with females after transduction of male germ cells. Eleven C57 / B1 / 6J young males were then selected to test that transduced male germ cells could transmit the transgene integrated by the retrovirus to the next generation. Sixteen of these mice were given 14 days prior to performing in vivo transduction microsurgery according to the in vivo method for integrating exogenous genetic material into the vertebrate genome as described above.
Treated with a bolus of busulfan (IP; 4 μg / g body weight), three received the same dose one week prior to in vivo transduction. Two other mice were not pretreated with busulfan prior to the in vivo transduction procedure. Lentiviral particles were introduced into seminiferous tubules. Fourteen weeks later, B6D2F1 females were cohabited with males. The transduced males yielded at least two consecutive littermates. Litters were gestated at 14, 15, 19 and 20 weeks of transduction. All males gave birth to transgenic offspring, except one that died immediately after surgery. (Table 3).

[0078]

[Table 3]

PCR and Southern blot analysis of DNA from progeny embryos. Approximately 12.5 day old embryos were screened for the presence of the transgene by polymerase chain reaction (PCR) and Southern blot analysis. For PCR, a GFP-specific primer was used, and for Southern blot analysis, a radiolabeled GFP cDNA probe was used (FIG. 3). DNA was purified from embryos using the Gentra purification system. The presence of the transgene was confirmed using a PCR amplification method using the following GFP-specific primers: (A) Positive primer: 5'-GGT GAG CAA GGG CGA GGA GCT-3 '(SEQ ID NO: 1) (B) Reverse primer: 5'-TCG GGC ATG GCG GAC TTG AAG A-3 '(SEQ ID NO: 2) PCR cycle conditions: denaturation 94 ° C for 1 minute, annealing 60 ° C for 1 minute, and extension 72 ° C for 3 minutes. The PCR was run for 35 cycles, yielding a specific GFP product 470 base pairs in length. Each cycle step can be shortened by 1 second.
Generated a significant 470 bp PCR amplification product. Southern blot analysis also revealed the same embryo DN
Performed on A extract. DNA was cut with Bamfll-Xhol, run on a 0.8% agarose gel, and blotted onto Hydrobond XL paper overnight at 20x SSC. The blot was hybridized overnight at 65 ° C. using the ³² P radiolabeled BamlU-Xhol GFP fragment isolated from the pHR ′ plasmid. This blot is at 65 ° C
Wash with 0.1% SDS in 2x SSC, 0.1% SDS in 1x SSC, 0.1% SDS in 0.1x SSC
Min.) And then exposed to X-ray film. PCR and Southern blot analysis showed that the highest proportion of transgenic offspring was obtained in littermates within 15 weeks. The results are summarized in Table 3. By week 20, the proportion of transgenic progeny in all treatment groups had decreased, indicating that no self-renewing spermatogonia were transduced, but rather a population of differentiated spermatogonia. Means that. Once daughter cells from this population matured and left the testis, they were not regenerated (Huckins, C. and Oakberg, W.
F. [1978]). In Table 3, the ratio is the number of transgenic offspring relative to the total number of embryos in litters.

Although pretreatment with busulfan enhances transduction of spermatogonia, mice that were not treated with busulfan also developed transgenic offspring. Male germ cells take 60 days to differentiate from spermatogonia, undergo meiosis and form spermatozoa (Russell, LD, et al., Histological and histopathological evaluation of testis. Cache Rive.
r Press [1990]). Because gestation is> 60 days after transduction, the transgenic progeny are likely to have been gestated by the differentiated daughter cells of the transduced spermatogonia. EGFP expression is driven by the CMV promoter and
It was confirmed that it was expressed in testis cells of primary males after a day. The infected animals did not show any toxic side effects, except that one animal died 17 weeks after surgery (V
erma, IM and Somia, N. [1997]). While the foregoing examples are illustrative, they do not illustrate all of the present invention, but rather present the following claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1 illustrates the steps of microinjecting a gene delivery mixture into mammalian (mouse) testes. FIG. 1 (a) shows a preferred site of microinjection into testicular efferent tubules; in mammals, efferent tubes communicate with the lumen of all seminiferous tubules. FIG. 1 (b) shows testicular efferent tubules supported by a 1 mm diameter pipette tip. FIG. 1 (c) shows mouse testes perfused with bromophenol after being injected into testicular efferent tubules. FIG. 1 (d) shows bubbles in the testis, confirming that the delivery of the virus particles was a satisfactory result.

