Gene-engineering construction with structural gene of human lactoferrin under control of regulatory elements of bovine -casein gene with reporter gene EGFP under cytomegalovirus promoter and its influence on viability of pre-implantation rabbit embryo
With the use of PCR-amplification the fragment of plasmid pсEGFP with gene of modified green fluorescent protein (EGFP) under cytomegalovirus (cmv) promoter and polyadenilation site (PolyA site BGH) was obtained. This fragment was cloned into intermediary vector pGEMT. The plasmid so obtained was named as pGEMTcmvEGFP. Isolated and purified fragment cmv-EGFP-PolyA site BGH was cloned into plasmid pas1Lf on site NotI. So, the plasmid was obtained, named as pas1LfGr,  which contained gene-engineering construction with two various genes: lactoferrin gene under regulatory elements of bovine gene of -casein and gene of modified green fluorescent protein under cytomegalovirus promoter. The whole construction was excised with restrictase ClaI (Bsu15I) and purified. After micro injection into pronucleus of rabbit's zygote the fluorescence was revealed in 35.4% of embryos and only 3.2% of microinjected embryos developed to stage of blastocyst (1/31). The data obtained suggest that there was significant decrease in survivability of rabbit's zygotes after in vitro micro injection into pronucleus.