生物技术通报 ›› 2022, Vol. 38 ›› Issue (6): 272-278.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1432
刘静静(), 刘晓蕊, 李琳, 王盈, 杨海元(), 戴一凡()
收稿日期:
2021-11-15
出版日期:
2022-06-26
发布日期:
2022-07-11
作者简介:
刘静静,女,硕士研究生,研究方向:转基因大动物;E-mail: 基金资助:
LIU Jing-jing(), LIU Xiao-rui, LI Lin, WANG Ying, YANG Hai-yuan(), DAI Yi-fan()
Received:
2021-11-15
Published:
2022-06-26
Online:
2022-07-11
摘要:
利用CRISPR/Cas9系统建立催产素受体(oxytocin receptor,OXTR)基因敲除的巴马猪胎儿成纤维细胞系(porcine fetal fibroblasts,PFFs),为构建OXTR基因敲除巴马猪模型提供供体细胞。首先,对人和猪的OXTR基因进行了生物学信息分析。其次,利用在线设计工具设计了2个靶向猪OXTR外显子区的sgRNA,并克隆至pX330骨架质粒中,最后将打靶质粒和Neomycin抗性质粒共转染至PFFs中,用G418药物筛选出抗性单克隆细胞,测序鉴定其基因型。生物信息学分析显示人和猪OXTR基因进化距离较近,氨基酸序列一致性达到91%,相似性为93%,三维结构的RMSD值为0.009。成功构建OXTR基因的Cas9/sgRNA打靶载体,转染PFFs细胞后,药物筛选获得OXTR基因敲除的单克隆细胞并测序证实了基因突变型。人和猪OXTR基因具有高度同源性;构建的Cas9/sgRNA表达载体高效实现了OXTR基因编辑,成功获得基因敲除的单细胞克隆,为后续构建OXTR敲除的巴马猪模型奠定了前期基础。
刘静静, 刘晓蕊, 李琳, 王盈, 杨海元, 戴一凡. 利用CRISPR/Cas9技术建立OXTR基因敲除猪胎儿成纤维细胞系[J]. 生物技术通报, 2022, 38(6): 272-278.
LIU Jing-jing, LIU Xiao-rui, LI Lin, WANG Ying, YANG Hai-yuan, DAI Yi-fan. Establishment of Porcine Fetal Fibroblasts with OXTR-knockout Using CRISPR/Cas9[J]. Biotechnology Bulletin, 2022, 38(6): 272-278.
名称 Primer name | 引物序列 Primer sequence(5'-3') | 产物大小 Fragment length |
---|---|---|
OXTR-F | ACCTGCCCAAGAAGTCTCAG | 748 bp |
OXTR-R | CAGCAGCAGGTAAGTGGAAG |
表1 OXTR基因exon 2序列扩增引物
Table 1 Amplification primers for exon 2 sequene of OXTR gene
名称 Primer name | 引物序列 Primer sequence(5'-3') | 产物大小 Fragment length |
---|---|---|
OXTR-F | ACCTGCCCAAGAAGTCTCAG | 748 bp |
OXTR-R | CAGCAGCAGGTAAGTGGAAG |
图2 人/猪OXTR的一级、二级、三级结构分析 A:人/猪OXTR氨基酸序列同源性分析,相同氨基酸用蓝色阴影表示;B:人/猪蛋白质二级结构,红色表示a螺旋,绿色表示β折叠,蓝色表示β转角;C:人/猪蛋白质三维建模结果,红色为人类OXTR结构,绿色为猪OXTR结构
Fig.2 Analysis of primary,secondary,and tertiary structures of OXTR between humans and pigs A:Amino acid sequence homology analysis of human and pig OXTR,and the identical amino acids are blue shaded. B:Secondary structures of human and pig proteins. Red indicates alpha-helix;green indicates beta-sheet;and blue indicates beta-turn. C:Tertiary structures of human and pig proteins. Red indicates the structure of human OXTR,and green indicates the structure of pig OXTR
图3 猪OXTR关键残基和结构域的鉴别 A:猪OXTR的结构域和催化残基示意图;B:OXTR的多重序列比对结果,#表示与配体结合的关键氨基酸残基
Fig. 3 Identification of domains and key residues of porcine OXTR A:Schematic diagram of domains and catalytic residues of pig OXTR. B:Multiple sequence alignment of OXTR,# denotes critical amino acid residues binding to ligand
图4 OXTR基因靶点和重组载体测序 A:巴马猪OXTR基因靶点示意图,灰色表示非编码区,黑色表示编码区;B:pX330载体示意;C、D:重组载体示意图,黑色划线处为插入序列
Fig.4 Target of OXTR gene and sequencing of recombinant vectors A:Schematic diagram of OXTR gene target in Bama miniature pig. Gray indicates the non-coding area and black indicates the coding areas. B:Schematic diagram of pX330 vector. C and D:Sequencing map of recombinant vector. Black underlined is the insertion sequence
sgRNA | 序列Sequence(5'-3') |
---|---|
sgRNA-1-F | CACCGGTGCTGCCTCAGCTACTGT |
sgRNA-1-R | AAACACAGTAGCTGAGGCAGCACC |
sgRNA-2-F | CACCGAACTTGCGGCTCAAGACTG |
sgRNA-2-R | AAACCAGTCTTGAGCCGCAAGTTC |
表2 OXTR基因靶向位点的sgRNA寡核苷酸序列
Table 2 Oligonucleotide sequences of sgRNAs at OXTR targeting sites
sgRNA | 序列Sequence(5'-3') |
---|---|
sgRNA-1-F | CACCGGTGCTGCCTCAGCTACTGT |
sgRNA-1-R | AAACACAGTAGCTGAGGCAGCACC |
sgRNA-2-F | CACCGAACTTGCGGCTCAAGACTG |
sgRNA-2-R | AAACCAGTCTTGAGCCGCAAGTTC |
编号 ID name | 目的片段 Target fragment | 突变型 Indels |
---|---|---|
WT | CAGGTGCTGCCTCAGCTACTGTGGG//GGCAGAACTTGCGGCTCAAGACTGCG | WT |
1 | CAGGTGCTGCCTCAGCTAC-----// ----------------------CTGCGG | -411 bp |
2 | CAGGTGCTGCCTCAGCTAC-----// ----------------------CTGCGG | -411 bp |
3 | CAGGTGCTGCCTCAGCTAC-----// ----------------------CTGCGG | -411 bp |
4 | CAGGTGCTGCCTCAGCTACTG---// -------------------------CGG | -412 bp |
5 | CAGGTGCTGCCTCAGCTACTCTTGA//AGCGGAAGGTGATATCCCACACTGCGG | -289,+289 bp |
表3 OXTR 敲除PFFs的基因型
Table 3 Genotypes of OXTR-knockout PFFs
编号 ID name | 目的片段 Target fragment | 突变型 Indels |
---|---|---|
WT | CAGGTGCTGCCTCAGCTACTGTGGG//GGCAGAACTTGCGGCTCAAGACTGCG | WT |
1 | CAGGTGCTGCCTCAGCTAC-----// ----------------------CTGCGG | -411 bp |
2 | CAGGTGCTGCCTCAGCTAC-----// ----------------------CTGCGG | -411 bp |
3 | CAGGTGCTGCCTCAGCTAC-----// ----------------------CTGCGG | -411 bp |
4 | CAGGTGCTGCCTCAGCTACTG---// -------------------------CGG | -412 bp |
5 | CAGGTGCTGCCTCAGCTACTCTTGA//AGCGGAAGGTGATATCCCACACTGCGG | -289,+289 bp |
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