Lop to PGCs that exclusively colonize the genital ridge. Conventional mating

July 13, 2017

Lop to PGCs that exclusively colonize the genital ridge. Conventional mating combined with IVF of PH chimeric males produced nearly 200 offspring all of which were paternally derived from the introduced stem cells, with no offspring derived from the PH recipient detected. Our data demonstrate: i) the PH strategy is effective for a number of distinct ESC genetic backgrounds, and ii) that production of hostderived offspring by PH males is absent and/or not significant. Further and importantly for production considerations, we also show that sperm of PH chimeras can be cryopreserved and used subsequently in IVF. These results validate the PH strategy as an effective mouse management tool, facilitating the rapid expansion of ESC-derived germline without interference from competing host-derived gametes. Testes of fertile PH sexually mature males often showed a continuum of ESC derived SSC colonization of the seminiferous tubules, ranging from apparent full colonization, partial colonization, to apparently empty seminiferous tubules (Figure 2). Although we did not fully explore the relationship between seminiferous tubules colonization, sperm count, sperm quality and fertility, it was apparent that in most cases even partial colonization of testes is sufficient to provide fertility. This is consistent with previous published work using busulfan SSC depleted mice and SSC recolonization. These studies also demonstrated a threshold effect in SSC colonization and resulting sperm Somatostatin-14 counts with apparently ,20 of normal sperm counts representing a threshold value conferring fertility [25]. This effect may also be linked to possible epididymal storage and accumulation of sperm over time, which may in part LY2409021 manufacturer compensate for reduced sperm production. Below this threshold there may not be enough healthy/viable sperm accumulated to confer fertility, however sperm isolation followed by IVF may overcome this. We also evaluated the effectiveness of PH versus conventional blastocysts in creation of new mouse models by comparing germline transmission of eleven IKMC C57BL/ 6N-derived ESC clones, with both conventional and PH blastocysts as microinjection hosts. The PH-derived chimerassuccessfully transmitted 9/11 of the ESC lines tested, while conventional hosts transmitted only 6/11. Further, when the best conventional host chimeras were bred, less than 35 of offspring in the first litters were derived from the ESC germline. In comparison, 100 of the offspring from the first litters of the PH-derived chimeras were confirmed to be ESCderived. With both approaches ,50 of germline transmitting animals carried the modified alleles. Together, these data reveal that the PH approach uses less total resources while providing higher efficiency and probability of germline transmission of ESC lines. Germline transmission of two of the IKMC ESC lines was obtained by using PH male chimeras as sperm donors for IVF. We believe that their fertility failure by natural mating was due to reduced sperm count due to poor colonization of the testis. The ability to isolate sperm from PH chimeras and cryopreserve it or use it directly in IVF provides further operational options. These include rescue of low level transmitting ESC (low sperm counts), as well as rapid offspring expansion directly from chimeras, significantly reducing time normally spent on breeding. This approach can also provide better logistical control of downstream operations and secures a cryopreserved stock. Addit.Lop to PGCs that exclusively colonize the genital ridge. Conventional mating combined with IVF of PH chimeric males produced nearly 200 offspring all of which were paternally derived from the introduced stem cells, with no offspring derived from the PH recipient detected. Our data demonstrate: i) the PH strategy is effective for a number of distinct ESC genetic backgrounds, and ii) that production of hostderived offspring by PH males is absent and/or not significant. Further and importantly for production considerations, we also show that sperm of PH chimeras can be cryopreserved and used subsequently in IVF. These results validate the PH strategy as an effective mouse management tool, facilitating the rapid expansion of ESC-derived germline without interference from competing host-derived gametes. Testes of fertile PH sexually mature males often showed a continuum of ESC derived SSC colonization of the seminiferous tubules, ranging from apparent full colonization, partial colonization, to apparently empty seminiferous tubules (Figure 2). Although we did not fully explore the relationship between seminiferous tubules colonization, sperm count, sperm quality and fertility, it was apparent that in most cases even partial colonization of testes is sufficient to provide fertility. This is consistent with previous published work using busulfan SSC depleted mice and SSC recolonization. These studies also demonstrated a threshold effect in SSC colonization and resulting sperm counts with apparently ,20 of normal sperm counts representing a threshold value conferring fertility [25]. This effect may also be linked to possible epididymal storage and accumulation of sperm over time, which may in part compensate for reduced sperm production. Below this threshold there may not be enough healthy/viable sperm accumulated to confer fertility, however sperm isolation followed by IVF may overcome this. We also evaluated the effectiveness of PH versus conventional blastocysts in creation of new mouse models by comparing germline transmission of eleven IKMC C57BL/ 6N-derived ESC clones, with both conventional and PH blastocysts as microinjection hosts. The PH-derived chimerassuccessfully transmitted 9/11 of the ESC lines tested, while conventional hosts transmitted only 6/11. Further, when the best conventional host chimeras were bred, less than 35 of offspring in the first litters were derived from the ESC germline. In comparison, 100 of the offspring from the first litters of the PH-derived chimeras were confirmed to be ESCderived. With both approaches ,50 of germline transmitting animals carried the modified alleles. Together, these data reveal that the PH approach uses less total resources while providing higher efficiency and probability of germline transmission of ESC lines. Germline transmission of two of the IKMC ESC lines was obtained by using PH male chimeras as sperm donors for IVF. We believe that their fertility failure by natural mating was due to reduced sperm count due to poor colonization of the testis. The ability to isolate sperm from PH chimeras and cryopreserve it or use it directly in IVF provides further operational options. These include rescue of low level transmitting ESC (low sperm counts), as well as rapid offspring expansion directly from chimeras, significantly reducing time normally spent on breeding. This approach can also provide better logistical control of downstream operations and secures a cryopreserved stock. Addit.