Compositions and methods for producing genetically modified animals

Abstract

Methods, compositions and non-human animals and parts thereof are disclosed for improving germ line transmission of genetic modifications. More particular, the present invention discloses methods and compositions for producing non-human embryos with a disrupted or disruptable fertility gene, which can be used as hosts for the development of donor pluripotent cells, including genetically modified donor pluripotent cells, into germ cells and gametes. Also disclosed are methods and compositions for producing from such embryos chimeric non-human animals with a disrupted fertility gene and for breeding the chimeric non-human animals with cognate non-human animals that comprise a fertility gene that lacks a disruption to produce non-human animals having substantially all gametes and/or germ cells derived from the donor pluripotent cells. The present invention also discloses non-human gametes, germ cells, embryos and animals for use in the subject methods.

What is claimed is: 
     
         1 . A pre-implantation nonhuman host embryo (e.g., blastocyst) comprising in its germ line a disrupted fertility gene. 
     
     
         2 . A pre-implantation non-human host embryo according to  claim 1 , wherein bath alleles of the fertility gene are disrupted. 
     
     
         3 . A pre-implantation non-human host embryo according to  claim 1  or  claim 2 , wherein the fertility gene is located on a sex chromosome, 
     
     
         4 . A pre-implantation non-human host embryo according to any one of  claims 1  to  3 , wherein the sex chromosome is a X chromosome. 
     
     
         5 . A pre-implantation non-human host embryo according to any one of  claims 1  to  3 , wherein the sex chromosome is a Y chromosome. 
     
     
         6 . A pre-implantation non-human host embryo according to any one of  claims 1  to  5 , wherein the disrupted fertility gene inhibits male fertility. 
     
     
         7 . A pre-implantation non-human host embryo according to any one of  claims 1  to  6 , wherein the fertility gene modulates the fertility of sperm. 
     
     
         8 . A pre-implantation non-human host embryo according to any one of  claims 1  to  7 , wherein the fertility gene modulates spermatogenesis. 
     
     
         9 . A pre-implantation non-human host embryo according to any one of  claims 1  to  8 , wherein the fertility gene is GILZ. 
     
     
         10 . A pre-implantation non-human host embryo according to  claim 9 , further comprising a donor pluripotent cell that comprises a fertility gene lacking a disruption. 
     
     
         11 . A pre-implantation non-human host embryo according to  claim 10 , wherein the pluripotent cell is a male pluripotent cell. 
     
     
         12 . A pre-implantation non-human host embryo according to  claim 10  or  claim 11 , wherein the pluripotent cell is a stem cell. 
     
     
         13 . A pre-implantation non-human host embryo according to  claim 12 , wherein the stem cell is an embryonic stem (ES) cell. 
     
     
         14 . A pre-implantation non-human host embryo according to any one of  claims 8  to  13 , wherein the pluripotent cell comprises a genetic modification in its genome. 
     
     
         15 . A method of generating a non-human animal, the method comprising introducing the pre-implantation non-human host embryo according to any one of  claims 1  to  14  into a pseudopregnant non-human animal and gestating the pre-implantation non-human host embryo under conditions suitable for development of the embryo, thereby generating a non-human animal. 
     
     
         16 . A non-human host embryo comprising a disrupted fertility gene as defined in any one of  claims 1  to  9  and a donor pluripotent cell as defined in any one of  claims 10  to  14 . 
     
     
         17 . A surrogate or foster non-human animal comprising a non-human host embryo according to  claim 16 . 
     
     
         18 . A method of generating a chimeric non-human animal, the method comprising introducing the pre-implantation non-human host embryo according to any one of  claims 10  to  14  into a pseudopregnant non-human animal and gestating the pre-implantation non-human host embryo under conditions suitable for development of the embryo, thereby generating a chimeric non-human animal. 
     
     
         19 . A method of producing a non-human host embryo that comprises a disrupted fertility gene, the method comprising: crossing: 1) a first animal strain carrying a conditional infertility transgene that comprises a disruptable fertility gene (“conditional infertility strain”); with 2) a second animal strain carrying an infertility-activating transgene that comprises a gene that disrupts the disruptable fertility gene (“infertility-activating strain”), thereby generating transgenic non-human host embryos that comprise germ cells having a disrupted fertility gene. 
     
     
         20 . A method according to  claim 18 , wherein female members of the first animal strain are crossed with male members of the second animal strain. 
     
