Construction of Nalm6-Cas9 Cell Line for Genome-Wide Translocation Sequencing.
10.19746/j.cnki.issn.1009-2137.2022.05.013
- Author:
Qing-Cheng LI
1
;
Jun-Bing HUANG
1
;
Hong-Man XUE
1
;
Mo YANG
2
;
Cheng-Ming ZHU
2
;
Chi-Kwong LI
3
;
Jun-Chao DONG
4
;
Chun CHEN
5
Author Information
1. Department of Pediatrics， The Seventh Affiliated Hospital， Sun Yat-sen University， Shenzhen 518107， Guangdong Province， China.
2. Scientific Research Center， The Seventh Affiliated Hospital， Sun Yat-sen University， Shenzhen 518107， Guangdong Province， China.
3. Division of Hematology/Oncology/BMT， Department of Paediatrics， The Chinese University of Hong Kong， Hong Kong 999077， China.
4. Zhongshan School of Medicine， Sun Yat-sen University， Guangzhou 510080， Guangdong Province， China.
5. Department of Pediatrics， 2Scientific Research Center， The Seventh Affiliated Hospital， Sun Yat-sen University， Shenzhen 518107， Guangdong Province， China,E-mail: chenchun69@ 126.com.
- Publication Type:Journal Article
- Keywords:
Cas9 protein;
Nalm6 cell lines;
high-throughput genome-wide;
lentivirus;
therapy-related leukemia;
translocation sequencing
- MeSH:
CRISPR-Associated Protein 9/genetics*;
CRISPR-Cas Systems;
Genetic Vectors;
HEK293 Cells;
Humans;
Lentivirus/genetics*;
Plasmids
- From:
Journal of Experimental Hematology
2022;30(5):1384-1390
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:In order to conduct high-throughput genome-wide translocation sequencing based on CRISPR/Cas9, Nalm6-cas9 monoclonal cell line expressing Cas9 protein was constructed by lentivirus transduction.
METHODS:Lentiviral vectors LentiCas9-Blast, pSPAX2, and pMD2.G were used to co-transfect HEK293T cells to obtain recombinant lentivirus. After Nalm6 cells were infected with the recombinant lentivirus, the cells were screened by Blasticidin, and multiple monoclonal cell lines expressing Cas9 protein were obtained by limited dilution. Western blot was used to detect the expression level of Cas9 protein in monoclonal cell lines, and cell count analysis was used to detect the proliferation activity of monoclonal cell lines. LentiCRISPRV2GFP-Δcas9, LentiCRISPRV2GFP-Δcas9-AF4, LentiCRISPRV2GFP-Δ cas9-MLL plasmids were constructed, and transfected with pSPAX2 and pMD2.G, respectively. T vector cloning was used to detect the function of Cas9 protein in Nalm6-Cas9 monoclonal cell line infected with virus.
RESULTS:Western blot showed that Nalm6-Cas9_1-6 monoclonal cell line had high expression of Cas9 protein. Cell count analysis showed that high expression of Cas9 protein in Nalm6-Cas9_1-6 monoclonal cell line did not affect cell proliferation activity. The Nalm6-Cas9_1-6 monoclonal cell line had high cleavage activity, and the editing efficiency of AF4 and MLL genes was more than 90% which was determined by T vector cloning.
CONCLUSION:Nalm6-Cas9_1-6 monoclonal cell line stably expressing highly active Cas9 protein was obtained, which provided a basis for exploring the translocation of MLL in therapy-related leukemias based on CRISPR/Cas9 genome-wide high-throughput genome-wide translocation sequencing.
