Targeting of gene expression by siRNA in CML primary cells.
Development of array methods contributes to elucidation of many genes expressed during oncogenesis. Our array-based analyses of gene expression in patients with chronic myeloid leukemia (CML) revealed several genes (MMP8, MMP9, PCNA, JNK2, MAPK p38) with significant increased expression. We suppose that the genes may be implicated in the disease development and a siRNA-suppression can elucidate their functions in leukemogenesis. One of the crucial requirements for this purpose is a high efficiency of siRNA delivery into CML primary cells. Using fluorescein-labeled siRNAs we systematically tested a variety of physical and chemical non-vector based transfection methods in order to evaluate which of them gave the most suitable transfer. Chemically synthesized siRNAs against mentioned genes were transfected into the cells and level of knockdown was determined by real time RT-PCR. Chemical transfection reagents (Oligofectamine, Metafectene, siPORT Amine) commonly used to transfect siRNAs in CML cell lines showed very low siRNA delivery in CML primary cells-mRNA levels decreased at the most to 76%. Electroporation achieved better results (suppression to 63%) but it was associated with high degree of cell death (more than 60%). In the study we obtained the best transfection efficiency using nucleofector technology. Gene expressions ranged 22-37% that remained from original levels. According to our results, nucleofection appears to be the only suitable non-viral method for siRNA delivery into the hard-to-transfect CML primary cells.
Merkerova
Michaela
M
Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20, Prague 2, Czech Republic.
Klamova
Hana
H
Brdicka
Radim
R
Bruchova
Hana
H
D000328:Adult; D000368:Aged; D018929:Cell Culture Techniques; D005260:Female; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D034741:RNA, Small Interfering