Transfection, storage and transfer of male germ cells for generation of transgenic species & genetic therapies

Abstract

Disclosed is an in vivo method of incorporating exogenous genetic material into the genome of a vertebrate, which involves administering to a male vertebrate's testis a gene delivery mixture comprising a viral vector, such as a retroviral vector, to deliver a polynucleotide encoding a desired trait or product. Also disclosed is an in vitro method of incorporating exogenous genetic material into the genome of a vertebrate, in which germ cells are obtained from a donor male vertebrate and are genetically modified in vitro, before being transferred to a recipient male vertebrate. After the transfer, the male vertebrate bearing the genetically modified germ cells is bred with a female vertebrate such that a transgenic progeny is produced that carries the polynucleotide in its genome. Also disclosed are non-human transgenic vertebrates produced in accordance with the method, including transgenic progeny. A transgenic cell derived from the transgenic vertebrate is also disclosed, being a germ cell, such as a spermatozoan or ovum, a precursor cell of either of these, or a somatic cell. A method of producing a non-human transgenic vertebrate animal line comprising native germ cells carrying in their genome at least one xenogeneic polynucleotide is disclosed, as is vertebrate semen containing the transgenic male germ cells useful in practicing the method.

1 - 60 . (canceled) 
     
     
         61 . An in vivo method of incorporating exogenous genetic material into the genome of a non-human mammal, said method comprising:
 (a) injecting into a vas efferens, or rete of a testis of a male non-human mammal a gene delivery mixture comprising a retroviral vector that comprises at least one polynucleotide encoding a desired gene product and optionally a gene encoding a desired gene produce and optionally a gene encoding a genetic selection marker, operatively linked to a promoter sequence such that a transcriptional unit is formed, under conditions effective to reach within the testis a male germ cell selected from the group consisting of spermatogonial stem cells, type B spermatogonial, primary spermatocytes, preleptotene spermatocytes, leptotene spermatocytes, zygotene spermatocytes, pachytene spermatocytes, secondary spermatocytes, spermatids, and spermatozoa;   (b) allowing the polynucleotide encoding a desired gene product to be incorporated into the genome of the male germ cell, so that a genetically modified male gamete is produced by the male non-human mammal; and   (c) breeding the male non-human mammal with a female non-human mammal of its species, such that the genetically modified gamete is united with a female gamete and a transgenic progeny is produced thereby, that carries the polynucleotide encoding the desired gene product in its somatic cells.   
     
     
         62 . The method of  claim 61 , wherein the retroviral vector is selected from the group consisting of Moloney murine leukemia virus vectors and pseudotyped lentiviral vectors. 
     
     
         63 . The method of  claim 61 , wherein the retroviral vector is a lentiviral vector. 
     
     
         64 . The method of  claim 61 , wherein the retroviral vector is a pseudotyped Moloney murine leukemia virus vector. 
     
     
         65 . The method of  claim 61 , wherein breeding is accomplished by natural mating of the male non-human mammal and female non-human mammal. 
     
     
         66 . The method of  claim 61 , wherein the retroviral vector further comprises a gene encoding a genetic selection marker operatively linked to a male germ cell-specific promoter. 
     
     
         67 . The method of  claim 61 , wherein breeding is accomplished by artificial insemination of the female non-human mammal with semen comprising the genetically modified male gamete. 
     
     
         68 . The method of  claim 61 , wherein breeding is accomplished by in vitro fertilization of an ovum of the female non-human mammal with the genetically modified male gamete. 
     
     
         69 . The method of  claim 61 , wherein breeding is accomplished by intracytoplasmic sperm injection, subzonal insemination, or partial zona dissection, resulting in fertilization of an ovum of the female non-human mammal with the genetically modified male gamete. 
     
     
         70 . The method of  claim 61 , wherein the polynucleotide encoding a desired gene product is operatively linked to a constitutive promoter. 
     
     
         71 . The method of  claim 61 , wherein the polynucleotide encoding a desired gene product is operatively linked to a cytokine-inducible promoter. 
     
     
         72 . The method of  claim 61 , wherein the polynucleotide encoding a desired gene product is operatively linked to a tissue-specific promoter. 
     
     
         73 . The method of  claim 61 , wherein the polynucleotide encoding a desired gene product is operatively linked to a developmentally or temporally regulated promoter. 
     
     
         74 . The method of  claim 61 , wherein the polynucleotide comprises a gene encoding a genetic selection marker, and said gene is operatively linked to a constitutive promoter. 
     
     
         75 . The method of  claim 61 , wherein the polynucleotide encoding a desired gene product is operatively linked to an exogenously inducible promoter. 
     
     
         76 . The method of  claim 61 , wherein the polynucleotide comprises a gene encoding a genetic selection marker, and said gene is operatively linked to a tissue-specific promoter. 
     
     
         77 . The method of  claim 61 , wherein the polynucleotide comprises a gene encoding a genetic selection marker, and said gene is operatively linked to a developmentally or temporally regulated promoter. 
     
     
         78 . The method of  claim 61 , wherein the polynucleotide encoding the desired gene product encodes a human gene product. 
     
     
         79 . The method of  claim 61 , wherein the genetic selection marker is a fluorescent protein or light-emitting protein. 
     
     
         80 . The method of  claim 61 , wherein the gene delivery mixture is injected into the vas efferens of the testis. 
     
     
         81 . The method of  claim 61 , wherein the gene delivery mixture is injected into a seminferous tubule of the testis.