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

doi:10.11843/j.issn.0366-6964.2025.10.021

摘要/Abstract

摘要：

本研究旨在建立具有稳定传代能力的野猪诱导多能干细胞系(induced pluripotent stem cells，iPSCs)，并将外源基因定点整合于iPSCs的X染色体，为野猪的资源保存和开发利用提供技术支撑。利用PiggyBac转座子系统将8种多能性因子导入野猪成纤维细胞，诱导建立iPSCs系，并鉴定其多能性；分别采用电转染(3种程序)、化学转染试剂lipofectamine 3000(Lip 3000)和jetPRIME将表达绿色荧光蛋白(green fluorescent protein，GFP)的质粒转入野猪iPSCs，通过流式细胞仪分析转染效率筛选最优转染方案；结合CRISPR/Cas9和不依赖于同源重组的定点整合技术，将筛选获得的高效sgRNA与GFP表达元件共转染野猪iPSCs，PCR扩增目的片段，并测序鉴定GFP的定点整合。本研究将人源OCT4、SOX2、KLF4、C-MYC、NANOG、LIN28、NR5A2和miR302/367导入野猪成纤维细胞，诱导后20 d获得野猪iPSCs，其核型正常，碱性磷酸酶染色阳性，能稳定表达多能性因子OCT4、SOX2和NANOG，可体外形成拟胚体，并标记到三胚层的标志蛋白(β Ⅲ-微管蛋白、α-平滑肌肌动蛋白和GATA6)。对于不同的电转染程序，CG104程序的细胞转染效率为(9.73±0.23)%，显著高于CA201((7.34±0.03)%)和CB150程序组((6.69±0.10)%)；而使用jetPRIME阳离子聚合物的细胞转染效率为(17.9±0.36)%，极显著高于Lip 3000((14.07±0.85)%)。成功构建分别携带sgRNA与GFP表达元件的两个质粒，共转染野猪iPSCs后，PCR和测序结果显示，GFP表达元件定点插入至X染色体的预定位置，且能够稳定表达。本研究将野猪成纤维细胞诱导为可稳定传代的iPSCs，并通过不依赖同源重组的定点整合技术将GFP敲入iPSCs的X染色体，为野猪的基因编辑和家猪育种提供技术借鉴。

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

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 resources. 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 expressing green fluorescent protein (GFP) were transferred into wild boar iPSCs by electroporation (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 integration 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 pluripotency 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 inserted into the predetermined position of X chromosome and could be stably expressed. In conclusion, wild boar iPSCs were derived from fibroblasts. And GFP was knocked into the X chromosome of iPSCs by site-directed integration technology independent of homologous recombination. This study provided technical reference for gene editing in wild boar and breeding of domestic pig.

Key words: wild boar, induced pluripotent stem cells, electroporation, liposomes, site-directed integration of X chromosome

中图分类号:

S828.3

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.

ZHOU Xinyi, YANG Lidan, GAO Chen, WEI Xinhua, HUO Haonan, ZOU Huiying, YU Dawei, DU Weihua. Establishment of Wild Boar Induced Pluripotent Stem Cell Line with X Chromosome-Targeted Integration of GFP Cassette[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5007-5017.

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引物名称 Primer name	引物序列(5′→3′) Primers sequence
HIT-EF1α-GFP-F	GACACTATAGAACTCGACCACTAGTTTTGAGATCGAGTGCCGCATCACGGGCTCGAG-
	TCGTCGTGAGGCTCCGGTGCCCG
HIT-EF1α-GFP-R	GGCACTCGATCTCAAAGGCCGGCGCGCCTAAGATACATTG
PH-I-sgRNA1-F	CACCGTTGAACTCATGCGGAAAGGG
PH-I-sgRNA1-R	AAACCCCTTTCCGCATGAGTTCAAC
PH-I-sgRNA2-F	CACCGGCACACTCAGGGTCCAGTA
PH-I-sgRNA2-R	AAACTACTGGACCCTGAGTGTGCC
PH-I-F	GAGGACCAAATTCTCACTTCAAC
PH-I-R	TAGGGGTCAAATCAGAGCTACAG
EF1α-R	TAGGCACCGGTTCAATTGCCGA

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