Transgenic Research Of Fat Metabolism Genes Mediated By PiggyBac Transposon

As the world’s second consumption meat, beef get more and more pro-consumer’s gaze. Thus, it isof graet effort to improve the beef quality and nutrition for breeders. Currently, transgenic technology isone of the most important methods for beef cattle breeding. They are hot topics to find a safe andefficient transfer vector and useful target genes in transgenic research. PiggyBac（PB）transposons is auseful tool for transgenic insect, mouse and etc., however, the studies about PB transposons intransgenic cattle were few. Fatty acid binding protein3(FABP3) is a candidate gene for intramuscularfat (IMF) deposit; Ω-3fatty acid desaturase encoded by fat-1gene can catalyze the conversion of ω-6polyunsaturated fatty acids (PUFAs) to ω-3PUFAs that were good at improving the health of human.The study was to probe the transposition of PB transposon in bovine genome and verify the function ofFABP3and Bfat-1(optimized gene of fat-1) gene. The results of the study were as follows:1. Donor plasmid of PB[CMV-EGFP] and helper-dependent plasmid of pcDNA-PBase wereconstructed and transferred into bovine ear tissue fibroblasts by amaxa basic nucleofector kit forprimary mammalian fibroblasts. The results showed that the cell colonies of experimental grouptransfected with PB co-transposition system were9times higher than the control group transfectedwithout PB transposase (PBase).2. Genomic DNA of transgenic cells was extracted and integration sites of transposon5′Bac weredetected by genome walking technology. Eight integration sites were obtained in bovine genome and5sites were mapped on chromosomes1,2,11and X chromosome. The results showed that PB transposonwas inserted into TTAA location and5′tended to be inserted into intergenic non-regulatory regionthat rich in GC (62.5%).3. Six potential single nucleotide polymorphisms (SNPs) were detected in Simmental andXuelong Black cattle populations for FABP3gene by DNA-pool sequencing. And T67C locus was notsignificantly associated with backfat thickness, marbling score and shear force in the experimentalpopulation (P>0.05).4. FABP3gene was cloned by RT-PCR using bovine muscle tissue; Bfat-1gene that expresshighly in cattle genome was codon optimized gene of C. elegans fat-1gene based on the codonfrequencies of11cattle muscle protein genes. And the nucleic acid, protein structure and function ofthe two candidate genes were predicted by the bioinformatics methods.5. Four bovine FABP3transgenic mice were made by microinjection. The blood and urinebiochemical analysis, the systemic fat tissue magnetic resonance imaging (MRI) scanning of transgenicmale mice and wild-type mice were tested. The results confirmed initially that FABP3gene can promotefat deposition.6. PB[CMV-EGFP-Bfat-1] plasmid was injected into mice by hydrodynamic tail vein injectionmethod. Bfat-1can transient express in mouse liver. After24h,48h and72h, ω-6/ω-3PUFAs ratio of18and20carbon (except48h) decreased significantly in liver (P<0.05); only the ratio of20carbondecreased significantly in muscle (P<0.05). 7. PB[CMV-EGFP-FABP3] and pcDNA-PBase circle plasmide were transferred into bovine fetalfibroblasts by amaxa basic nucleofector kit for primary mammalian fibroblasts. And transgenic cellswere obtained by G-418. It will provide experimental material for the production of FABP3transgeniccattle in the future.