Construction of HMGA2 knockout model of human papillary thyroid cancer cell line TPC-1 / 中华内分泌外科杂志

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]-EGFPT2APuro- 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 T2APuro-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.