G were produced at the expected rate and appeared to be grossly normal. Coat color was agouti or less frequently black. As PH males grew towards sexual maturity it became obvious that their testes were of reduced size (,12 volume of wild type), suggesting an absence of germ cell colonization; see Figure 1 panels A and B. Upon examination of mature F1 males vasa deferentia and epididymides, no sperm were observed (n = 5). Histological examination of testis confirmed the absence of sperm production and of detectable spermatogonial stem cells (SSC); see Figure 2 panels A and B. As expected, these males did not produce any offspring when mated (n = 5). These data demonstrate that this combination of strains leads to F1 males devoid of competing germ cells. F1 PH females produced 24195657 by this same cross displayed nearly complete infertility, with only vestigial ovaries and associated fat pad remaining (data not shown). However, during the course of these experiments we observed 2 of 99 PH mated females that did produce three litters of three to five offspring. These proved by SNP genotyping to be maternal host gamete derived. These data suggest that there are rare sporadic failures of cre-driven STOP excision in female PH mice which can lead to low level of host germ cell colonization and occasional “leakage”. No such failures have been observed in males (.200 PH males mated) and all further studies used only male PH animals. Title Loaded From File Attempts to use the reciprocal cross, i.e. Vasa-Cre females6R26RDTA males resulted in no offspring. Previous studies suggested that Cre protein is present in the oocyte of Vasa-Cre females and this would mediate a recombination event shortly after fertilization resulting in 24195657 by this same cross displayed nearly complete infertility, with only vestigial ovaries and associated fat pad remaining (data not shown). However, during the course of these experiments we observed 2 of 99 PH mated females that did produce three litters of three to five offspring. These proved by SNP genotyping to be maternal host gamete derived. These data suggest that there are rare sporadic failures of cre-driven STOP excision in female PH mice which can lead to low level of host germ cell colonization and occasional “leakage”. No such failures have been observed in males (.200 PH males mated) and all further studies used only male PH animals. Attempts to use the reciprocal cross, i.e. Vasa-Cre females6R26RDTA males resulted in no offspring. Previous studies suggested that Cre protein is present in the oocyte of Vasa-Cre females and this would mediate a recombination event shortly after fertilization resulting in 1315463 lethal expression of DTA [16].Conventional Host vs PH, Comparative Germline Transmission of Genetically Modified ESCsTo determine if the PH approach improved the rate and efficiency of germline transmission from genetically modified ESCs over that of conventional hosts, we conducted comparative microinjection tests. Eleven different C57BL/6N-derived genetically modified ESC lines were obtained from the International Knockout Mouse Consortium (IKMC) (see Table 2). For the evaluation of germline transmission from chimeras using conven-Figure 1. Dissected Testis. Testis were dissected from 8?2 week old sexually mature males; A) normal wild type C57Bl/6J mice, B) PH testis, where germ cells ablated, C) PH testis colonized (partially) by 129 F1 ESC line R1 derived germ cells. Scale bar equals 10 mm. doi:10.1371/journal.pone.0067826.gImproved Germ Line of Embryonic Stem CellsFigure 2. Sections of testis from wild type and F1 animals. Sections of testis at 56and 206, scale bar 100 micrometer: A+B) wild type C57Bl/ 6J testis, shows normal colonization of the testis seminiferous tubules with characteristic spermatogonia, spermatocytes, round spermatids and elongating spermatids; C+D) PH male, non colonized testis, these animals were sterile having no sperm in the vasa deferentia or epididymis, the seminiferous tubules are almost exclusively filled with Sertoli cells and are apparently devoid of sperm and earlier germ cell progenitors; E+F) PH male, partially colonized with differentiated derivatives of Balb/cJ derived ESC line PB150.18, shows partial colonization of the seminiferous tubules, this animal was fertile however, this phenotype was at times associated with reduced fertility (data not shown); G+H) PH male, well colonized testis with differentiated der.