FIG. 2 shows transduction by a pseudotyped lentiviral vector expressing green fluorescent protein (GFP) in a Zeiss 410 confocal image (wavelength 488 nm; 19 stacked images) of a frozen section of mouse testis. Shows testis cells. FIG. 2 (a) shows transduced Sertoli cells expressing GFP. FIG. 2 (b) shows transduced spermatogonia; GFP expression is observed in the cytoplasm surrounding the large dark nucleus.

FIG. 3 shows DNA analysis from three consecutive litters of offspring arising from one male treated according to an in vivo method of integrating exogenous genetic material into the vertebrate genome.
The top panel shows GFP isolated on agarose gel from embryonic DNA of 22 offspring.
Shows specific PCR amplification products. Although this run lacked amplification from fetal No. 2, the FCR assay confirmed the presence of the transgenic reporter gene. The lower panel shows a Southern blot analysis of the same DNA. Southern blots were probed with radiolabeled GFP DNA fragment.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/10 A61P 3/10 5/50 5/50 7/04 7/04 9/10 9/10 9 / 12 9/12 15/08 15/08 25/14 25/14 25/18 25/18 25/24 25/24 37/06 37/06 C12N 5/10 C12N 15/00 ZNAA 15/09 ZNA 5 / 00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ) , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), A E, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI , GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA , UG, UZ, VN, YU, ZA, ZW (72) Winston, Robert, London, UK, Denman Drive 11F term (reference) 4B024 AA01 AA10 AA20 CA01 CA06 CA12 DA02 DA03 EA02 FA02 FA10 GA13 HA17 4B065 AA90X AA93X AA95X AA97X AB01 AC14 BA05 CA43 CA44 4C084 AA13 BA35 CA 17 CA25 CA53 NA14 ZA01 ZA12 ZA18 ZA42 ZA45 ZA53 ZA68 ZA81 ZB08 ZC35 4C086 AA01 AA02 EA16 MA56 MA63 MA66 ZB01 ZB21 ZC80 Vertebrate semen containing transgenic male germ cells that are useful will also be published.

Claims (60)

[Claims] 1. An in vivo method of incorporating exogenous genetic material into the genome of a vertebrate, said method comprising: a virus comprising in the testes of a male vertebrate at least one polynucleotide encoding a desired trait or product. A gene delivery mixture comprising the vector is administered and the promoter sequence is operative to form a transcription unit under conditions such that the polynucleotide effectively reaches sperm cells or progenitor cells in the testis. And the progenitor cells are spermatogonia stem cells, B-type spermatogonia, primary spermatocytes, pre-filament spermatocytes, fine-filament spermatocytes, phagocytic spermatocytes, and the thick silk stage. Selected from the group consisting of spermatocytes, secondary spermatocytes, and spermatids; incorporating the polynucleotide encoding the desired trait or product into the genome of the sperm or progenitor cell Thus, a genetically modified male gamete is produced by the male vertebrate; and the transgenic breeding the male vertebrate with a female vertebrate of that species, thereby carrying the polynucleotide in its genome. Producing offspring. 2. An in vitro method for integrating at least one polynucleotide encoding a desired trait into the genome of a vertebrate, the method comprising: from a donor male vertebrate, sperm or spermatogonial stem cell, type B sperm; A group consisting of a progenitor cell, a primary spermatocyte, a pre-filament stage spermatocyte, a filament stage spermatocyte, a phagocytic spermatocyte, a thick stage spermatocyte, a secondary spermatocyte, and a spermatocyte Obtaining a sperm cell or progenitor cell selected from the group consisting of: a polynucleotide encoding a desired trait or product, rather than an immortalizing molecule, and a gene encoding a genetic selection marker in vitro. In the presence of a gene delivery mixture comprising a viral vector,
