Kit for transfection, storage and transfer of male germ cells for generation of transgenic species

Abstract

A composition for in vivo transfection of vertebrate male germ cells comprises a nucleic acid or transgene, and a gene delivery system, and optionally a protective internalizing agent, such as an endosomal lytic agent, a virus or a viral component, which is internalized by cells along with the transgene and which enhances gene transfer through the cytoplasm to the nucleus of the male germ cell. A pharmaceutical preparation and a transfer kit utilize the composition. A method for introducing a polynucleotide into vertebrate male germ cells comprises the administration of the composition to a vertebrate. A method for isolating or selecting transfected cells utilizes a reporter gene, and a method for administering transfected male germ cells utilizes male germ cells which have been transfected in vitro.

What is claimed:  
     
         1 . An in vivo method of incorporating a polynucleotide into a male vertebrate's germ cells, comprising 
 administering to a male vertebrate's gonads a transfection mixture comprising at least one polynucleotide encoding a desired trait or product, and at least one transfecting agent, and optionally a genetic selection marker, and under conditions effective to reach the vertebrate's germ cells or precursors thereof; and    allowing the polynucleotide encoding a desired trait or product to be taken up by, and released into, the germ cells or precursors thereof.    
     
     
         2 . The method of  claim 1 , further comprising allowing the incorporation of the released polynucleotide into the genome of the germ cells.  
     
     
         3 . The method of  claim 1  wherein the transfecting agent is selected from the group consisting of liposomes, viral vectors, transferrin-polylysine enhanced viral vectors, retroviral vectors, lentiviral vectors, and uptake enhancing DNA segments, or comprises a mixture of any members of said group.  
     
     
         4 . The method of  claim 3 , wherein the transfecting agent comprises a viral vector selected from the group consisting of retroviral vectors, adenoviral vectors, transferrin-polylysine enhanced adenoviral vectors, human immunodeficiency virus vectors, lentiviral vectors, Moloney murine leukemia virus-derived vectors, mumps vectors, and virus-derived DNAs that facilitate polynucleotide uptake by and release into the cytoplasm of germ cells, or comprises an operative fragment of- or mixture of any members of said group.  
     
     
         5 . The method of  claim 1 , wherein the transfecting agent comprises an adenovirus vector having endosomal lytic activity, and the polynucleotide is operatively linked to the vector.  
     
     
         6 . The method of  claim 1 , wherein the transfecting agent comprises a lipid transfecting agent.  
     
     
         7 . The method of  claim 1 , wherein the transfecting agent further comprises a male-germ-cell-targeting molecule.  
     
     
         8 . The method of  claim 7 , wherein the male-germ-cell-targeting molecule is specific for targeting spermatogonia, and is a c-kit ligand.  
     
     
         9 . The method of  claim 1 , where the transfection mixture further comprises an immunosuppressing agent.  
     
     
         10 . The method of  claim 9 , wherein the immunosuppressing agent is selected from the group consisting of cyclosporin and corticosteroids, and the agent is administered systemically.  
     
     
         11 . The method of  claim 1 , wherein the transfection mixture is administered by injection.  
     
     
         12 . The method of  claim 11 , where injection comprises percutaneous injection into the vertebrate's testis.  
     
     
         13 . The method of  claim 1 , wherein the transfection mixture is administered into the vertebrate's testis.  
     
     
         14 . The method of  claim 13 , wherein the transfection mixture is directly administered into the vertebrate's vas efferens.  
     
     
         15 . The method of  claim 13 , wherein the transfection mixture is directly administered into a seminiferous tubule of the vertebrate's testis.  
     
     
         16 . The method of  claim 1 , wherein the transfection mixture is directly administered into the rete of the vertebrate's testis.  
     
     
         17 . The method of  claim 1 , wherein the vertebrate is a mammal.  
     
     
         18 . The method of  claim 17 , wherein the mammal is a human.  
     
     
         19 . The method of  claim 17 , wherein the mammal is selected from the group consisting of human and non-human primates, farm mammals, and marine mammals.  
     
     
         20 . The method of  claim 19 , wherein the farm mammal is selected from the group consisting of swine, equines, ovines and bovines.  
     
     
         21 . The method of  claim 1 , wherein the vertebrate is a bird selected from the group consisting of ducks, geese, turkeys and chickens.  
     
