Determining GFP activity in HCT-8 cells infected with Cryptosporidium parvum by reverse transcription polymerase chain reaction and nested PCR

Abstract

This study focusses on the determination of GFP activity in HCT-8 cells infected with Cryptosporidium parvum measured by RT-PCR and Nested PCR. Our findings demonstrate that the level of expression for green fluorescent protein reporter vector was undetecteable under fluorescent microscope at 48 h postelectroporation in the presence of 25 μg closed-circular construct. Realtime melting curves and quantitation analysis of RT PCR and Nested PCR were used to show low mRNA expression of GFP. The methods used here allow detection of GFP activity in HCT-8 cells. Here we describe the preliminary study which forms the basis for future transient transformation of C. parvum. These approaches may contribute to our understanding of the feasibility of transforming C. parvum, since the ability to transiently transform C. parvum should lead to expanded research on this important parasite.