Construction and characterization of QKI knockout GC1-spg cell strain with CRISPR/CAS9
- VernacularTitle:利用CRISPR/CAS9技术构建QKI敲除GC1-spg细胞株及其生物学功能检测
- Author:
Shun-Shun ZHONG
1
;
Kai LI
;
Yang YANG
;
Shi-Ying MIAO
;
Lin-Fang WANG
;
Wei SONG
Author Information
1. 中国医学科学院基础医学研究所北京协和医学院基础学院医学分子生物学国家重点实验室
- Keywords:
CRISPR/CAS9;
GC1-spg;
QKI;
spermatogenesis
- From:
Basic & Clinical Medicine
2018;38(5):589-593
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate whether QKI protein plays any important role in the process of spermatogene-sis.Constructing GC1-spg cell strain which knocked out QKI by the technology of CRISPR/Cas9,and detecting its effect on the proliferation and differentiation of QKI protein in vitro.Methods The plasmid PX330 was used to construct QKI knockout recombinant plasmid, then transfected it to GC1-spg wild-type cells and selected by puromycin.GC1-spg knock-QKI cell strain was identified by Western blot and gene sequencing; The wild-type and knockout cell strain was cultured normally,then detected the growth curve by cell counting kit(CCK8),and using quantitative PCR to get the changes of meiotic-related gene differentiation.Results QKI knockout GC1-spg cell strain was successfully constructed.Compared with the control group,the growth of QKI knockout cell strain was significantly decreased(P<0.05), and the expression of meiosis related molecular marker gene of c-kit, Mtl5 and Pspa2 was significantly decreased(P<0.05).Conclusions QKI proteins can affect reproductive sper-matogenesis by acting on proliferation and differentiation.
