Construction Of Expression Vector Of Human Lysozyme Gene/Lactoferrin Gene And Development Of Bovine Transgenic Cloned Embryos Cultured In Vitro

Expression sequence of human lactoferrin(LTF) and internal ribozyme entry site (IRES)were successfully connected by double enzyme digestion and directed cloned, andrecombination plasmid pIL was constructed. Mammary gland specific expression plasmidpEBHIL of human lysozyme and human LTF was constructed by BamHI restrictive enzymedigestion. plasmid pEBHIL was introduced to cow mammary gland epithelial cells afterliposome transfection. Positive cells were determined by G418and PCR, and they were usedas donor cells to produce transgenic cloned embryos by nuclear transplantation. Exogenousgenes in transgenic cloned embryos were detected by PCR and observation of expression ofgreen fluorescent protein(GFP),aiming to transfer positive cloned embryos and producetransgenic animals. Results show as follows:1. Human LTF gene (2255bp) and IRES sequence (986bp) were separately cloned byPCR. Human LTF gene was cloned at T-site of pMD18-T Vector after PCR products wererecovered and purified, and named the plasmid phLTF. IRES was cloned at T-site of pGEM-TEasy Vector and the positive recombination plasmid was markered as pIRES. Plasmid phLTFand pIRES were digested by Xba I and Nhe I separately. Objective fragments were recoveredand connected with T4DNA ligase, and then transformed to Bacterium coli DH5α. Positiverecombination plasmid was detected and named as pIL. pIL and pEBH were digested byBamH I. Objective fragments were connected with T4DNA ligase and connective directionwas identified by BamH I digestion. Mammary specific expression vector was establishedand markered pEBHIL which contained regulatory elements(cow β-caseinum5′and3′regulatory sequence), objective genes(human lysozyme gene and hLTF gene), IRES, reportgene(GFP gene), and antibiotics gene(neo+gene).2. Expression vector pEBHIL containing human lysozyme gene and human LTF genewas introduced to cow mammary gland epithelial cells after liposome transfection. Positivecells were determined by G418and PCR, after GFP expression observed by fluorescentmicroscope, and proliferated and induced by hormone. The results of Western Blottinganalysis on cell supermatant showed that transfected cells han expressed and secreted human lysozyme (MW14.7ku)and human LTF (MW40ku). Transgenic cells were cultured in vitrofor15generations and the chromosomes of transgenic cells of generation3,5,7,9,11,13wereanalyzed. The results indicated that cells cultured in vitro didn't transform, therefore theywere provided as donor cells for the production of transgenic bovine by nucleartransplantation.3. The construction efficiency was analyzed concerning enucleation method of oocytesat MⅡ, synchronization of nuclei donor cells, mammary gland epithelial cells of transgenicor not, thansgenic nuclei donor cells of freezing-thawing or freezing-free and passagegenerations of transgenic mammary gland epithelial cells. The results showed that there was asignificantly higher enucleation rate for DM assistant enucleation(DM) as compared withblind aspiration method(BAM()19.5%vs11.0%,p<0.05). It was better for contact inhibitionto induce bovine mammary gland epithelial cells synchronization than that of serumstarvation(19.5%vs11.8%,p<0.05). The construction rate of cloned embryos was notsignificantly different between mammary gland epithelial cells of transgenic or not used asnuclei donor cells(16.8%vs18.7%,p>0.05). Freezing-free transgenic bovine mammarygland epithelial cells used as donor cells made a higher construction rate compared withfreezing-thawing cells(16.8%vs9.2%,p<0.05). There was no difference among the groups ofpositive transgenic mammary gland epithelial cells of generations3,5,7and9used as donorcells to construct cloned embryos(p>0.05).4. The foreign objective genes were combined in the chromosomes of the transgeniccloned embryos successfully through PCR detecting. GFP expression was observed intransgenic cloned embryos by fluorescence microscopy. The results indicated that GFPexpressed at2-cell stage of some cloned embryos, with little expression amount and weakfluorescence, and expressed cells increased, GFP expression enhanced gradually withculturing time. These results help to explain that foreign gene could express in early embryostage and GFP gene could applied as report gene to detect whether foreign gene combine andexpress in the embryos of their early stages.