通过微量细胞鉴定快速生产基因编辑猪

目的建立快速筛选猪的基因编辑阳性成纤维细胞系的方法以提高利用体细胞核移植产生基因编辑克隆猪的效率。方法通过PCR、T7EN1酶切和Sanger测序分析不同数量的已知猪基因编辑阳性细胞(20、50、100、150和200个细胞)的基因型，确定最小细胞数用于鉴定未知的基因编辑阳性细胞系以证明该方法的可行性。结果除20细胞组外，其他所有细胞组中均扩增出2条明显的T7EN1酶切条带(175和555 bp)。定量数据显示：20细胞组和50细胞组之间的条带灰度值存在极显著差异(P<0.01)，因此50细胞可用于基因编辑阳性细胞系鉴定。利用CRISPR/Cas9系统构建IPO13打靶载体，转染猪胎儿成纤维细胞形成细胞克隆点后计数50个细胞来筛选阳性细胞系，再通过体细胞核移植快速生产了IPO13敲除猪，从而证实通过微量细胞鉴定快速生产基因编辑猪的可行性。筛选基因编辑阳性细胞系的一般方法大约需要33.7 d，新建立的方法细胞筛选周期仅为18.9 d (减少15 d)，获得阳性细胞系的成功率也提高了约4倍。结论本研究建立了一种通过微量细胞鉴定快速生产基因编辑克隆猪的方法，该方法可行可靠。

Viable offspring derived from fetal and adult mammalian cells

PurposeTo establish a rapid selection method of porcine gene-editing (GE) positive fibroblast lines to improve the efficiency of gene-editing cloned (GEC) pig production by somatic cell nuclear transfer (SCNT).MethodDifferent numbers of known porcine GE positive cells (20, 50, 100, 150 and 200 cells) were analyzed for genotyping by PCR, T7EN1 digestion and sequencing to determine the minimum cell number. The minimal cell number was used for the identification of unknown GE cell lines, thereby to validate the feasibility of this method.ResultPCR products harboring the targeted region were amplified from all number of cells, but the distinct cleavage bands by T7EN1 (175 and 555 bp) were not observed in the 20 cells group. Moreover, band densities had significant difference between 20 and 50 cell groups (P<0.01), thus the 50-cell group could be used for genotyping to obtain GE positive cells. Then, we designed the IPO13 targeting vectors by using CRSIPR/Cas9 system. Fifty cells were counted to identify the positive cell lines after transfection and formation of cell colonies. Subsequently, we rapidly produced the IPO13 knockout pigs by SCNT, thus verified the feasibility of rapid production of gene-editing pigs by identifying minimum numbers of positive fibroblasts for SCNT. The general method of screening GE positive cell lines needs approximately 33.7 days. However, the selecting cycle of newly established method was only 18.9 days (reduced by 15 days) and the success rate of obtaining positive cell lines was also increased by four times.ConclusionThis study established a method of rapid production of gene editing cloned pigs by identifying minimum numbers of positive fibroblasts, which is feasible and reliable.