关键词: BMP15基因, CRISPR/Cas9, RGS双荧光替代性报告载体

Abstract:

As Chinese have raised most pigs and consumed most pig products in the world, improving the fertility of sow is of economic benefits to the pig industry in China. The sheep BMP15 (bone morphogenetic protein 15) gene has been identified as a major gene for controlling ovulation rates and prolific traits, which are key factors affecting the fertility of livestock. As similar natural occurring mutations in the porcine BMP15 gene have not yet been reported, we speculated that introducing the same prolific sheep mutations into the porcine BMP15 gene by using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system could be promising for the generation of pig breeds with improved fertility. In this study, we described the targeted disruption of the BMP15 gene in the porcine embryonic fibroblasts (PEFs) of Landrace pig. The results showed that only 5% cells in the population were targeted by CRISPR/Cas9 as revealed by the T7E1 assay. Then we co-transfected a RFP-GFP surrogate (RGS) reporter with the CRISPR/Cas9 expression vector into PEFs, followed by sorting the cells with dual fluorescent signals through fluorescence-activated cell sorter (FACS). In the sorted cell population, the percentage of cells with gene targeted modification by CRISPR/Cas9 was improved to 18%. Our study proved that the RGS surrogate reporter system is useful for improving gene targeting efficiency on BMP15 in PEF by CRISPR/Cas9. Our work thus builds a useful basis for the future generation of BMP15 gene-edited pigs through the somatic cell nuclear transfer technique and further investigations on the regulatory functions of BMP15 in porcine reproductive traits.

Key words: BMP15, CRISPR/Cas9, RGS surrogate reporter system