Construction of HMGA2 knockout model of human papillary thyroid cancer cell line TPC-1
10.3760/cma.j.cn.115807-20210705-00207
- VernacularTitle:人甲状腺乳头状癌细胞系TPC-1的HMGA2基因敲除模型构建
- Author:
Hong YONG
1
;
Xiaojuan LI
;
Shan JIN
;
Yousheng LIU
;
Yuntu WU
;
Yinbao BAI
;
La TA
Author Information
1. 内蒙古医科大学附属医院甲状腺乳腺外科，呼和浩特 010050
- Keywords:
CRISPR/Cas9;
Human papillary thyroid cancer cell line TPC-1;
Thyroid carcinoma;
HMGA2
- From:
Chinese Journal of Endocrine Surgery
2022;16(4):421-425
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a TPC-1 cell model that stably knocks out the HMGA2 by using CRISPR/Cas9 gene editing technology. Methods:Recombinant pLV[2gRNA]-EGFP:T2A:Puro- U6> {hHMGA2 [gRNA# A1]*}- U6>{hHMGA2 [gRNA#A2]*} of lentiviral plasmid vector was constructed: targeting HMGA2 Dual-gRNA sequence was designed, the synthesized Dual-gRNA fragment into pLV [2gRNA]-EGFP was cloned: T2A:Puro-U6 vector, extract a single clone for sequencing verification. the constructed recombinant plasmid vector with lentivirus was packed, and TPC-1 cells were infected, puromycin was used to obtain HMGA2 knock-out single clone, PCR and sequencing verification were performed, and real-time fluorescent quantitative qPCR was used to detect HMGA2 mRNA in cells Knockout efficiency. Results:After sequencing verification, pLV [2gRNA]-EGFP targeting HMGA2: T2A: Puro-U6>{hHMGA2 [gRNA#A1]*}-U6>{hHMGA2 [gRNA #A2]*} plasmid was successfully constructed; A single clone was picked for PCR identification and gene sequencing, TPC-1 cells were successfully obtained with HMGA2 gene completely knocked out; TPC-1 cells with HMGA2 knocked out were detected by real-time fluorescent quantitative qPCR, and they did not express HMGA2 mRNA.Conclusion:CRISPR/Cas9 gene editing technology enables us to construct a human papillary thyroid cancer cell line TPC-1 cell model with stable knockout of HMGA2.
