Transformation of Digested Genomic DNA of Cryptosporidium parvum with Different Restriction Enzymes to Green Fluorescent Protein Reporter (GFP) Vector

Abstract

The focus of this study is to develop methods for transformation of random genomic fragment of Cryptosporidium parvum DNA with GFP vector. We propose to insert upstream and downstream intergenic regions from C. parvum genes into plasmid carriying the green fluorescent protein reporter. For this objective, random genomic fragment of C. parvum DNA was digested NcoI-SpeI; Fat I-XbaI and BspHI-XbaI, and fragments between 750 and 23000 bp were gel purified and cloned into a GFP plasmid vector. Colonies were randomly selected and subjected to automated sequencing by using one or two primers flanking the cloning site. Even though sequence of the C. parvum genome very nearly was completed, genetic transformation is needed to translate genomic sequence the function of unknown genes and identifying regulatory sequences. We described here the preliminary study which form the basis of future for C. parvum gen transformation and to construct gene manipulation techniques.