Applying a genetic modification to the vertebrate at or below the body temperature, at a time effective for the polynucleotide to be integrated into the genome of the cell; a genetic selectable marker expressed in the genetically modified cell. Used to isolate or select the genetically modified cells; thus, isolating or selecting the genetically modified cells so that the cells remain in the testicular seminiferous tubules. Transferring into the testes of the recipient male vertebrate so as to produce a male gamete that has been genetically modified therein, and so that the recipient male vertebrate is transferred to the female vertebra of that species. Breeding with an animal, thereby producing transgenic progeny that carries the polynucleotide in the genome. 3. The transfection factor selected from the group consisting of a liposome, a lipid transfection factor, a transferrin-polylysine enhanced virus vector, a retrovirus vector, and a lentivirus vector, wherein the gene delivery mixture further comprises:
Alternatively, the method of claim 2, comprising a mixture consisting of any member of the group. 4. The method of claim 2, wherein the polynucleotide encoding the desired trait or product is expressed in said cell by a germ cell-specific promoter. 5. The method of claim 2, wherein the polynucleotide encoding the desired trait or product is in the form of a complex with a viral vector. 6. The method of claim 3, wherein said transfection factor comprises a lipid transfection factor. 7. The method of claim 2, wherein said transfection factor further comprises a male germ cell targeting molecule. 8. The male germ cell targeting molecule comprises a c-kit ligand; and the genetic selectable marker is a c-kit promoter, a b-Myb promoter, a c-raf-1.
Promoter, vasa promoter, ATM promoter, RBM promoter, DAZ
Promoter, XRCC-1 promoter, cyclin A1 promoter, HSP 90 (
8. The method of claim 7, comprising a gene expressing a detectable product transcribed from a spermatogonia-specific promoter, selected from the group consisting of a heat shock gene promoter and a FRMI promoter. 9. The method according to claim 2, wherein the gene encoding the genetic selection marker is expressed by a germ cell-specific promoter. 10. The virus vector may be a retrovirus vector, an adenovirus vector, a transferrin-polylysine enhanced adenovirus vector, a human immunodeficiency virus vector, a lentivirus vector, a Moloney murine leukemia virus-derived vector, a mumps vector, and a polynucleotide. A virus selected from the group consisting of viral-derived DNA that promotes uptake and release of germ cells into the cytoplasm, or comprising a mixture of members of any of the groups. The described method. 11. The method according to claim 1, wherein the viral vector is a retroviral vector, and a polynucleotide encoding a desired trait or product is expressed in one or more cells of the progeny. The method described in. 12. The method according to claim 11, wherein the retroviral vector is a Moloney murine leukemia virus-derived vector or a pseudotyped lentiviral vector. 13. An in vivo method for incorporating exogenous genetic material into the genome of a vertebrate, the method comprising: providing a retrovertebrate testis of a male vertebrate comprising at least one polynucleotide encoding a desired trait or product. Administering a gene delivery mixture comprising a viral vector and, optionally, a gene encoding a genetic selectable marker, wherein the mixture contains, in the testes, sperm or spermatogonial stem cells, B-type spermatogonia, primary spermatocytes, Effect on progenitor cells selected from the group consisting of prefilament-phase spermatocytes, fine-filament-phase spermatocytes, mitotic-phase spermatocytes, thicker-phase spermatocytes, secondary spermatocytes, and sperm cells Operably linked to a promoter sequence so as to form a transcription unit under conditions that will ultimately result in the transcription of the sperm; Alternatively, a male gamete, integrated into the genome of the progenitor cell, and thus genetically modified, is produced by a male vertebrate; and the male vertebrate is crossed with a female vertebrate of that species, resulting in Producing a transgenic progeny carrying said polynucleotide. 