     
         22 . The method of  claim 1 , wherein the vertebrate is selected from the group consisting of wild and domesticated vertebrates.  
     
     
         23 . A gene therapy method, comprising the method of  claim 1 , wherein the polynucleotide encoding a desired trait or product is derived from the same species as the male vertebrate.  
     
     
         24 . A non-human transgenic vertebrate produced by the method of  claim 1 , or progeny thereof, wherein the polynucleotide encoding a desired trait or product is derived from any genome.  
     
     
         25 . The non-human transgenic vertebrate of  claim 24 , comprising native germ cells carrying in their genome at least one xenogeneic polynucleotide.  
     
     
         26 . The non-human transgenic vertebrate of  claim 25 , wherein the polynucleotide comprises at least one biologically functional gene.  
     
     
         27 . The non-human transgenic vertebrate of  claim 24 , being a male.  
     
     
         28 . The progeny resulting from breeding the non-human transgenic vertebrate of  claim 27 , with a female of the same species.  
     
     
         29 . A non-human vertebrate, carrying in its germ cells at least one xenogeneic polynucleotide sequence, said non-human vertebrate being obtained by breeding the vertebrate of  claim 24 , or progeny thereof, with a member of the opposite sex of the same species, and selecting the bred progeny for the presence of the transfected xenogeneic polynucleotide.  
     
     
         30 . The non-human vertebrate of  claim 29 , which is selected from the group consisting of mammals and birds.  
     
     
         31 . The non-human vertebrate of  claim 30 , which is a mammal selected from the group consisting of humans and non-human primates, canines, felines, swine, farm and marine mammals, pachyderms, equines, murine, ovines and bovine, or a bird selected from the group consisting of ducks, geese, turkeys and chickens.  
     
     
         32 . The vertebrate of  claim 31 , wherein the mammal is selected from the group consisting of wild and domesticated mammals.  
     
     
         33 . The vertebrate of  claim 31 , wherein the mammal is a farm or marine animal.  
     
     
         34 . The vertebrate of  claim 30 , wherein the mammal is selected from the group consisting of a bull and a pig, and the bird is a chicken.  
     
     
         35 . A germ cell, obtained from the vertebrate of  claim 25 .  
     
     
         36 . Vertebrate male germ cells, obtained by a method comprising the method of  claim 1;  raising the transfected male vertebrate; and collecting male germ cells produced by the male vertebrate.  
     
     
         37 . The vertebrate male germ cells of  claim 36 , wherein the method for obtaining them further comprises breeding the transfected vertebrate to produce progeny, and then collecting the germ cells produced by a male progeny.  
     
     
         38 . Vertebrate semen, comprising the germ cell of  claim 35 .  
     
     
         39 . Vertebrate semen, comprising the germ cells obtained from the vertebrate of  claim 25 .  
     
     
         40 . A method of producing a non-human vertebrate animal line comprising native germ cells carrying in their genome at least one xenogeneic polynucleotide, comprising 
 breeding of the vertebrate of  claim 25 , with a member of the opposite sex of the same species; and selecting progeny for the presence of said polynucleotide.    
     
     
         41 . A method of isolating or selecting a male germ cell transfected with at least one polynucleotide encoding a desired trait or product and at least one genetic selection marker, comprising 
 the method of  claim 1 , wherein the transfection mixture comprises at least one genetic selection marker; and    isolating or selecting a transfected male germ cell with the aid of the genetic selection marker.    
     
     
         42 . A method of transferring maturing male germ cells transfected with at least one polynucleotide encoding a desired trait or product to the testis of a recipient male vertebrate, comprising 
 isolating or selecting maturing male germ cells carrying at least one polynucleotide encoding a desired trait or product and at least one polynucleotide encoding a genetic selection marker, from a donor male vertebrate by the method of claim  41 ;    administering the germ cells, thus isolated or selected, to a testis of a recipient male vertebrate; and    allowing the administered germ cells to lodge in a seminiferous tubule of the recipient male vertebrate.    
     
     
         43 . A method of transferring autologous germ and support cells to the testis of a vertebrate, comprising the method of  claim 42 , wherein the donor vertebrate is the same as the recipient vertebrate.  
     
     
         44 . The method of  claim 41 , further comprising the step of incorporating into the genome of the germ cell the polynucleotide encoding a desired trait or product.  
     
