Project description:Pluripotent stem cells (PSCs) provide a powerful tool to produce transgenic animals for biomedical research. However, impaired PSC contribution to chimerism and most notably the germline oftentimes impedes production of genetically modified animals, rendering techniques that expediate PSC contribution to the germline highly desirable. Blastocyst complementation denotes a technique which purposes to generate organs, tissues or cells in animal chimeras via microinjection of PSCs into genetically compromised blastocyst-stage embryos. Here we report on successful blastocyst complementation of the male germline in adult chimeras following microinjection of mouse or rat PSCs into mouse blastocysts mutated for Tsc22d3, an essential gene for spermatozoa production. Microinjection of mouse embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into Tsc22d3-KnockOut (KO) blastocyst-stage embryos gave rise to intraspecies chimeras embodying functional spermatozoa, which were solely derived from microinjected PSCs. Furthermore, microinjection of rat ESCs into Tsc22d3-KO mouse embryos gave rise to viable mouse-rat chimeras that exhibited extensive contribution of rat cells to various organs. Notably, multiple mouse-rat chimeras showed contribution of rat ESCs to the male germline, solely harboring rat spermatids and spermatozoa that were rat ESC-derived and could fertilize rat oocytes. Collectively, this study reports a method for exclusive production of functional germ cells of one species in another via blastocyst complementation with PSCs. Implications of this study extend to development of transgenic rats via sterile mice, and may further assist efforts to generate xenogeneic gametes from endangered animal species.
Project description:Pluripotent stem cells (PSCs) provide a powerful tool to produce transgenic animals for biomedical research. However, impaired PSC contribution to chimerism and most notably the germline oftentimes impedes production of genetically modified animals, rendering techniques that expediate PSC contribution to the germline highly desirable. Blastocyst complementation denotes a technique which purposes to generate organs, tissues or cells in animal chimeras via microinjection of PSCs into genetically compromised blastocyst-stage embryos. Here we report on successful blastocyst complementation of the male germline in adult chimeras following microinjection of mouse or rat PSCs into mouse blastocysts mutated for Tsc22d3, an essential gene for spermatozoa production. Microinjection of mouse embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into Tsc22d3-KnockOut (KO) blastocyst-stage embryos gave rise to intraspecies chimeras embodying functional spermatozoa, which were solely derived from microinjected PSCs. Furthermore, microinjection of rat ESCs into Tsc22d3-KO mouse embryos gave rise to viable mouse-rat chimeras that exhibited extensive contribution of rat cells to various organs. Notably, multiple mouse-rat chimeras showed contribution of rat ESCs to the male germline, solely harboring rat spermatids and spermatozoa that were rat ESC-derived and could fertilize rat oocytes. Collectively, this study reports a method for exclusive production of functional germ cells of one species in another via blastocyst complementation with PSCs. Implications of this study extend to development of transgenic rats via sterile mice, and may further assist efforts to generate xenogeneic gametes from endangered animal species.
Project description:Pluripotent stem cells (PSCs) provide a powerful tool to produce transgenic animals for biomedical research. However, impaired PSC contribution to chimerism and most notably to the germline oftentimes impedes production of genetically modified animals, rendering techniques that expediate PSC contribution to the germline highly desirable. Blastocyst complementation denotes a technique which purposes to generate organs, tissues or cells in animal chimeras following microinjection of PSCs into genetically compromised blastocyst-stage embryos. Here, we report on successful blastocyst complementation of the male germline in adult chimeras via microinjection of mouse or rat PSCs into mouse blastocysts mutated for Tsc22d3, an essential gene for spermatozoa production. Microinjection of mouse embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into Tsc22d3-KnockOut (KO) blastocyst-stage embryos gave rise to intraspecies chimeras embodying functional spermatozoa, which were solely derived from the microinjected PSCs. Furthermore, microinjection of rat ESCs into Tsc22d3-KO mouse embryos gave rise to viable mouse-rat chimeras that exhibited extensive contribution of rat cells to various tissues and organs. Notably, multiple mouse-rat chimeras showed contribution of rat ESCs to the male germline, solely harboring rat spermatids and spermatozoa that were rat ESC-derived and could fertilize rat oocytes. Collectively, this study reports on a method for exclusive germ cell production of one species in another via blastocyst complementation with PSCs. Implications of this study may extend to development of transgenic rat gametes in sterile mice and could further assist efforts to generate germ cells from endangered animal species.
Project description:Pluripotent stem cells (PSCs) provide a powerful tool to produce transgenic animals for biomedical research. However, impaired PSC contribution to chimerism and most notably the germline oftentimes impedes production of genetically modified animals, rendering techniques that expediate PSC contribution to the germline highly desirable. Blastocyst complementation denotes a technique which purposes to generate organs, tissues or cells in animal chimeras via microinjection of PSCs into genetically compromised blastocyst-stage embryos. Here we report on successful blastocyst complementation of the male germline in adult chimeras following microinjection of mouse or rat PSCs into mouse blastocysts mutated for Tsc22d3, an essential gene for spermatozoa production. Microinjection of mouse embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into Tsc22d3-KnockOut (KO) blastocyst-stage embryos gave rise to intraspecies chimeras embodying functional spermatozoa, which were solely derived from microinjected PSCs. Furthermore, microinjection of rat ESCs into Tsc22d3-KO mouse embryos gave rise to viable mouse-rat chimeras that exhibited extensive contribution of rat cells to various organs. Notably, multiple mouse-rat chimeras showed contribution of rat ESCs to the male germline, solely harboring rat spermatids and spermatozoa that were rat ESC-derived and could fertilize rat oocytes. Collectively, this study reports a method for exclusive production of functional germ cells of one species in another via blastocyst complementation with PSCs. Implications of this study extend to development of transgenic rats via sterile mice, and may further assist efforts to generate xenogeneic gametes from endangered animal species.
Project description:Generation of functional rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells [scRNAseq_rat]
Project description:Generation of functional rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells [scRNAseq_mouse]
Project description:Generation of functional rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells [bulk RNA-seq]
Project description:Blastocyst complementation denotes a technique that aims to generate organs, tissues, or cell types in animal chimeras via injection of pluripotent stem cells (PSCs) into genetically compromised blastocyst-stage embryos. Here, we report on successful complementation of the male germline in adult chimeras following injection of mouse or rat PSCs into mouse blastocysts carrying a mutation in Tsc22d3, an essential gene for spermatozoa production. Injection of mouse PSCs into Tsc22d3-Knockout (KO) blastocysts gave rise to intraspecies chimeras exclusively embodying PSC-derived functional spermatozoa. In addition, injection of rat embryonic stem cells (rESCs) into Tsc22d3-KO embryos produced interspecies mouse-rat chimeras solely harboring rat spermatids and spermatozoa capable of fertilizing oocytes. Furthermore, using single-cell RNA sequencing, we deconstructed rat spermatogenesis occurring in a mouse-rat chimera testis. Collectively, this study details a method for exclusive xenogeneic germ cell production in vivo, with implications that may extend to rat transgenesis, or endangered animal species conservation efforts.
Project description:We report the application of single cell RNAseq analysis of bone marrow cells from mouse-mouse intraspecies blastocyst complementation and mouse-rat interspecies blastocyst complementation.