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Construction of Recombinant Adenovirus Vector Carrying Human RARα Gene and Its Effect on NB4 Human Promyelocytic Leukemia Cells
Jiang Kailing1,2, Zhong Liang2, Yang Xiaoqun2, Wang Hui2, Ma Pengpeng2, Zhu Xinyu2, Liu Beizhong1,2*
1Central Laboratory of Yongchuan Hospital, Chongqing Medical University, Chongqing 402160, China; 2Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine,Chongqing Medical University, Chongqing 400016, China
Abstract: In this research， we constructed recombinant adenovirus Ad-RARα and determined its effect on NB4 human promyelocytic leukemia cells. RARα gene was amplified by PCR using cDNA of NB4 cells as a template and inserted into shuttle plsmid pAdTrace-TO4. The constructed recombinant shuttle plasmid pAdTrace-TO4-RARα was digested with Pme I， and transformed to competent E.coli BJ5183 containing adenovirus backbone plasmid pAdEasy-1. The obtained recombinant adenovirus plasmid Ad-RARα was digested with Pac I and transfected to AD293 cells for packaging. The obtained recombinant adenovirus Ad-RARα was subjected to three cycles of amplification， then determined titer and identified by PCR and sequencing. NB4 cells were infected by the recombinant adenovirus， with infection rate of 70%. Protein expression， proliferation and differentiation in NB4 cells of RARα were detected by Western blot， CCK-8 and flow cytometry respectively. Cell cycle was measured by Annexin V/PI. Recombinant adenovirus Ad-RARα reached a titer of 5.2×105 pfu/mL after three cycles of amplification and recombinant adenovirus Ad-RARα was constructed correctly as proved by PCR and sequencing， and the RARα gene in Ad-RARα was successfully expressed in NB4 cells. Recombinant adenovirus Ad-RARα was constructed successfully and RARα gene could be expressed in NB4 cells. The proliferation of infected NB4 cells was enhanced. Physiological concentration of ATRA (all-trans-retinoic acid) can induce differentiation effectively. All of these results lay the foundation to further study for the role of this gene in acute myeloid leukemia development.
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