     
         45 . The method of  claim 41 , wherein the transfected male germ cell comprises an undifferentiated male germ cell.  
     
     
         46 . The method of  claim 41 , wherein transfection is conducted under conditions of temperature of about 25° C. to about 38° C.  
     
     
         47 . The method of  claim 41 , wherein the transfecting agent is selected from the group consisting of liposomes, viral vectors, transferrin-polylysine enhanced viral vectors, retroviral vectors, lentiviral vectors, and other uptake enhancing DNA segments, or comprises a mixture of any members of said group.  
     
     
         48 . The method of  claim 47 , wherein the transfecting agent comprises a viral vector selected from the group consisting of retroviral vectors, adenoviral vectors, transfernin-polylysine enhanced adenoviral vectors, human immunodeficiency virus vectors, lentiviral vectors, Moloney murine leukemia virus-derived vectors, mumps vectors, and virus-derived DNAs that facilitate polynucleotide uptake by and release into the cytoplasm of germ cells, or said transfecting agent comprises an operative fragment of- or mixture of any members of said group.  
     
     
         49 . The method of  claim 47 , wherein the transfecting agent comprises an adenovirus vector having endosomal lytic activity, and the polynucleotide is operatively linked to the vector.  
     
     
         50 . The method of  claim 41 , wherein the polynucleotide encoding a desired trait or product is in the form of a complex with a viral vector.  
     
     
         51 . The method of  claim 41 , wherein the transfecting agent comprises a lipid transfecting agent.  
     
     
         52 . The method of  claim 42 , wherein the transfecting agent further comprises an agent selected from the group consisting of a male-germ-cell-targeting molecule and at least one genetic selection marker.  
     
     
         53 . The method of  claim 52 , wherein the male-germ-cell-targeting molecule is specifically targeted to spermatogonia and comprises a c-kit ligand; and 
 the genetic selection marker comprises a gene encoding a detectable product. expression of said gene being driven by a spermatogonia-specific promoter, said promoter being selected from the group consisting of c-kit promoter, b-Myb promoter, c-raf-1 promoter, ATM (axataia-telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, and FRMI (from fragile X site) promoter.    
     
     
         54 . The method of  claim 41 , wherein the vertebrate is a mammal.  
     
     
         55 . The method of  claim 54 , wherein the mammal is a human.  
     
     
         56 . The method of  claim 54 , wherein the mammal is selected from the group consisting of human and non-human primates and farm and marine mammals.  
     
     
         57 . The method of  claim 42 , wherein the polynucleotide encoding a desired trait or product is derived from the same species of vertebrate as the recipient vertebrate.  
     
     
         58 . The method of  claim 42 , wherein the vertebrate is selected from the group consisting of wild and domesticated vertebrates.  
     
     
         59 . The method of  claim 41 , wherein the polynucleotide encoding a desired trait or product is derived from a mammal selected from the group consisting of human and non-human primates, canines, felines, swines, farm mammals, pachyderms, marine mammals, equines, murine, ovine and bovine, or from a bird selected from the group consisting of ducks, geese, turkeys and chickens.  
     
     
         60 . The method of  claim 59 , wherein the polynucleotide is derived from a human.  
     
     
         61 . A non-human transgenic vertebrate, comprising native germ cells carrying in their genomes at least one xenogeneic polynucleotide, said transgenic vertebrate being the recipient male vertebrate of the method of  claim 42 , or progeny thereof.  
     
     
         62 . The non-human transgenic vertebrate of  claim 61 , wherein the polynucleotide comprises at least one biologically functional gene.  
     
     
         63 . The non-human transgenic vertebrate of  claim 62 , being a male.  
     
     
         64 . The non-human transgenic vertebrate of  claim 63 , harboring native male germ cells transfected with a xenogeneic polynucleotide.  
     
     
         65 . The progeny resulting from breeding the non-human transgenic vertebrate of  claim 63  or progeny thereof, with a female of the same species.  
     
     
         66 . A non-human vertebrate, carrying in its germ cells at least one xenogeneic polynucleotide sequence, obtained by breeding the vertebrate of  claim 61  or progeny thereof, with a member of the opposite sex of the same species, and selecting the bred progeny for the presence of the transfected xenogeneic polynucleotide.  
     
