[Specific silencing of PCNA gene expression in leukemic cell lines using siRNA].
BACKGROUND
Increased expression of PCNA gene was detected in chronic myeloid leukemia (CML) patients in our laboratory. The gene may participate in the disease development. The aim of the study was to develop appropriate conditions for PCNA-siRNA transfection into K562 and MOLM-7 cell lines (which both have the up-regulated PCNA gene) and to silence the increased expression.


RESULTS
Key parameters of successful siRNA delivery into the cells are type and quantity of transfection reagent, cells and siRNA concentration or cultivation time before an expression analysis. Transfection reagents ExGene 500 (Fermentas), Metafectene (Biontex), Oligofectamine (Qiagen) and siPORT Amine (Ambion) were tested. Transfection efficiency was monitored by fluorescence microscopy of fluorescein labeled siRNA. Gene silencing was determined at mRNA level by real-time PCR and at protein level by western blots. As the most suitable reagent was chosen Oligofectamine, which achieved 70% decrease of PCNA mRNA level. Further, 50 nM siRNA concentration, 1 x 10(6) cells/ml and amount of Oligofectamine 4 microl per 1 ml of transfected cells were selected. The best cultivation time after siRNA delivery was 48 h.


CONCLUSIONS
Based on the results of this study, transfection method for siRNA delivery into the K562 and MOLM-7 cell lines was proposed. The procedure can be transferred also on further selected genes potentially involved in CML and afterwards it will be possible to monitor the impact of siRNA-inhibition on expression profile. In the future siRNAs against some over-expressed genes would be used for gene therapy of CML.
Merkerová
M
M
Ustav hematologie a krevní transfuze, Praha. michaela.merkerova@uhkt.cz
Bruchová
H
H
Brdicka
R
R
D045744:Cell Line, Tumor; D015973:Gene Expression Regulation, Leukemic; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D006801:Humans; D020014:K562 Cells; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics*; D034741:RNA, Small Interfering; D014162:Transfection
D018809:Proliferating Cell Nuclear Antigen; D034741:RNA, Small Interfering