Studies On Expression And Function Of Interferon-a Gene In The Transgenic Cells And Nuclear Transfer Embroys Of Cattle

This study was carried out to produce transgenic dairy cattle by somatic cell nuclear transfer(SCNT),which contains Lactoferricin gene and interferon α gene to resist the mastitis and the foot and mouthdisease. The results were as follows:A vector with bovine Lactoferrincin B cassette containing a goat β casein regulatory sequence andhuman interferon α cassette containing the immediate early promoter of CMV,and with a EGFP gene asreporter and a neomycin resistance gene for selecting, was constructed in this experiment.Bovine fetal fibroblast (BFFb) cells were isolated by digesting and attaching tissue explants from theskin of bovine fetus at the age of40days. The BFFb cells were transfected with the vector by FuGENETransfection Reagent and Amaxa Nucleofector.The transgenic cell lines were obtained after the selectionof G418by the PCR to amplify the fragment of target gene. Also, we detected the interferon α proteinin transgenic cells by Western Blot.Antiviral ability of the transgenic cells were detected by infecting the cells with vesicular stomatitis virus(VSV) and titrating VSV in the supernatants. The virus titer was reduced markedly in transgenic cellscompared with the control group. Then the cell viability was estimated by dye exclusion method and theresults showed that77percent transgenic cells infected with VSV eventually underwent apoptosis whileonly less than11percent control cells were apoptosis. The expression of antiviral genes were analyzedby quantitative real time RT–PCR.The results showed that mRNA of OAS、PKR and P53wereinduced to express highly in the transgenic cells.Transgenic SCNT embryos were produced by a new SCNT method that combines the micro extrusionand zona free fusion with a higher fusion rate than micro injection. Foreign genes have been integratedwithin the genome of the transgenic SCNT embryos by PCR test and interferon α protein has beenexpressed in the transgenic SCNT embryos detected by immunohistochemical analysis. There is nosignificantly difference in the fusion rate, cleavage rate and blastocyst rate between two differentactivation methods. Forthy six transgenic SCNT embryos were transplanted into the surrogate cows and5cows had been pregnant. Two cloned calf were born and the PCR identification results showed thevector had integrated into one of the SCNT calf genome. Fibroblast cell line was established from eartissue of the transgenic SCNT calf and used as donor cells for re cloned.