幼年特发性关节炎10p11.21遗传位点敲除的单克隆细胞系的建立  

Establishment of a monoclonal cell line with deletion of juvenile idiopathic arthritis associated genetic locus at 10p11.21

作　　者：张四鹏 李津 ZHANG Si-peng;LI Jin(Department of Cell Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学基础医学院细胞生物学系,天津300070

出　　处：《天津医科大学学报》2022年第4期372-377,共6页Journal of Tianjin Medical University

基　　金：天津市自然科学基金(18JCYBJC42700)。

摘　　要：目的:建立幼年特发性关节炎10p11.21遗传位点敲除的单克隆细胞系。方法:首先利用自身免疫病致病变异的遗传和表观遗传精细定位平台鉴定10p11.21遗传位点可能的因果变异;通过全表型组关联研究(PheWAS)数据库确定这一位点的因果变异与其他疾病的相关性;荧光素酶报告基因实验来验证候选因果变异是否具有转录调控功能;利用CRISPR/Cas9技术将因果变异所在基因组区域进行敲除。结果:生物信息学分析的结果显示10p11.21遗传位点的单核苷酸多态性(SNP)rs7100025的PICS值为0.7057,可能是这一位点的因果变异;PheWAS数据库检索结果显示rs7100025与多种免疫疾病具有显著相关性;rs7100025具有调控基因转录的功能,正向插入包含G等位基因和A等位基因的rs7100025序列可以提高荧光素酶表达水平,并且G等位基因相较于A等位基因效果更加明显;成功设计构建了删除rs7100025所在基因组区域的guide RNA质粒,并在Jurkat细胞系中将rs7100025位点进行敲除。筛选得到的单克隆细胞系的基因型在Sanger测序中得以验证。结论:精细定位幼年特发性关节炎10p11.21遗传位点可能的因果变异rs7100025(PICS值为0.7057),并成功建立这一变异敲除的单克隆Jurkat细胞系,敲除的片段大小为456 bp。Objective:To establish a monoclonal cell line with deletion of juvenile idiopathic arthritis associated genetic locus at 10p11.21.Methods:Fine-mapping was performed on the platform which integrates genetic and epigenetic annotations of pathogenic variants in autoimmune diseases to determine the likely causal variants at the 10p11.21 locus.The PheWAS database was searched for the association between the candidate causal variant of this locus and other diseases.Luciferase reporter gene assay was conducted to examine whether the likely causal variant has transcription regulatory ability.The CRISPR/Cas9 genome editing technology was employed to knock out the genomic region where the causal variant is located from the CD4+T cells.Results:Fine-mapping result showed that the PICS value of the single nucleotide polymorphism(SNP)rs7100025 at the 10p11.21 locus was 0.7057,which may be the causal variation of this locus.PheWAS-database searching results showed that rs7100025 is associated with a variety of immune diseases.rs7100025 has the function of regulating gene transcription.Forward insertion of rs7100025 sequence containing G allele and A allele was able to promote transcription in the luciferase reporter assay,and the G allele demonstrated a stronger transcription regulatory activity than the A allele.Guide-RNA construct was designed and constructed targeting the flanking genomic region of rs7100025.The genomic locus containing rs7100025 was deleted from the genomic DNA of Jurkat cells.The genotype of the selected monoclonal cell line was verified by Sanger sequencing.Conclusion:The likely causal variant rs7100025 at the 10p11.21 juvenile idiopathic arthritis associated genetic locus(PICS value was 0.7057)are identified,and a monoclonal Jurkat cell line with this variant deleted and the knockout fragment size of 456 BP are successfully established.

关 键 词：全基因组关联分析 因果变异的精细定位 荧光素酶报告基因 基因编辑 单克隆细胞系 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]