Establishment of <italic style="font-style: italic">PIN</italic>1 knockout porcine fetal fibroblast cell lines <italic style="font-style: italic">via</italic> CRISPR/Cas9 mediated gene-targeting

Wanyi WANG; Yilin YUAN; Lin LI; Ying WANG; Yifan DAI; Haiyuan YANG

doi:10.16016/j.2097-0927.202112217

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Establishment of PIN1 knockout porcine fetal fibroblast cell lines via CRISPR/Cas9 mediated gene-targeting

Basic Medicine | 更新时间：2026-02-13

- Establishment of PIN1 knockout porcine fetal fibroblast cell lines via CRISPR/Cas9 mediated gene-targeting
- Journal of Army Medical University Vol. 44, Issue 13, Pages: 1349-1355(2022)
- 作者机构：
  
  211166 南京，南京医科大学基础医学院，江苏省异种移植重点实验室
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81970164)
- DOI：10.16016/j.2097-0927.202112217
  CLC： R329.2;R394-33;R394.2
- Received：24 December 2021，
  
  Revised：2022-03-21，
  
  Published：15 July 2022
- 稿件说明：
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Wanyi WANG, Yilin YUAN, Lin LI, et al. Establishment of PIN1 knockout porcine fetal fibroblast cell lines via CRISPR/Cas9 mediated gene-targeting[J]. Journal of Army Medical University, 2022, 44(13): 1349-1355.
DOI：

Wanyi WANG, Yilin YUAN, Lin LI, et al. Establishment of PIN1 knockout porcine fetal fibroblast cell lines via CRISPR/Cas9 mediated gene-targeting[J]. Journal of Army Medical University, 2022, 44(13): 1349-1355. DOI： 10.16016/j.2097-0927.202112217.

摘要

目的

利用成簇的规律间隔短回文重复序列和相关蛋白9(clustered regular interval short palindromic repeats/CRISPR-associated protein 9，CRISPR/Cas9)基因编辑技术敲除长白猪胎儿成纤维细胞(porcine fetal fibroblasts，PFFs)的

1基因，以构建

1基因敲除长白猪胎儿成纤维细胞系。

方法

对人和猪的

1基因进行同源性分析。利用在线工具设计2个靶向猪

1基因第二个外显子区的sgRNAs，并克隆至pX330骨架质粒中。将构建成功的打靶质粒和Neomycin抗性质粒共转染至PFFs中，用G418药物筛选出抗性单细胞克隆，测序鉴定其基因型并从转录和翻译水平鉴定

1的表达。

结果

同源性分析显示人和猪PIN1蛋白的氨基酸序列一致性和相似性均为98%，二者三维结构的均方根偏差(RMSD)值为0.014。获得15个

1基因敲除的纯合单克隆细胞系，并且证实PIN1蛋白在翻译水平被完全敲除。

结论

利用双sgRNAs引导的CRSIPR/Cas9系统在PFFs中高效实现

1基因的双等位基因敲除，成功建立

1基因敲除的长白猪胎儿成纤维细胞系。

Abstract

Objective

To knock out

1 gene of porcine fetal fibroblasts (PFFs) by clustered regular interval short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in order to establish

1 knockout porcine fetal fibroblast cell lines.

Methods

[Firstly

the homology of the

1 gene between humans and pigs was analyzed. Secondly

2 sgRNAs targeting the second exon region of the porcine

1 gene were designed using online tools (

http://crispor.tefor.net/

) and then cloned into the pX330 (addgene #42230) skeleton plasmid. Finally

the

1 targeting plasmid and Neomycin resistant plasmid were co-transfected into PFFs. G418 screening was used to obtain resistant single-cell colonies. Sanger sequencing

qRT-PCR

and Western blotting were adopted to determine the

1 genotypes and expression levels

respectively.

Results

The bioinformatics analysis showed that the amino acid sequence consistency and similarity of human and pig PIN1 proteins were 98%

and the root-mean-square deviation (RMSD) value of their 3-dimensional structures was 0.014. Fifteen homozygous monoclonal cell lines with

1 gene knockout were obtained

and the PIN1 protein was disrupted entirely in those

1 knockout colonies at translational level.

Conclusion

The

1 gene is efficiently knocked out in PFFs by a double sgRNAs-guided CRISPR/Cas9 system

and the porcine fetal fibroblast cell lines with

1 gene knocked out are successfully established.

关键词

Keywords

references

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⁰

Establishment of PIN1 knockout porcine fetal fibroblast cell lines via CRISPR/Cas9 mediated gene-targeting

DOI：10.16016/j.2097-0927.202112217

摘要

Abstract

关键词

Keywords

references