CAJ | 학술논문

IGF2(Insulin-like growth factor 2)基因作为最复杂多样的生长因子之一，对猪胎儿发育以及出生后生长发育和肌肉生成起着非常重要的作用。通过基因组编辑技术对我国本地猪种的 IGF2基因作精确的遗传修饰，对于提高本地猪种的瘦肉率具有重要的育种意义。文章在蓝塘猪胎儿成纤维细胞(Porcine fetal fibroblasts, PEF)中检测了锌指核酸酶(Zinc finger nucleases, ZFN)和CRISPR/Cas9对IGF2基因的打靶效率，结果表明CRISPR/Cas9对IGF2基因的切割效率最高可达9.2%，显著高于ZFN的切割效率(<1%)，但两者均未达到作为体细胞核移植(Somatic nuclear transfer, SCNT)供体细胞所需的打靶效率。应用SSA (Single-strand annealing)报告载体筛选技术来富集IGF2基因被ZFN和CRISPR/Cas9修饰过的PEF细胞，结果表明，该技术可使CRISPR/Cas9的打靶效率提高5倍左右，对ZFN的打靶效率具有更大的增强作用。
IGF2(Insulin-like growth factor 2)기인작위최복잡다양적생장인자지일，대저태인발육이급출생후생장발육화기육생성기착비상중요적작용。통과기인조편집기술대아국본지저충적 IGF2기인작정학적유전수식，대우제고본지저충적수육솔구유중요적육충의의。문장재람당저태인성섬유세포(Porcine fetal fibroblasts, PEF)중검측료자지핵산매(Zinc finger nucleases, ZFN)화CRISPR/Cas9대IGF2기인적타파효솔，결과표명CRISPR/Cas9대IGF2기인적절할효솔최고가체9.2%，현저고우ZFN적절할효솔(<1%)，단량자균미체도작위체세포핵이식(Somatic nuclear transfer, SCNT)공체세포소수적타파효솔。응용SSA (Single-strand annealing)보고재체사선기술래부집IGF2기인피ZFN화CRISPR/Cas9수식과적PEF세포，결과표명，해기술가사CRISPR/Cas9적타파효솔제고5배좌우，대ZFN적타파효솔구유경대적증강작용。
IGF2 (Insulin-like growth factor 2) is a major growth factor affecting porcine fetal and postnatal de-velopment. We propose that the precise modification of IGF2 gene of Chinese indigenous pig breed——Lantang pig by genome editing technology could reduce its backfat thickness, and increase its lean meat content. Here, we tested the genome editing activities of zinc finger nucleases (ZFNs) and CRISPR/Cas9 system on IGF2 gene in the Lantang porcine fetal fibroblasts (PEF). The results indicated that CRISPR/Cas9 presented cutting efficiency up to 9.2%, which was significantly higher than that generated by ZFNs with DNA cutting efficiency lower than 1%. However, even by using CRISPR/Cas9, the relatively lower percentage of genetically modified cells in the transfected popula-tion was not satisfied for somatic nuclear transfer (SCNT). Therefore, we used a SSA (Single-strand annealing) re-porter system to enrich genetically modified cells induced by ZFN or CRISPR/Cas9. T7 endonuclease I assay re-vealed that this strategy improved genome editing activity of CRISPR/Cas9 by 5 folds, and was even more effective for improving genome editing efficiency of ZFN.