利用CRISPR-Cas9构建syncytinA条件敲除小鼠

Establishment of SyncytinA Conditional Knockout Mouse by CRISPR-Cas9

目的构建syncytinA(合胞素A)条件敲除小鼠,为进一步研究syncytinA在胎盘形成过程中发挥的融合及非融合作用及研究子痫前期病理模型提供基础。方法在用ES细胞打靶完成syncytinA外显子上游loxp同源重组基础上,利用CRISPR-Cas9得到syncytinA-loxp小鼠。构建syncytinA-loxp转基因载体及sg RNA,通过原核显微注射方法将构建好的doner、Cas9及sg RNA一并注射到C57小鼠受精卵中,并移植入同期受孕代孕受体ICR输卵管中获得子代小鼠。用PCR方法检测子代鼠尾基因型,loxp阳性小鼠与WT交配获得syncytinA-loxp小鼠。为了检测syncytinA-loxp能否被敲除,通过与prime1cre及zp3cre交配获得syncytinA-/-,PCR及q-PCR检测syncytinA是否被敲掉。结果经PCR及q-PCR方法检测,我们成功得到syncytinA-/-胚胎。结论 syncytinA条件敲除小鼠构建成功,为更好的研究它在胎盘及子痫前期中的功能提供了基础。

Objective To establish syncytinA conditional knock out mice to investigate the function of syncytinA in the placental development and preeclampsia pathology. Method After successful incorporation of loxp synA exon by homologous recombination in ES cells,we used CRISPR-Cas9 technology to construct the syncytinA-loxp mice.We construct syncytinA-loxp transgenic vector and sg RNA,they were injected with Cas9 into fertilized eggs of C57 by prokaryotic microinjection methods,and these eggs were transplanted into pseudopregnant mice. The genotype of transgenic mice was identified by PCR. Then loxp positive mice were mated with WT( wild type) to get syncytinAloxp mice. In order to detect whether syncytinA knockout. Syncytin A-/-was obtained by mating with prime1 cre and zp3 cre. Syncytin A-/-was identified by PCR and q-PCR. Conclusion Syncytin A conditions knockout mouse was successfully constructed in present study.