Transgene manipulation in rainbow trout using Cre recombinase
Transgene manipulation in rainbow trout using Cre recombinase
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DOI：
10.1007/s12562-014-0742-x
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发表时间：
2014-07-01
影响因子：
1.9
通讯作者：
Yoshizaki, Goro
中科院分区：
文献类型：
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作者：
Kume, Sachi;Katayama, Naoto;Yoshizaki, Goro
Cre/loxP介导的细胞靶向被认为是养殖鱼类生物技术的有力工具。作为在鲑科动物中建立细胞靶向的第一步，我们分析了虹鳟鱼中Cre/loxP系统的功能。我们首先建立了稳定的转基因菌株携带DsRed基因，这是侧翼的loxP位点，并进一步剪接与EGFP基因。通过显微注射Cre互补RNA（cRNA）到转基因鳟鱼的受精卵中，评估Cre/loxP系统的功能。结果表明，所有的胚胎至少在其部分细胞中显示出绿色荧光。虽然20个胚胎中有19个包含显示绿色和红色荧光的细胞，但剩余的胚胎仅显示绿色荧光。使用设计用于识别两个loxP位点之外的序列的引物的聚合酶链反应（PCR）显示，除了长的完整片段之外，19个个体携带长度与loxP切除片段相等的短片段。剩下的绿色胚胎只携带这一小段。对短片段的DNA测序显示，它缺乏loxP位点侧翼的DNA片段，并且剪接片段不包含任何序列重排。这些结果表明Cre/loxP系统在虹鳟鱼中是功能性的。
Cre/loxP-mediated cell targeting is considered to be a powerful tool for biotechnology in farmed fish. As a first step toward establishing cell targeting in salmonids, we analyzed the functionality of the Cre/loxP system in rainbow trout. We first established stable transgenic strains carrying the DsRed gene, which was flanked by loxP sites and further spliced with the EGFP gene. By microinjecting Cre complementary RNA (cRNA) into fertilized eggs of the transgenic trout, the functionality of the Cre/loxP system was evaluated. The results showed that all of the embryos exhibited green fluorescence in at least some of their cells. While 19 out of 20 embryos comprised cells showing both green and red fluorescence, the remaining embryo showed only green fluorescence. Polymerase chain reaction (PCR) using primers designed to recognize sequences outside of the two loxP sites revealed that, in addition to long intact fragments, the 19 individuals carried short fragments that were equivalent in length to the loxP-excised fragments. The remaining green embryo carried only this short fragment. DNA sequencing of the short fragment revealed that it lacked the DNA fragments flanking the loxP sites and the spliced fragments did not contain any sequence rearrangements. These results suggest that the Cre/loxP system is functional in rainbow trout.