Page 7 - 南京医科大学学报自然科学版

第42卷第11期 南京医科大学学报（自然科学版）
2022年11月 Journal of Nanjing Medical University（Natural Sciences） ·1499 ·

·基础研究·

利用CRISPR/Cas9技术构建ACE2基因修饰的猪胎儿成纤维细

胞系

张成霖，方 斌，刘晓蕊，李 琳，王 盈，杨海元 ，戴一凡 *
*
南京医科大学江苏省异种移植重点实验室，江苏 南京 211166

［摘 要］ 目的：利用 CRISPR/Cas9 基因编辑技术构建血管紧张素转换酶 2（angiotensin converting enzyme 2，ACE2）基因编辑
的长白猪胎儿成纤维细胞（porcine fetal fibroblast，PFF）细胞系，为构建新型冠状病毒即严重急性呼吸综合征冠状病毒 2 型（se⁃
vere acute respiratory syndrome coronavirus type 2，SARS⁃CoV⁃2）不易感猪模型提供实验材料。方法：①利用生物信息学方法对
大鼠和鸡的ACE2氨基酸序列进行比对，将大鼠ACE2与SARS⁃CoV⁃2 S蛋白相互作用的关键氨基酸残基替换为鸡ACE2的氨
基酸残基，设计嵌合的ACE2基因并构建ACE2基因敲入供体。②设计并合成靶向猪ACE2基因的sgRNA，克隆到pX330载体
上构建ACE2基因打靶载体。③将ACE2打靶载体和ACE2基因敲入供体以及Neomycin抗性质粒共转染至长白猪PFF细胞，筛
选G418抗性单细胞克隆并进行测序鉴定。结果：根据氨基酸序列比对结果，将大鼠ACE2蛋白的第19、31、34、35、42、353、354位
氨基酸残基替换为鸡ACE2的氨基酸残基，设计合成了嵌合的ACE2基因。G418抗性单细胞克隆基因型分析结果显示获得了
阳性单细胞克隆。结论：利用CRISPR/Cas9技术成功获得了内源ACE2基因敲除、嵌合ACE2敲入的长白公猪PFF细胞系，为构
建SARS⁃CoV⁃2不易感猪模型奠定了基础。
［关键词］ ACE2；CRISPR/Cas9；SARS⁃CoV⁃2；猪胎儿成纤维细胞
［中图分类号］ R394.33 ［文献标志码］ A ［文章编号］ 1007⁃4368（2022）11⁃1499⁃08
doi：10.7655/NYDXBNS20221101

Establishment of ACE2⁃modified porcine fetal fibroblasts via CRISPR/Cas9 system

* *
ZHANG Chenglin，FANG Bin，LIU Xiaorui，LI Lin，WANG Ying，YANG Haiyuan ，DAI Yifan
Jiangsu Key Laboratory of Xenotransplantation，Nanjing Medical University，Nanjing 211166，China

［Abstract］ Objective：This study aims to establish angiotensin converting enzyme 2（ACE2）gene⁃modified porcine fetal fibroblast
（PFF） cell lines via CRISPR/Cas9 system ⁃ mediated gene modification，which could serve as donor cells for the subsequent
construction of a severe acute respiratory syndrome coronavirus type 2（SARS⁃CoV⁃2）insensitive pig model. Methods：①The ACE2
amino acid sequences of rats and chickens were compared by bioinformatic analysis. The ACE2 amino acid residues，which play a
crucial role in the interaction between ACE2 and SARS⁃CoV⁃2 S protein，were replaced by the amino acid residues of chicken’s ACE2
at the corresponding sites. The chimeric ACE2 gene was synthesized and cloned into the knock⁃in vector. ②The sgRNA targeting the
porcine ACE2 gene was designed，synthesized，and cloned into the pX330 vector to construct the ACE2 gene targeting vector. ③The
ACE2 targeting vector，the ACE2 gene knock⁃in donor vector，and a neomycin⁃resistant plasmid were co⁃transfected into PFP. G418
was used to screen the resistant single⁃cell colonies，and sequencing was performed to identify the correct ones. Results：According to
the amino acid sequence alignment，the rat ACE2 amino acid residues at positions 19 th，31st，34 th，35 th，42 nd，353 rd and 354 th
were replaced by the amino acid residues of chicken ACE2. The chimeric ACE2 gene was synthesized，and the ACE2 knock⁃in vector
was successfully constructed. Genotype analysis of G418 resistant colonies showed that single⁃cell colonies with expected modification
were successfully obtained. Conclusion：The PFF cells with endogenous ACE2 knock ⁃ out and chimeric ACE2 knock ⁃ in are
successfully generated using CRISPR/Cas9⁃mediated gene modification，which will lay a foundation for the construction of SARS⁃CoV⁃2
insusceptible pig models.
［Key words］ ACE2；CRISPR/Cas9；SARS⁃CoV⁃2；PFF
［基金项目］ 国家自然科学基金（81874144，81970164） ［J Nanjing Med Univ，2022，42（11）：1499⁃1506］
∗
通信作者（Corresponding author），E⁃mail：hyyang@njmu.edu.cn；daiyifan@njmu.edu.cn