Dual over-expression of P2X7 receptor and intracellular domain of Notch1 in leukemia cells.
10.7534/j.issn.1009-2137.2013.03.002
- Author:
Li FENG
1
;
Xiao YANG
;
Jin-Feng LIAO
;
Sha-Yan CHEN
;
Wen-Li FENG
;
Yong-Min LIN
;
Qian REN
;
Guo-Guang ZHENG
Author Information
1. Chinese Academy of Medical Sciences, Tianjin, China.
- Publication Type:Journal Article
- MeSH:
Gene Expression;
Genetic Vectors;
Humans;
K562 Cells;
RNA, Messenger;
genetics;
Receptor, Notch1;
genetics;
Receptors, Purinergic P2X7;
genetics;
Retroviridae;
genetics
- From:
Journal of Experimental Hematology
2013;21(3):544-549
- CountryChina
- Language:Chinese
-
Abstract:
This study aimed to construct the dual expression vectors of wide type or N187D mutant P2X7 receptor and intracellular domain of Notch1 (ICN1) linked by 2A peptide to coexpress them in leukemia cells so as to lay a foundation for further investigating the role of P2X7 in development of leukemia. Overlap PCR was used to construct the dual expression vectors encoding wide type or N187D mutant type P2X7 receptor and ICN1 linked by the self-cleaving 2A sequence. The results showed that stable expressing cell lines were obtained by retroviral infection followed by cell sorting after DNA sequence analysis. RT-PCR, Western blot, intracellular free calcium concentration analysis were used to verify the functionally successful construction of K562 cell line expressing P2X7 receptor alone or with ICN1. DNA sequence analysis revealed that all construction were right. The infection efficiency of packaged constructed virus ranged from 40% to 70% for K562 cells. Stable infected cell line was obtained by cell sorting. RT-PCR analysis revealed that P2X7 receptor and/or ICN1 could be detected at high level in their stable infected cell lines, respectively. Western blot analysis also showed that P2X7 receptor was highly expressed in cell line infected by virus with P2X7 receptor. Sustained increase in intracellular free calcium concentration ([Ca(2+)]i) could be observed in K562 cells overexpressing either type of P2X7 receptor upon stimulation with BzATP. It is concluded that the wide type or N187D mutant P2X7 receptor and ICN1 are simultaneously and functionally over-express in leukemia cells, which lay a foundation for further studying the role of P2X7 receptor in the development of leukemia.