     
         21 . A breeding pair of non-human animals, comprising: 1) a first breeding partner comprising a fertility gene that is disruptable by a fertility gene disruptor molecule (“a disruptable fertility gene”), wherein the disruptable fertility gene is operably connected to a promoter, and (2) a second breeding partner comprising a disruptor transgene that comprises a nucleotide sequence encoding the fertility gene disruptor molecule (“a disruptor nucleotide sequence”), wherein the disruptor nucleotide sequence is operably linked to a promoter. 
     
     
         22 . A breeding pair according to  claim 21 , wherein the fertility gene disruptor is a recombinase and the disruptable fertility gene is operably connected to recombinase recognition sites that mediate disruption of the fertility gene in the presence of the recombinase. 
     
     
         23 . A breeding pair according to  claim 21 , wherein the fertility gene disruptor is an inhibitory RNA molecule that inhibits the expression of the fertility gene, suitably by antisense suppression or RNA interference. 
     
     
         24 . A breeding pair according to  claim 21 , wherein the fertility gene disruptor is an antibody that is immuno-interactive with a polypeptide product of the fertility gene. 
     
     
         25 . A breeding pair according to any one of  claims 21  to  24 , wherein the disruptor nucleotide sequence is conditionally expressible. 
     
     
         26 . A breeding pair according to  claim 25 , wherein the disruptor transgene comprises an expression-modulating element operably linked to the disruptor nucleotide sequence, wherein the element conditionally inhibits expression of the disruptor nucleotide sequence. 
     
     
         27 . A breeding pair according to  claim 26 , wherein the expression-modulating element inhibits transcription of the disruptor nucleotide sequence under a first condition and disruption of the expression-modulating element permits or enhances transcription of the disruptor nucleotide sequence under a second condition. 
     
     
         28 . A breeding pair according to  claim 26  or  claim 27 , wherein the expression-modulating element comprises an inhibitor nucleotide sequence (e.g., a transcription terminator) that inhibits expression of the disruptor nucleotide sequence and that is operably linked to recombinase recognition sites, wherein the recombinase recognition sites mediate disruption of the inhibitor nucleotide sequence in the presence of a recombinase, and wherein the first breeding partner comprises an activator transgene that comprises a coding sequence for the recombinase, operably connected to a promoter. 
     
     
         29 . A breeding pair according to any one of  claims 21  to  28 , wherein the first breeding partner is female and the second breeding partner is male. 
     
     
         30 . A breeding pair according to  claim 28  or  claim 29 , wherein the first breeding partner is homozygous for the activator transgene. 
     
     
         31 . A breeding pair according to any one of  claims 21  to  30 , wherein the second breeding partner is homozygous for the disruptor transgene. 
     
     
         32 . A breeding pair of non-human animals, comprising: (1) a first breeding partner comprising a disruptable transgene that comprises a disruptable fertility gene that is operably linked to a promoter and to recombinase recognition sites that mediate disruption of the fertility gene in the presence of a recombinase; and (2) a second breeding partner comprising a disruptor transgene that comprises a disruptor nucleotide sequence encoding a fertility gene disruptor molecule, which is operably connected to a promoter, wherein the fertility gene disruptor molecule comprises the recombinase. 
     
     
         33 . A breeding pair according to  claim 32 , wherein the first breeding partner is female and the second breeding partner is male. 
     
     
         34 . A breeding pair according to  claim 32  or  claim 33 , wherein the first breeding partner is homozygous for the disruptable transgene and/or the second breeding partner is homozygous for the disruptor transgene. 
     
     
         35 . A breeding pair of non-human animals, comprising: (1) a first breeding partner comprising a first disruptable transgene that comprises a fertility gene operably connected to a promoter and to first recombinase recognition sites, which mediate disruption of the disruptable fertility gene in the presence of a first recombinase, and a first disruptor transgene that comprises a coding sequence for a second recombinase operably linked to a promoter, wherein the second recombinase specifically recognizes second recombinase recognition sites, and (2) a second breeding partner comprising a second disruptable transgene that comprises a fertility gene operably connected to a promoter and to second recombinase recognition sites, which mediate disruption of the disruptable fertility gene in the presence of the second recombinase, and a second disruptor transgene that comprises a coding sequence for the first recombinase operably linked to a promoter, wherein the first recombinase specifically recognizes the first recombinase recognition sites. 
     
     
         36 . A breeding pair according to  claim 35 , wherein the first breeding partner is homozygous for the first disruptable and first disruptor transgenes and/or the second breeding partner is homozygous for the second disruptable and second disruptor transgenes. 
     