     
         67 . The non-human vertebrate of  claim 66 , which is selected from the group consisting of mammals and birds.  
     
     
         68 . The non-human vertebrate of  claim 67 , which is a mammal selected from the group consisting of humans and non-human primates, canines, felines, swine, farm and marine mammals, pachyderms, equines, murine, ovines and bovine, and a bird selected from the group consisting of ducks, geese, turkeys and chickens.  
     
     
         69 . The non-human vertebrate of  claim 67 , which is a bird selected from the group consisting of ducks, geese, turkeys and chickens.  
     
     
         70 . The non-human vertebrate of  claim 67 , wherein the mammal is a farm or marine mammal.  
     
     
         71 . The non-human vertebrate of  claim 68 , wherein the mammal is a bull.  
     
     
         72 . The non-human vertebrate of  claim 68 , wherein the mammal is a pig.  
     
     
         73 . The non-human vertebrate of  claim 66 , which is selected from the group consisting of wild and domesticated animals.  
     
     
         74 . A germ cell obtained from a vertebrate of claims  24  or  61  comprising a native germ cell carrying in its genome at least one xenogeneic polynucleotide.  
     
     
         75 . Vertebrate semen comprising the germ cell of  claim 74 .  
     
     
         76 . A gene therapy method, comprising the method of  claim 42 , wherein the polynucleotide encoding a desired trait or product is derived from the same species of vertebrate as the recipient vertebrate.  
     
     
         77 . A non-human transgenic vertebrate produced by the method of  claim 42 , wherein the polynucleotide encoding a desired trait or product is derived from any genome.  
     
     
         78 . An in vitro method of incorporating at least one polynucleotide encoding a desired trait into a maturing male germ cell, comprising 
 obtaining a maturing male germ cell from a vertebrate;    transfecting the germ cell in vitro with at least one polynucleotide encoding a desired trait in the presence of a gene delivery mixture comprising at least one transfecting agent, and optionally a polynucleotide encoding a genetic selection marker, at about or below the vertebrate's body temperature and for a transfection-effective period of time; and    allowing the polynucleotide encoding a desired trait to be taken up by, and released into the germ cell.    
     
     
         79 . The method of  claim 78 , further comprising allowing the incorporation of the released polynucleotide into the genome of the germ cell.  
     
     
         80 . The method of  claim 78 , wherein the encoding a desired trait is incorporated into the vertebrate germ cell's genome.  
     
     
         81 . The method of  claim 78 , wherein the maturing male germ cell comprises a spermatogonia or other undifferentiated male germ cell.  
     
     
         82 . The method of  claim 78 , wherein the transfection is conducted under conditions of temperature of about 25° C. to about 38° C.  
     
     
         83 . The method of  claim 78 , wherein the transfecting agent is selected from the group consisting of liposomes, viral vectors, transferrin-polylysine enhanced viral vectors, retroviral vectors, lentiviral vectors, and other uptake enhancing DNA segments, or comprises a mixture of any members of said group.  
     
     
         84 . The method of  claim 83 , wherein the transfecting agent comprises a viral vector selected from the group consisting of retroviral vectors, adenoviral vectors, transferrin-polylysine enhanced adenoviral vectors, human immunodeficiency virus vectors, lentiviral vectors, Moloney murine leukemia virus-derived vectors, mumps vectors, and virus-derived DNAs that enhance polynucleotide uptake by and release into the cytoplasm of germ cells, or said transfecting agent comprises an operative fragment of- or mixture of any members of said group.  
     
     
         85 . The method of  claim 84 , wherein the transfecting agent comprises an adenovirus vector having endosomal lytic activity, and the polynucleotide encoding a desired trait is operatively linked to the vector.  
     
     
         86 . The method of  claim 78 , wherein the polynucleotide encoding a desired trait is in the form of a complex with a viral vector.  
     
     
         87 . The method of  claim 78 , wherein the transfecting agent comprises a lipid transfecting agent.  
     
     
         88 . The method of  claim 78 , wherein 
 the transfecting agent further comprises an agent selected from the group consisting of a male-germ-cell-targeting molecule and at least one genetic selection marker; and    the method further comprises isolating or selecting a maturing male germ cell carrying at least one polynucleotide encoding a desired trait or product and at least one polynucleotide encoding a genetic selection marker, from a donor male vertebrate with the aid of the genetic selection marker.    
     
