Transient transformation of Cryptosporidium parvum

The goal of the present research is to develop a genetic transformation system for Cryptosporidium parvum. Genetic transformation is an essential tool for analyizing gene function and regulation, but it is not avaliable for C. parvum. Establish a transient transformasyon system for C. parvum, electroporetic transformation of sporozoites with plasmids carrying C. parvum intergenic regions flanking a reporter ( Prot L11 on BX538351 segment 2/4, between 84361- 85501 bp ); detection of transformants in cell culture by fluorescent microscopy or reverse transcription PCR. In this study, transformation efficiency was optimized by testing different plasmid constructs (pCMV-DsRed and pEYFP-Mem expression vectors) and electroporation conditions It was showed that all of the using excystation condition, TA/cytomix:RPMI-1640media (1:1) was the best condition and determined 80% of excystation rate. Electroporated sporozoites giving 1 pulse infected HCT-8 cell culture much more efficiently than the other pulse. pCMV-DsRed and pEYFP-Mem Vectors were transfected into HCT-8 cell line by using both electroporation and lipofectamine methods. Transfected pCMV-DsRed and pEYFP-Mem Vectors expressions at 80 voltage, 50 capacitance, none resistance of electroporation condition observed by fluorescent microscopy. However when giving 1000 voltage, 50 capacitance, 50 resistance in electroporator, transfected pCMV-DsRed Vector and pEYFP-Mem Vector and Crypto-construct GFP Vector expressions could not be observed visually by fluorescent microscopy. There was no visually C. parvum (Crypto-construct GFP Vector) expression in both of elektroporation condition by fluorescent microscopy. In order to show incase of avaliable a little GFP mRNA expression of C. parvum vector in transfected HCT-8 cells by two electroporation conditions was used RT-PCR, Nested PCR. Key words: Cryptosporidium, Cell culture, Electroporation, Lipofectamine, Real time PCR