14. The method according to claim 13, wherein the retroviral vector is a Moloney murine leukemia virus-derived vector or a pseudotyped lentiviral vector. 15. The method according to claim 1, wherein the mating is achieved by natural mating of said male and female vertebrates. 16. The method of claim 1 or claim 13, wherein said viral vector further comprises a gene encoding a genetic selectable marker operably linked to a male germ cell specific promoter. 17. The method of claim 1, 2, or 13, wherein the mating is accomplished by artificial insemination of a female vertebrate with semen containing a genetically modified male gamete. 18. The method of claim 1, 2, or 13, wherein the mating is accomplished by in vitro fertilization of a female vertebrate egg with a genetically modified male gamete. 19. Mating is accomplished by intracytoplasmic sperm injection, subzonal insemination, partial decapitation, thereby allowing fertilization of a female vertebrate egg by a genetically modified male gamete. 14. The method according to any of claims 1, 2, or 13 resulting from the method. 20. The method of any of claims 1, 2, or 13, wherein the method further comprises culturing the transgenic progeny to sexual maturity. 21. The method of claim 20, wherein the method further comprises breeding the sexually mature transgenic offspring with a homologous homologous animal to produce a second or subsequent generation of the transgenic offspring. The described method. 22. The vector comprises a genetic selection marker operably linked to the promoter sequence, and a second or subsequent generation of transgenic progeny is present in the one or more cells. 22. The method of claim 21, wherein the method is identified by expression of said genetic selectable marker. 23. The method according to claim 1, wherein the gene delivery mixture is administered into the testes of a vertebrate by transdermal injection. 24. The method of claim 1 or 13, wherein the gene delivery mixture is administered directly into a vertebrate testicular export tubule. 25. The method according to claim 1, wherein the gene delivery mixture is administered directly into the seminiferous tubules of a vertebrate testis. 26. The method of claim 1 or 13, wherein the gene delivery mixture is administered directly into the testis network of a vertebrate testis. 27. The polynucleotide of claim 1, wherein the polynucleotide encoding the desired trait or product is operably linked to a constitutive promoter.
14. The method according to any of 13. 28. The method according to claim 1, wherein the polynucleotide encoding the desired trait or product is operably linked to a cytokine-inducible promoter. 29. The polynucleotide encoding the desired trait or product is operably linked to a tissue-specific promoter.
14. The method according to any one of 2 and 13. 30. The polynucleotide of claim 1, 2 or 13, wherein said polynucleotide encoding the desired trait or product is operably linked to a developmentally or temporally regulated promoter. The method described in Crab. 31. The method of any one of claims 1, 2, and 13, wherein the gene encodes a genetic selectable marker, said gene being operably linked to a constitutive promoter. 32. The polynucleotide encoding the desired trait or product is operably linked to an exogenous inducible promoter.
14. The method according to any one of 2 and 13. 33. The polynucleotide of claim 1, wherein the polynucleotide comprises a gene encoding a genetic selectable marker, wherein the gene is operably linked to a tissue-specific promoter. The method described in. 34. The polynucleotide of claim 1, wherein the polynucleotide comprises a gene encoding a genetic selectable marker, wherein the gene is operably linked to a developmentally or temporally regulated promoter. 14. The method according to any of items 2 and 13. 35. The vertebrate is a mammal or a bird.
