Abstract:
Electroporation in combination with the red and yellow fluorescent protein (RFP and YFP, respectively) reporter systems results in transient gene expression in HCT-8 cell lines infected and uninfected with Cryptosporidium parvum. Conditions are described allowing efficient electroporation of HCT-8 cell monolayers in a commercial electroporation device (180 kV/cm voltage, 250 A mu F capacitance, and 0 ohm abroken vertical bar resistance resulting in a time constant of 0.4 ms.). Fluorescent microscopy showed that uninfected HCT-8 cell monolayers achieved higher gene transfer and expression efficiencies than HCT-8 cells infected with C. parvum under similar conditions. Real-time melting curve and quantitation analysis of reverse transcription polymerase chain reaction amplicons are presented for the detection of all mRNA sample levels. Our findings demonstrate that C. parvum infection of HCT-8 cells may negatively affect the transient expression of RFP and YFP plasmids. The focus of this study was to achieve transient expression of reporter genes in HCT-8 cell lines and provide the basis for further analyses of gene regulation in protozoan. These approaches may provide to understanding the feasibility of gene transfer and expression efficiencies HCT-8 cell cultures by RFP and YFP containing the CMV promoter; they could serve as tools for gene transfer in mammalian cell.