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Generation of B6‐Ddx4em1(CreERT2)Utr,a novel CreERT2 knock‐in line,for germ cell lineage by CRISPR/Cas9
Authors:Hoai Thu Le  Yoshikazu Hasegawa  Yoko Daitoku  Kanako Kato  Saori Miznuo‐Iijima  Tra Thi Huong Dinh  Yumeno Kuba  Yuki Osawa  Natsuki Mikami  Kento Morimoto  Shinya Ayabe  Yoko Tanimoto  Kazuya Murata  Ken‐ichi Yagami  Satoru Takahashi  Seiya Mizuno  Fumihiro Sugiyama
Abstract:Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock‐out mouse studies. In this study, we established a novel knock‐in mouse line, B6‐Ddx4 em1(CreERT2)Utr, which expresses CreERT2 recombinase under the control of the endogenous DEAD‐box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen‐treated B6‐Ddx4 em1(CreERT2)Utr::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6‐Ddx4 em1(CreERT2)Utr is beneficial for studying female and male germ cell development.
Keywords:Cre/loxP     Ddx4  genome editing  oogenesis  spermatogenesis
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