畜牧兽医学报2025，Vol.56Issue(10)：5007-5017,11.DOI:10.11843/j.issn.0366-6964.2025.10.021
X染色体定点整合GFP元件的野猪诱导多能性干细胞系的建立
Establishment of Wild Boar Induced Pluripotent Stem Cell Line with X Chromosome-Targeted Integration of GFP Cassette
摘要
Abstract
This study aimed to establish induced pluripotent stem cells(iPSCs)of wild boar with stable passage ability,and integrate exogenous genes into the X chromosome of iPSCs,which provides technical support for the conservation,development and utilization of wild boar re-sources.Eight pluripotency factors were introduced into wild boar fibroblasts by the PiggyBac transposon system to establish the iPSCs line and the pluripotency was identified.Plasmids ex-pressing green fluorescent protein(GFP)were transferred into wild boar iPSCs by electropora-tion(3 procedures)and chemical transfection reagents lipofectamine 3000(Lip 3000)and jetPRIME,respectively.And the optimal transfection protocol was screened with analysis of transfection efficiency by flow cytometry.Combined with CRISPR/Cas9 and site-directed inte-gration technology independent of homologous recombination,the sgRNAs with high-efficiency were co-transfected with GFP expression elements into wild boar iPSCs.Then target fragments were amplified by PCR and the site-directed integration of GFP was identified by sequencing.In this study,human OCT4,SOX2,KLF4,C-MYC,NANOG,LIN28,NR5A2 and miR302/367 were introduced into wild boar fibroblasts.Twenty days post-induction,wild boar iPSCs were obtained with normal karyotype,positive alkaline phosphatase staining,stable expression of plu-ripotency factors OCT4,SOX2 and NANOG.Additionally,embryoid bodies,labeled with three germ layers marker(β Ⅲ tubulin,α-smooth muscle actin and GATA6),were observed in vitro.For different electroporation procedures,the cell transfection efficiency using CG104((9.73±0.23)％)was significantly higher than that of CA201((7.34±0.03)％)and CB150((6.69±0.10)％).The cell transfection efficiency with jetPRIME cationic polymer was(17.9±0.36)％,which was significantly higher than that of Lip 3000((14.07±0.85)％).After two plasmids with sgRNA or GFP expression elements were constructed successfully,they were co-transfected into wild boar iPSCs.PCR and sequencing results showed that GFP expression elements were in-serted into the predetermined position of X chromosome and could be stably expressed.In con-clusion,wild boar iPSCs were derived from fibroblasts.And GFP was knocked into the X chro-mosome of iPSCs by site-directed integration technology independent of homologous recombina-tion.This study provided technical reference for gene editing in wild boar and breeding of domestic pig.关键词
野猪/诱导多能干细胞/电转染/脂质体/X染色体定点整合Key words
wild boar/induced pluripotent stem cells/electroporation/liposomes/site-directed integration of X chromosome分类
农业科技引用本文复制引用
周心仪,杨利丹,高晨,魏新华,霍浩楠,邹惠影,余大为,杜卫华..X染色体定点整合GFP元件的野猪诱导多能性干细胞系的建立[J].畜牧兽医学报,2025,56(10):5007-5017,11.基金项目
国家重点研发计划(2024YFD1301002) （2024YFD1301002）
中央级公益性科研院所基本科研业务费专项(2023-YWF-ZYSQ-04) （2023-YWF-ZYSQ-04）
中国农业科学院科技创新工程项目(ASTIP-IAS06) （ASTIP-IAS06）
