Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.
Graduate Program in Development, Disease Models & Therapeutics, Baylor College of Medicine, Houston, TX, USA.
Biol Reprod. 2023 Aug 10;109(2):184-191. doi: 10.1093/biolre/ioad063.
The development of oocytes occurs over a broad time frame, starting at the earliest stages of embryogenesis and continuing into adulthood. Conditional knockout technologies such as the Cre/loxP recombination system are useful for analyzing oocyte development at specific stages, but not every time frame has appropriate Cre drivers, for instance, during oocyte meiotic initiation through early prophase I in the embryo. Here, we generated a novel knockin mouse line that produces a bicistronic transcript from the endogenous Stra8 locus that includes a "self-cleaving" 2A peptide upstream of cre. This allows for high efficiency cleavage and production of both proteins individually and results in expression of cre in both male and female gonads at the biologically relevant stage. Fluorescent reporter analysis confirms that this line recapitulates endogenous Stra8 expression in both sexes and does not affect fertility of heterozygous nor homozygous mice. This line, named Stra8P2Acre, adds to the repertoire of germ-cell specific cre driver lines and, importantly, allows for deletion of target genes during key embryonic oocyte developmental stages, including early events in meiosis. Summary Sentence Generation of a novel cre recombinase knockin to the Stra8 locus allows production of Stra8 and cre without affecting fertility.
卵母细胞的发育经历了一个广泛的时间框架,从胚胎发生的最早阶段开始,并持续到成年期。条件性敲除技术,如 Cre/loxP 重组系统,可用于分析特定阶段的卵母细胞发育,但并非每个时间框架都有合适的 Cre 驱动子,例如在胚胎中卵母细胞减数分裂起始到早期细线期。在这里,我们生成了一种新型的 knockin 小鼠品系,该品系从内源性 Stra8 基因座产生一个双顺反子转录本,其中包括 Cre 上游的“自我切割” 2A 肽。这允许高效切割和单独产生两种蛋白质,并导致 cre 在雄性和雌性性腺中以生物学上相关的阶段表达。荧光报告分析证实,该品系在两性中均能重现内源性 Stra8 的表达,且不影响杂合子和纯合子小鼠的生育能力。该品系命名为 Stra8P2Acre,增加了生殖细胞特异性 cre 驱动子系的范围,重要的是,允许在关键的胚胎卵母细胞发育阶段(包括减数分裂的早期事件)删除靶基因。总结句子 生成一种新型的 Cre 重组酶 knockin 到 Stra8 基因座,可在不影响生育能力的情况下产生 Stra8 和 Cre。