Establishment of a HL-60 cell line with stable RYBP silencing

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Author:
ZHOU Jia-yan(Department of Hematology, Guangzhou First Municiple People Hospital, the Affiliated Guangzhou Medical College, Guangzhou 510180, China)
DENG Hui(Department of Hematology, Guangzhou First Municiple People Hospital, the Affiliated Guangzhou Medical College, Guangzhou 510180, China)
WANG Chun-li(Department of Hematology, Guangzhou First Municiple People Hospital, the Affiliated Guangzhou Medical College, Guangzhou 510180, China)
ZHANG Yu-ping(Department of Hematology, Guangzhou First Municiple People Hospital, the Affiliated Guangzhou Medical College, Guangzhou 510180, China)
YING Yi(Department of Hematology, Guangzhou First Municiple People Hospital, the Affiliated Guangzhou Medical College, Guangzhou 510180, China)
XU Yan-li(Department of Hematology, Guangzhou First Municiple People Hospital, the Affiliated Guangzhou Medical College, Guangzhou 510180, China)
WANG Shun-qing(Department of Hematology, Guangzhou First Municiple People Hospital, the Affiliated Guangzhou Medical College, Guangzhou 510180, China)
Journal Title:
Journal of Leukemia & Lymphoma
Issue:
Volume 21, Issue 10, 2012
DOI:
10.3760/cma.j.issn.1009-9921.2012.10.001
Key Word:
RYBP gene;RNA,short hairpin;HL-60 cells;Lentivirus;RNA interference

Abstract: Objective To establish stable HL-60 cell line with stable RYBP gene silencing using lentivirus-mediated RNA interference. Methods Five special shRNAs for RYBP gene were cloned to lentivirus vector.Recombinate lentivirus vectors were packed into lentivirus,which were used to infect HL-60 cells, and took empty vector and non-specific shRNA as control groups. Stable infected cells were selected with puromycin in 8 μg/ml concentration.The expression levels of RYBP were analyzed by Western blot,and confirmed the most effective RYBP shRNA.Then the level of mRNA was analyzed by real-time PCR.Results Stable infected cells were selected by puromycin successfully.Comparing to control groups,the expression of RYBP were reduced at different degrees (P < 0.01). And RYBP shRNA2 took the most silencing effect, the RYBP mRNA was decreased by more than 95 % (P < 0.05).Conclusion The shRNA2 targeting RYBP gene can effectively inhibit the expression of RYBP. HL-60 cell line with stable RYBP gene silencing were constructed successfully,which had provided experiment fundament for further studying the function of RYBP.

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