Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss
Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss
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虹鳟 Oncorhynchus mykiss 生殖细胞特异性切除 loxP 侧翼转基因
DOI：
10.1095/biolreprod.115.136929
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发表时间：
2016
影响因子：
3.6
通讯作者：
G.
中科院分区：
文献类型：
--
作者：
Katayama;N.;Kume;S.;Hattori-Ihara;S.;Sadaie;S.;Hayashi;M.;Yoshizaki;G.
Cre/loxP-介导的DNA切除生殖细胞谱系可能会大大有助于研究鲑科动物的生殖细胞生物学，这是新兴的模式物种在这一领域。然而，细胞类型特异性Cre/loxP系统尚未成功开发用于任何鲑科物种。因此，我们研究了Cre/loxP介导的生殖细胞特异性基因切除和转基因激活虹鳟鱼的可行性。双转基因（wTg）后代是通过将携带cre的转基因雄性与携带hsc-LRLG基因的转基因雌性交配获得的; cre由虹鳟鱼血管调节区驱动，hsc-LRLG基因由虹鳟鱼休克同源物71启动子、两侧有两个oxP位点的DsRed基因和Egfp基因组成。PCR分析、荧光成像和组织学分析表明，只有在wTg鱼的生殖细胞中才发生了loxP侧翼序列的切除和Egf的激活。但后代检测显示，loxP侧翼序列在生殖细胞中的切除效率较低（≤3.27%）。相反，来自两个不同cre转基因雄性的wTg鱼经常在生殖细胞中切除loxP侧翼序列（≤89.25%）。因此，我们第一次展示了通过Cre/loxP系统在虹鳟鱼中成功的生殖细胞特异性转基因操作。我们预计，这项技术将适用于研究细胞功能，通过细胞靶向，细胞谱系追踪，并产生细胞类型特异性的条件基因敲除和单独开发不育虹鳟鱼在水产养殖。
Cre/loxP-mediated DNA excision in germ cell lineages could contribute substantially to the study of germ cell biology in salmonids, which are emerging as a model species in this field. However, a cell type-specific Cre/loxPsystem has not been successfully developed for any salmonid species. Therefore, we examined the feasibility of Cre/loxP-mediated, germ cell-specific gene excision and transgene activation in rainbow trout. Double-transgenic (wTg) progeny were obtained by mating a transgenic male carryingcrewith a transgenic female carrying thehsc-LRLGgene;crewas driven by rainbow troutvasaregulatory regions and thehsc-LRLGgene was made up of the rainbow troutheat-shock-cognate71promoter, theDsRedgene flanked by twoloxPsites, and theEgfpgene. PCR analysis, fluorescence imaging, and histological analysis revealed that excision of theloxP-flanked sequence and activation ofEgfpoccurred only in germ cells of wTg fish. However, progeny tests revealed that the excision efficiency ofloxP-flanked sequence in germ cells was low (≤3.27%). In contrast, the other wTg fish derived from two differentcre-transgenic males frequently excised theloxP-flanked sequence in germ cells (≤89.25%). Thus, we showed for the first time successful germ cell-specific transgene manipulation via the Cre/loxPsystem in rainbow trout. We anticipate that this technology will be suitable for studies of cell function through cell targeting, cell-linage tracing, and generating cell type-specific conditional gene knockouts and separately for developing sterile rainbow trout in aquaculture.