Construction and cell transfection of the targeting vectors of Jiv90 genes in swine | 猪Jiv90基因打靶载体的构建与细胞转染试验

Chinese. 构建猪Jiv90基因的同源打靶载体，并转染猪血管内皮细胞系(SUVEC)，对Jiv90基因的敲除效果进行初步验证。根据NCBI中公布的Jiv90基因序列，克隆得到Jiv90基因核心区两侧的基因序列，并以此为长短臂，构建了可定点敲除Jiv90基因的打靶载体。用该载体转染SUVEC，通过新霉素(G418)和丙氧鸟苷(GANC)的筛选得到稳定的细胞株，再利用PCR检测neo基因在细胞基因组上的整合情况，最后对筛选得到的neo阳性细胞进行猪瘟病毒(CSFV)感染试验，并用实时定量PCR(Real-time PCR)检测CSFV的增殖情况。以提取所得品质良好的SUVEC全基因组DNA为模板，克隆得到了针对Jiv90基因打靶的核心区两侧基因序列，基因片段长度分别为4 522 bp和1 944 bp。将长短臂与打靶骨架载体pA2T相连后，构建了关于猪Jiv90基因的同源打靶载体JKS。用JKS载体转染SUVEC，通过1 500 μg/mL G418和200 nmol/mL GANC正负筛选，得到了稳定转染JKS载体的SUVEC。对于稳定筛选后的试验组细胞，感染CSFV并进行Real-time PCR，结果表明，正常细胞中的CSFV核酸含量是转染JKS载体细胞的3.65倍。成功扩增了针对猪Jiv90基因的同源长短臂，并构建了猪Jiv90基因的打靶载体JKS，敲除Jiv90基因细胞中的CSFV核酸片段含量显著降低，说明Jiv90基因的敲除可降低细胞中猪瘟病毒的增殖量。

English. This research constructed the targeting vectors of Jiv90 genes and to have a preliminary verification of these vectors at the cellular level by transfecting them into the swine umbilical vein endothelial cells (SUVEC). Based on the published sequences of Jiv90 genes in GenBank, the sequences of both outsides of the core area of Jiv90 genes were cloned. These sequences were used as long and short arms for construction of targeting vectors to knock out the Jiv90 genes in SUVEC. The targeting vectors were used to transfect SUVEC and the knocked out SUVEC line was obtained by drugs screening of both G418 and GANC. Then the knocked out SUVEC line was tested through PCR to make sure that the neo genes were integrated. Furthermore, the SUVEC line with neo genes integrated was inoculated with the classical swine fever virus (CSFV) and measured with real-time PCR to testify their susceptibility with CSFV. The sequences of both outsides of the core area of Jiv90 genes were cloned from the extracting DNA of SUVEC with the use of long and short homologous arms in targeting vectors. In addition, the genetic fragments of the long and short arms were 4 522 bp and 1 944 bp respectively. The targeting vectors named JKS were successfully constructed by the skeleton vectors pA2T connected with both long and short homologous arms. With transfecting reagents, the JKS vectors were transfected into SUVEC. After drugs screening with both G418 and GANC, the stable cell lines were obtained. In addition, the screening concentration of G418 was 1 500 μg/mL and GANC 200 nmol/mL. These cells were tested through PCR to make sure that the neo genes were integrated. Then the cells were inoculated with the CSFV and measured with real-time PCR to testify their susceptibility with CSFV. The results indicated that the CSFV content of the normal cells was 3.65 times that of the SUVEC lines obtained by drug screening. The homologous arms were successfully cloned and the targeting vectors named JKS were successfully constructed. After transfecting SUVEC, the neo genes were integrated and real-time PCR tests showed that knocking out of the Jiv90 genes could affect the SUVEC when infected with CSFV.