     
         89 . The method of  claim 88 , wherein 
 the male-germ-cell-targeting molecule is specifically targeted to spermatogonia and comprises a c-kit ligand, and    the genetic selection marker comprises a gene expressing a detectable product, driven by a spermatogonia-specific promoter selected from the group consisting of c-kit promoter, b-Myb promoter, c-raf-1 promoter, ATM (axataia-telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, and FRMI (from fragile X site) promoter.    
     
     
         90 . The method of  claim 78 , wherein the vertebrate is a mammal.  
     
     
         91 . The method of  claim 90 , wherein the mammal is a human.  
     
     
         92 . The method of  claim 90 , wherein the mammal is selected from the group consisting of human and non-human primates and farm and marine mammals.  
     
     
         93 . The method of  claim 78 , wherein the polynucleotide encoding a desired trait is derived from the same vertebrate species as the maturing germ cell.  
     
     
         94 . The method of  claim 78 , wherein the vertebrate is selected from the group consisting of wild and domesticated vertebrates.  
     
     
         95 . The method of  claim 78 , wherein the polynucleotide encoding a desired trait is derived from a mammal selected from the group consisting of human and non-human primates, canines, felines, swines, farm mammals, pachyderms, marine mammals, equines, murine, ovine and bovine, or from a bird selected from the group consisting of ducks, geese, turkeys and chickens.  
     
     
         96 . The method of  claim 95 , wherein the polynucleotide is derived from a human.  
     
     
         97 . An non-human transgenic vertebrate, or its progeny, comprising a native germ cell carrying in its genome at least one xenogeneic polynucleotide, said polynucleotide having been incorporated into the genome of said germ cell through the method of  claim 78 .  
     
     
         98 . The non-human transgenic vertebrate of  claim 97 , wherein the polynucleotide comprises at least one biologically functional gene.  
     
     
         99 . The non-human transgenic vertebrate of  claim 98 , being a male.  
     
     
         100 . The non-human transgenic vertebrate of  claim 99 , harboring native male germ cells transfected with a xenogeneic polynucleotide.  
     
     
         101 . The progeny resulting from breeding the non-human transgenic vertebrate of  claim 99  or progeny thereof, with a female of the same species.  
     
     
         102 . A non-human vertebrate, carrying in its germ cells at least one xenogeneic polynucleotide sequence, said vertebrate obtained by breeding the vertebrate of  claim 98  or progeny thereof, with a member of the opposite sex of the same species, and selecting the bred progeny for the presence of the transfected xenogeneic polynucleotide.  
     
     
         103 . The non-human vertebrate of  claim 102 , which is selected from the group consisting of mammals and birds.  
     
     
         104 . The non-human vertebrate of  claim 103 , which is a mammal selected from the group consisting of humans and non-human primates, canines, felines, swine, farm and marine mammals, pachyderms, equines, murine, ovines and bovine, or a bird selected from the group consisting of ducks, geese, turkeys and chickens.  
     
     
         105 . The non-human vertebrate of  claim 104 , which is a bird selected from the group consisting of ducks, geese, turkeys and chickens.  
     
     
         106 . The non-human vertebrate of  claim 104 , wherein the mammal is a farm or marine mammal.  
     
     
         107 . The non-human vertebrate of  claim 104 , wherein the mammal is a bull.  
     
     
         108 . The non-human vertebrate of  claim 104 , wherein the mammal is a pig.  
     
     
         109 . The non-human vertebrate of  claim 102 , which is selected from the group consisting of wild and domesticated animals.  
     
     
         110 . A germ cell obtained from the vertebrate of  claim 97 , or its progeny.  
     
     
         111 . Vertebrate semen comprising a plurality of the germ cells obtained from the vertebrate of  claim 98 .  
     
     
         112 . A gene therapy method, comprising the method of claim  78 ; further comprising the step of introducing said transfected male germ cell into the testis of a recipient vertebrate, wherein the polynucleotide encoding a desired trait is derived from the same vertebrate species as the recipient vertebrate.  
     
     
         113 . A non-human transgenic vertebrate produced by the method of  claim 78 , wherein the polynucleotide encoding a desired trait is derived from any genome.  
     