     
         37 . A breeding pair of non-human animals, comprising: (1) a first breeding partner comprising a disruptable fertility gene operably connected to a promoter and an activator transgene that comprises a coding sequence for a recombinase operably linked to a promoter; and (2) a second breeding partner comprising a disruptor transgene that comprises a disruptor nucleotide sequence encoding an inhibitory RNA molecule (e.g., antisense RNA, siRNA, shRNA, etc.) that inhibits expression of the disruptable fertility gene, wherein the disruptor nucleotide sequence is operably linked to a promoter and to an expression-modulating element that comprises an inhibitor nucleotide sequence (e.g., a transcription terminator) that inhibits expression of the disruptor nucleotide sequence in the absence of the recombinase and that is operably linked to recombinase recognition sites that mediate disruption of the inhibitor nucleotide sequence in the presence of the recombinase. 
     
     
         38 . A breeding pair according to  claim 37 , wherein the first breeding partner is homozygous for the activator transgene and/or the second breeding partner is homozygous for the disruptor transgene. 
     
     
         39 . A breeding pair according to  claim 37  or  claim 38 , wherein the first breeding partner is female and the second breeding partner is male. 
     
     
         40 . A breeding pair of non-human animals, comprising: (1) a first breeding partner comprising a disruptable fertility gene operably connected to a promoter and an activator transgene that comprises a coding sequence for a recombinase operably linked to a promoter; and (2) a second breeding partner comprising a disruptor transgene that comprises a disruptor nucleotide sequence encoding an antibody that is immuno-interactive with a polypeptide product of the disruptable fertility gene, wherein the disruptor nucleotide sequence is operably linked to a promoter and to an expression-modulating element that comprises an inhibitor nucleotide sequence (e.g., a transcription terminator) that inhibits expression of the disruptor nucleotide sequence in the absence of a recombinase and that is operably linked to recombinase recognition sites that mediate disruption of the inhibitor nucleotide sequence in the presence of the recombinase. 
     
     
         41 . A breeding pair according to  claim 40 , wherein the first breeding partner is homozygous for the activator transgene and/or the second breeding partner is homozygous for the disruptor transgene. 
     
     
         42 . A breeding pair according to  claim 40  or  claim 41 , wherein the first breeding partner is female and the second breeding partner is male. 
     
     
         43 . A method for producing a non-human host embryo that comprises a disrupted fertility gene, the method comprising: (a) mating a first breeding partner of a breeding pair of non-human animals according to any one of  claims 21  to  42  with a second breeding partner of the breeding pair; and (b) producing a pre-implantation non-human embryo from a female member of the breeding pair, which embryo comprises in its germ line a disruption of the fertility gene. 
     
     
         44 . A method according to  claim 43 , further comprising culturing a non-human host embryo under conditions that allow formation of the pre-implantation non-human host embryo. 
     
     
         45 . A method according to  claim 43  or  claim 44 , wherein the first breeding partner is female and the second breeding partner is male. 
     
     
         46 . A method according to any one of  claims 43  to  45 , further comprising introducing a donor pluripotent cell as defined in any one of  claims 10  to  14  into the pre-implantation non-human embryo. 
     
     
         47 . A method according to  claim 46 , wherein the pluripotent cell comprises a genetic modification. 
     
     
         48 . A method according to  claim 46  or  claim 47 , wherein the pluripotent cell is a male pluripotent cell. 
     
     
         49 . A method of producing a chimeric non-human animal, the method comprising (1) transplanting a pre-implantation non-human embryo that comprises in its germ line a disruption of a fertility gene (e.g., as defined in any one of  claims 1  to  9 ) and that further comprises a donor pluripotent cell (e.g., as defined in any one of  claims 10  to  14 ); and (2) gestating the non-human host embryo of (1) under conditions suitable for development of the embryo, thereby generating a chimeric non-human animal with a disrupted fertility gene and the genetic modification in its germ line. 
     
     
         50 . A method according to  claim 49 , wherein the chimeric non-human animal is a male chimeric non-human animal. 
     
     
         51 . A method of producing a non-human animal that comprises a genetic modification in its genome, the method comprising breeding a male chimeric non-human animal produced according to the method of  claim 50  with a cognate female non-human animal that comprises in its genome the fertility gene lacking a disruption therein, to produce a non-human animal that comprises the genetic modification when the male chimeric non-human animal comprises germ cells or gametes derived from the donor pluripotent cell. 
     
     
         52 . A non-human animal (e.g., surrogate or foster non-human animal) having transplanted therein a preimplantation non-human host embryo according to any one of  claims 1  to  14 . 
     
     
         53 . A chimeric non-human animal that is derived from a pre-implantation non-human host embryo according to any one of  claims 1  to  14 . 
     
     
         54 . A non-human animal produced according to the method of  claim 51 .