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Updated: Aug 19, 2026
Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Published on: October 21, 2012
[Experimental study of K562 cell apoptosis induced by siRNA]
Bo-bin Chen1, Hua-hua Fan, Guo-wei Lin
1Department of Hematology, Huashan Hospital, Fudan University, Shanghai 200040, China.
Objectives:
To construct a siRNA expression vector pBCR6 that produces siRNA against bcr/abl mRNA and detect apoptosis rate of K562 cells after pBCR6 transfection.
Methods:
Template sequence for siRNA was designed, synthesized and inserted into an expression vector pSilencer1.0-U6. Restriction analysis and sequencing were performed to verify the pBCR6 vector. Then pBCR6 was transfected into K562 cells by X-tremeGene Q2. pSilencer1.0-U6 was used as the control. At different time point after transfection, apoptosis rate was determined by Tunel and Annexin V+ PI with FCM.
Result:
pBCR6 was verified by restriction analysis and sequencing. The apoptosis rate of K562 cells markedly increased at 48 and 72 hour after transfected with pBCR6, and increased in a time-dependent manner [the apoptosis rate of transfected K562 cells was (47.80 +/- 1.63)% at 72 hrs, whereas the control group was (6.67 +/- 0.37)%, P < 0.0001] No prominent change in apoptosis rate was found in the control.
Conclusion:
The siRNA expression vector against bcr/abl mRNA was successfully constructed. The pilot study showed that pBCR6 could effectively induce K562 cells apoptosis. siRNA may be a new tool for molecular target therapy for chronic myelogenous leukemia.
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