Establishment of C57/B6-L and A549 cell lines stably expressing circLAMP3
10.16303/j.cnki.1005-4545.2024.09.21
- VernacularTitle:稳定表达circLAMP3的C57/B6-L和A549细胞系的构建
- Author:
Fuzai CHEN
1
;
Conghui ZHAO
;
Xiaoxuan ZHANG
;
Chunping ZHANG
;
Jiacheng HUANG
;
Jilong CHEN
;
Shujie MA
Author Information
1. 福建农林大学动物科学学院福建省"一带一路"畜禽重大疫病防控联合实验室,福建 福州 350002
- Keywords:
circular RNA;
circLAMP3;
vector construction;
cell line
- From:
Chinese Journal of Veterinary Science
2024;44(9):2010-2016
- CountryChina
- Language:Chinese
-
Abstract:
This study aims to construct C57/B6-L and A549 cell lines that stably overexpress circu-lar RNA LAMP3(circLAMP3),laying the foundation for further research on the biological func-tions of circLAMP3.Total RNA was extracted and reverse transcripted into cDNA from C57/B6-L and A549 cells to amplify the full-length sequence of circLAMP3.Then,the fragments of cir-cLAMP3 were ligated into pLC5-ciR vector to obtain pLC5-Mouse-circLAMP3 and pLC5-Human-circLAMP3 recombinant plasmids.The lentiviruses expressing circLAMP3 were packaged on tran-sient transfected HEK293T cells.C57/B6-L and A549 cells were infected with lentiviruses to gen-erate cell lines overexpressing circLAMP3 through puromycin screening.To verify the overexpres-sion efficiency of circLAMP3 of cell lines,we performed the fluorescence microscopy,PCR amplifi-cation,quantitative PCR(qPCR),and Sanger sequencing experiments.The results indicated that the overexpression plasmids of pLC5-Mouse-circLAMP3 and pLC5-Human-circLAMP3 were suc-cessfully constructed.Strong green fluorescence was observed under a fluorescence microscopy.C57/B6-L and A549 cell lines showed a significant increase in the expression of circLAMP3 by PCR and qPCR methods.Sanger sequencing results showed that the junction site of circLAMP3 was correct.This study successfully constructed C57/B6-L and A549 cell lines overexpressing circLAMP3,providing biomaterials for further exploration of the biological function of circLAMP3 in influenza virus replication.