14. The method according to any of 13. 36. The mammal comprises a human, a non-human primate, a dog, a cat, a pig, a mouse, a rat, a gerbil, a hamster, a rabbit, a bark, a horse, a sheep, a pig, a cow, and a marine mammal. 36. The method of claim 35, wherein the method is selected from a group. 37. The method of claim 35, wherein the birds are selected from the group consisting of ducks, geese, turkeys, chickens, ostrich, emu, guinea fowl, pigeons, and quail. 38. A non-human transgenic male vertebrate produced by the method of any one of claims 1, 2 and 13, wherein the vertebrate defines a gene encoding a desired trait or product. A non-human transgenic male vertebrate, comprising native male germ cells that carry an exogenous polynucleotide in their genome, wherein the somatic cells in extratesticular tissues of the transgenic vertebrate are deficient in the polynucleotide. 39. The vertebrate, wherein the vertebrate is a non-human primate, dog, cat, pig, mouse, rat, gerbil, hamster, rabbit, dermis, horse, sheep, pig,
39. A mammal selected from the group consisting of cattle and marine mammals, or a bird selected from the group consisting of duck, goose, turkey, chicken, ostrich, emu, guinea fowl, pigeon, and quail. 2. The non-human transgenic male vertebrate according to 2. 40. The vertebrate is a progeny of the male vertebrate, wherein the progeny comprises one or more cells that carry an exogenous polynucleotide in their genome encoding a desired trait or product. A non-human transgenic vertebrate produced by the method according to any one of claims 1, 2, and 13. 41. The vertebrate is a group consisting of a non-human primate, a dog, a cat, a pig, a mouse, a rat, a gerbil, a hamster, a rabbit, a marine mammal, a macroderm, a horse, a sheep, a pig, and a cow. 41. The non-human transgenic male spine of claim 40, wherein the mammal is a mammal selected from the group consisting of a duck, goose, turkey, chicken, ostrich, emu, guinea fowl, pigeon, and quail. animal. 42. The polynucleotide of claim 40, wherein the polynucleotide comprises a heterologous coding sequence.
2. The non-human transgenic vertebrate according to 1. 43. The non-human transgenic vertebrate of claim 40, wherein said polynucleotide comprises at least one biologically functional gene. 44. The non-human transgenic vertebrate of claim 40, wherein said vertebrate comprises one or more cells expressing a genetic selectable marker. 45. The non-human transgenic vertebrate of claim 40, wherein said vertebrate is a male vertebrate. 46. A transgenic cell, wherein said cell is derived from the vertebrate of claim 40, and wherein said cell is a somatic cell. 47. A transgenic germ cell, wherein said cell is obtained from the vertebrate of claim 40. 48. The transgenic germ cell according to claim 47, wherein said cell is an egg. 49. The cell, wherein the cell is a sperm or a spermatogonial stem cell, a B-type spermatogonia, a primary spermatocyte, a pre-filament spermatocyte, a fine-filament spermatocyte, a phagocytic spermatocyte, A mitotic spermatocyte, a secondary spermatocyte, and a progenitor cell selected from the group consisting of sperm cells,
48. The transgenic germ cell of claim 47. 50. The cell is obtained from a male vertebrate according to claim 38.
Transgenic male germ cells. 51. The cell is obtained from a male vertebrate according to claim 45.
Transgenic male germ cells. 52. The cell according to claim 52, wherein the cell is a sperm or a spermatogonial stem cell, a B-type spermatogonia, a primary spermatocyte, a pre-fibrillary spermatocyte, a fine-filament spermatocyte, a phagocytic spermatocyte, A mitotic spermatocyte, a secondary spermatocyte, and a progenitor cell selected from the group consisting of sperm cells,
The transgenic germ cell according to claim 50 or 51. 53. A vertebrate semen comprising the plurality of transgenic germ cells of claim 52. 54. A method for producing a non-human transgenic vertebrate strain comprising a native germ cell that carries at least one heterologous or allogeneic polynucleotide in its genome, said method comprising: Breeding a vertebrate of the same species with a homologous isomer; and selecting a progeny having the polypeptide, the method comprising producing a transgenic non-human vertebrate strain. 55. The method according to claim 1, wherein the polynucleotide encoding the desired trait or product is derived from a human. 56. The method according to claim 1, wherein said genetic selection marker is a fluorescent protein or a luminescent protein. 57. The method of claim 2, wherein the support cells are co-administered to the testes with the isolated or selected germ cells. 58. The genetically modified Leydig and / or Sertoli cells are isolated or selected and co-administered with the isolated or selected germ cells into the testes of a recipient male vertebrate. 3. The method according to 2. 59. The method of claim 2, wherein the testes of the recipient male vertebrate are substantially reduced in number of cells before transferring the genetically modified germ cells into them. 60. The recipient male vertebrate is a donor male vertebrate.
The method according to claim 2.