Targeting of gene expression by siRNA in CML primary cells |
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Authors: | Michaela Merkerova Hana Klamova Radim Brdicka Hana Bruchova |
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Institution: | (1) Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20 Prague 2, Czech Republic |
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Abstract: | 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. |
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Keywords: | Chemical transfection CML Electroporation Nucleofection Primary cells siRNA |
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