Rapid Production of Gene-editing Pigs by Identifying Minimum Numbers of Positive Fibroblasts for SCNT

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.