Homozygous deletion 8p21 in prostate cancer
MANNI, VISA (2012)
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MANNI, VISA
2012
Biokemia - Biochemistry
Biolääketieteellisen teknologian yksikkö - Institute of Biomedical Technology
Hyväksymispäivämäärä
2012-06-01Tiivistelmä
Research background and aimsThe aim of this research project was to investigate the effect of the chromosome 8p21 homozygous deletion of genes BNIP3L and PPP2R2A on the phenotype of the prostate cancer cell model called VCaP. The chromosome 8p21 deletion has been known for a long time, but the contribution of these deletion genes has not been verified in prostate cancer before. VCaP is known to be difficult to transfect; hence the first aim was to find a functional transfection method.
MethodsDifferent transient transfection methods were studied to optimize the VCaP transfection protocol and to be able to transfect the deletion genes back into VCaP. Accordingly, plasmid reconstruction was prepared for virus transduction and the usability of two cell proliferation analysis methods were compared in VCaP.
ResultsLiposome-mediated transfection methods provided only modest results, not sufficient transfection efficiency for subsequent functional analysis. VCaP cells showed atypical morphology and severely suffered after transfection in many cases. Reverse transfection with polyethylenimine (PEI25) was found to be cytotoxic with all studied variables. Viral transduction preparations including vector ligation were inefficient, showing no positive clones of the correct vector size. Thus, proceeding to more efficient viral infection was not possible.
ConclusionsProstate cancer cell model VCaP was shown to be difficult to transfect. Adherent cells suffered in all studied transfection protocols. The best alternative for transfecting VCaP was shown to be a commercial liposome-mediated forward transfection reagent. VCaP cell line needs thorough optimization for each experiment and further studies to address the function of chromosome 8p21 deletion genes.
Asiasanat:prostate cancer
MethodsDifferent transient transfection methods were studied to optimize the VCaP transfection protocol and to be able to transfect the deletion genes back into VCaP. Accordingly, plasmid reconstruction was prepared for virus transduction and the usability of two cell proliferation analysis methods were compared in VCaP.
ResultsLiposome-mediated transfection methods provided only modest results, not sufficient transfection efficiency for subsequent functional analysis. VCaP cells showed atypical morphology and severely suffered after transfection in many cases. Reverse transfection with polyethylenimine (PEI25) was found to be cytotoxic with all studied variables. Viral transduction preparations including vector ligation were inefficient, showing no positive clones of the correct vector size. Thus, proceeding to more efficient viral infection was not possible.
ConclusionsProstate cancer cell model VCaP was shown to be difficult to transfect. Adherent cells suffered in all studied transfection protocols. The best alternative for transfecting VCaP was shown to be a commercial liposome-mediated forward transfection reagent. VCaP cell line needs thorough optimization for each experiment and further studies to address the function of chromosome 8p21 deletion genes.
Asiasanat:prostate cancer