     
         114 . A kit for the transfection and storage of a male vertebrate's germ cells, comprising a transfection mixture, said tranfection mixture comprising at least one transfecting agent, and optionally a genetic selection marker, whereby said kit may be used to transfect and store said germ cells in a viable condition.  
     
     
         115 . The kit of  claim 114 , wherein the transfecting agent is selected from the group consisting of liposomes, viral vectors, transferrin-polylysine enhanced viral vectors, retroviral vectors, lentiviral vectors, and uptake enhancing DNA segments, or comprises a mixture of any members of said group.  
     
     
         116 . The kit of  claim 114 , wherein the transfecting agent comprises a viral vector selected from the group consisting of retroviral vectors, adenoviral vectors, transferrin-polylysine enhanced adenoviral vectors, human immunodeficiency virus vectors, lentiviral vectors, Moloney murine leukemia virus-derived vectors, mumps vectors, DNAs that facilitate polynucleotide uptake by and release into the cytoplasm of germ cells, or comprises an operative fragment of- or mixture of any members of said group.  
     
     
         117 . The kit of  claim 114 , wherein the transfecting agent comprises an adenovirus vector having endosomal lytic activity, and the polynucleotide is operatively linked to the vector.  
     
     
         118 . The kit of  claim 114 , wherein the transfecting agent comprises a lipid transfecting agent.  
     
     
         119 . The kit of  claim 114 , wherein the transfecting agent further comprises a male-germ-cell-targeting molecule.  
     
     
         120 . The kit of  claim 119 , wherein the male-germ-cell-targeting molecule is specific for targeting spermatogonia and comprises a c-kit ligand.  
     
     
         121 . The kit of  claim 114 , where the transfection mixture further comprises an immunosuppressing agent.  
     
     
         122 . The kit of  claim 121 , wherein the immunosuppressing agent is selected from the group consisting of cyclosporin and corticosteroids.  
     
     
         123 . The kit of  claim 119 , wherein the male-germ-cell-targeting molecule is specifically targeted to spermatogonia and comprises a c-kit ligand; and 
 the genetic selection marker comprises a gene expressing a detectable product driven by a spermatogonia-specific promoter.    
     
     
         124 . The kit of  claim 119 , wherein the male-germ-cell-targeting molecule is specifically targeted to spermatogonia and comprises a c-kit ligand; and 
 the genetic selection marker comprises a gene expressing a detectable product, driven by a spermatogonia-specific promoter, said promoter selected from the group consisting of c-kit promoter, b-Myb promoter, c-raf-1 promoter, ATM (axataia-telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, and FRMI (from fragile X site) promoter.    
     
     
         125 . The kit of  claim 114 , wherein at least one polynucleotide comprises at least one polynucleotide sequence encoding a genetic selection marker.  
     
     
         126 . The kit of  claim 114 , further comprising a cryoprotectant.  
     
     
         127 . The germ cell as in any of claims  35 ,  36 ,  37 ,  74 , or  110 , wherein said germ cell has been cryopreserved in a viable and functional condition.  
     
     
         128 . A transgenic male germ cell produced by the method of any of claims  17 ,  18 ,  19 ,  20 ,  21 , or  22 , wherein the transgenic male germ cell has been cryopreserved in a viable and functional condition.  
     
     
         129 . A transgenic male germ cell produced by the method of any of claims  54 ,  55 ,  56 , or  58 , wherein the transgenic male germ cell has been cryopreserved in a viable and functional condition.  
     
     
         130 . A transgenic male germ cell produced by the method of any of claims  90 ,  91 ,  92 , or  94 , wherein the transgenic male germ cell has been cryopreserved in a viable and functional condition.  
     
     
         131 . The method of any of claims  1  or  78 , wherein the polynucleotide encoding a desired trait or product is operatively linked to a germ cell-specific promoter.  
     
     
         132 . The method of any of claims  41 ,  78 , or  88 , wherein the polynucleotide encoding a genetic selection marker is operatively linked to a germ cell-specific promoter.  
     
     
         133 . The method of  claim 42 , wherein support cells are co-administered to a testis along with isolated or selected germ cells.  
     
     
         134 . The method of  claim 42 , wherein transfected support cells are isolated or selected, and co-administered to a testis of a recipient male vertebrate along with said isolated or selected germ cells.