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CANCER GENE THERAPY: THE ESTIMATION OF REPORTER GENES DELIVERY TO B16-F10 MELANOMA CELLS Milena Czajka1, Agnieszka Zajkowska1, Natalia Stachowiak1, Maciej Małecki1 1 Department of Applied Pharmacy and Bioengineering, Medical University of Warsaw, Poland
INTRODUCTION
RESULTS
Nowadays, gene transfer development has advanced into the use of gene therapy as a novel strategy against various human cancers. Due to its particular set of features, melanoma represents a reasonable target for clinical research concerning various methods of gene transfer which have been developed recently. One of the current methodological challenges in the research of melanoma is discovering high transfection efficiency vectors. Viral and nonviral vectors are the most common in gene transfer systems. Higher transfection efficiencies are usually observed in case of viral vectors compared to non-viral vectors. However, their immunogenicity and toxicity are sometimes problematic. Therefore, they need to be avoided. Non-viral gene delivery has gained considerable importance as a therapeutic tool because of its safety profile, ease of preparation, ability to deliver large gene sizes, and its potential to be modified for tissue- or cell-targeting. In this study, we evaluated the transfection efficiency of pDNA encoding Gfp reporter gene driven by a cmv promoter.
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MATERIALS AND METHODS
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The selected transfection reagents characterized by different mechanisms of action (e.g. lipofection) were used to transfect of B16-F10 melanoma cells (ATCC® CRL-6475™). The cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco BRL, Life Technologies) supplemented with 2% FBS (Lonza, BioWhittaker). Cells were maintained at 37°C The pGFP transfection efficiency was also assessed with PCR, and qPCR methods using selected primers in a humidified 5% CO2 atmosphere. The cultured cells were observed at 48 h after transfection and transfection efficiencies were analyzed with the use of an inverted fluorescence microscope (IX Olympus) and Automated Cell Counter (ThermoFisher). The pGFP transfection efficiency was also assessed with standard PCR (data not shown), and Quantitative PCR methods using primers to itr sequence..
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G
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CONCLUSIONS Our study has shown the usefulness of non-viral vectors in introducing reporter genes into melanoma cells. The highest infection efficiency was observed for pGFP:Att formulation (as was documented both by GFP cell fluorescence and qPCR methods). According to the results, non-viral methods of gene delivery are attainable for melanoma cells. Therefore, the possibility of replacing viral with non-viral methods of gene delivery is a promising option. This work was supported by a grant from NCBiR Development (Strategmed1/233264/4/NCBR/2014, MentorEYE).
Figure. Transfection efficiency of B16-F10 cells measured by (A) fluorescence microscope , (B) automated cell counter, (C) qPCR method. The cells were transfected with six non –viral formulations consist of pAAV/GFP plasmid vector. The results are presented as the mean ± standard deviation. Significant difference between transfection reagents, p
INTRODUCTION
RESULTS
Nowadays, gene transfer development has advanced into the use of gene therapy as a novel strategy against various human cancers. Due to its particular set of features, melanoma represents a reasonable target for clinical research concerning various methods of gene transfer which have been developed recently. One of the current methodological challenges in the research of melanoma is discovering high transfection efficiency vectors. Viral and nonviral vectors are the most common in gene transfer systems. Higher transfection efficiencies are usually observed in case of viral vectors compared to non-viral vectors. However, their immunogenicity and toxicity are sometimes problematic. Therefore, they need to be avoided. Non-viral gene delivery has gained considerable importance as a therapeutic tool because of its safety profile, ease of preparation, ability to deliver large gene sizes, and its potential to be modified for tissue- or cell-targeting. In this study, we evaluated the transfection efficiency of pDNA encoding Gfp reporter gene driven by a cmv promoter.
A
MATERIALS AND METHODS
B
The selected transfection reagents characterized by different mechanisms of action (e.g. lipofection) were used to transfect of B16-F10 melanoma cells (ATCC® CRL-6475™). The cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco BRL, Life Technologies) supplemented with 2% FBS (Lonza, BioWhittaker). Cells were maintained at 37°C The pGFP transfection efficiency was also assessed with PCR, and qPCR methods using selected primers in a humidified 5% CO2 atmosphere. The cultured cells were observed at 48 h after transfection and transfection efficiencies were analyzed with the use of an inverted fluorescence microscope (IX Olympus) and Automated Cell Counter (ThermoFisher). The pGFP transfection efficiency was also assessed with standard PCR (data not shown), and Quantitative PCR methods using primers to itr sequence..
C
G
.
CONCLUSIONS Our study has shown the usefulness of non-viral vectors in introducing reporter genes into melanoma cells. The highest infection efficiency was observed for pGFP:Att formulation (as was documented both by GFP cell fluorescence and qPCR methods). According to the results, non-viral methods of gene delivery are attainable for melanoma cells. Therefore, the possibility of replacing viral with non-viral methods of gene delivery is a promising option. This work was supported by a grant from NCBiR Development (Strategmed1/233264/4/NCBR/2014, MentorEYE).
Figure. Transfection efficiency of B16-F10 cells measured by (A) fluorescence microscope , (B) automated cell counter, (C) qPCR method. The cells were transfected with six non –viral formulations consist of pAAV/GFP plasmid vector. The results are presented as the mean ± standard deviation. Significant difference between transfection reagents, p
