Modified PiggyBac Transpson Systems To Perform The Generation Of Transgenic Donor Nuclei For Bovine Somatic Cell Nuclear Transfer

Transposons, which are in the form of jumping movement in the host genome, are lackof activity in mammal animals. piggyBac (PB) transposon has been developed as atransposable and efficient gene transfer and gene screening tool in mammalian cells. Thereare some reports to confirm that PB transposition isn’t dependent on host factors. Comparedlinearized and viral vectors, PB is more effcient and safer for gene transfer. Thus, it has aboard application prospects in genetic engineering. In present study, PB transpson system hasbeen improved and employed to generate transgenic bovine fibroblastsor mammary epithelialcells as donor nuclei having developmental potential for SCNT. Taken together, theinformation from this study will supply some supports for transgenic animals mediated by PBtransposon. The main contents of this study are as follows:1. Construction of versatile PB transposon systems and identification of basicelements’ functionwe developed three types of PB transposon systems containing a dual plasmid system(DPS),a single plasmid system (SPS), and a DNA-mRNA combined system (DRPS) Thebasic elements of the donor plasmid includeda selectable-reporter gene expression cassette,two loxP sites in the same orientation, a multiple cloning site, and two chicken cHS4insulatorcore elements. To identify above functional elements and plasmids, results showed that threetype PB transpson systems have been successfully established.2. Evaluation for transposition and PBase activity of three PB transposon systemsWe characterized the basic properties of DPS, SPS and DRPS in HEK293cells.Resultsindicated that three PB transpson systems can mediated the PB element to integrate intoTTAA site in genome.We also compared the transposition efficacy between SPS andDPSwith methylene blue stainingand statistical analysis. The finding suggested thatSPStransposed more efficiently.Results discovered the loss of PBase activity in the DRPS,indicating that this system is much more biologically safe, as compared to DPS and SPS.3. Prokaryotic expression of PBaseand verification of its activity in HEK293cellsThe PBase with N-terminal fused TAT peptide was successfully obtained by prokaryotic expression. PBase were determined by Immunofluorescent staining (IF) in HEK293cells.Tail-PCR was used to detect the flanking sequences of PB integrate sites from purified cellgenome. After methylene blue staining for HEK293cell clones, Student’s t-test wasemployed to analyze the colony count.Polyacrylamide gel electrophoresis (PAGE) andwestern blotting (WB) were used to confirm the PBase expression. Results showed that thePBase was successfully expressed by the form of soluble protein. Simultaneously, we foundthat the PBase was localized in the nucleus from the result of immunofluorescent staining.Detection offlanking sequences and colony count indicated that the PBasewith prokaryoticexpression lost its natural activity and caused the failure of transposition.Hence,further studiesare necessary forthis PB transposon system lead to transposition in other eukaryotic cells.However, the comprehensive information from this study supplied a reliable method forforeign proteins mediated with TAT peptide across eukaryocyte membranes.4. Generation of transgenic bovine fibroblasts mediated by PB single plasmidsystemWe developed a bovine mammary specific expression cassette, which was inserted intothe PB single plasmid system pBNW-TP, for human LYZ and LTF gene co-expression.Primary bovine fibroblasts transfected with this plasmid were selected and identified.Moreover, transgenic bovine fibroblasts as donor nuclei were used to produce cloned embryoswith SCNT. In addition, we evaluated the function of bovineβcasein promoter (2.0kb) inBMECs. Results indicated that the LYZ/LTF expression element was inserted into the hostgenome with PB transposition. The transgenic cloned embryos generated with threetrasposable clonal cells had good developmental potentialin vitro. Furthermore, the activity of2.0kb bovineβcasein promoter is nomal.5. PB transposase mRNA combined Cre/loxP system to produceselectable-reporterfree transgenic bovine mammary epithelial cellsThe plasmid pBNW-TP1was used to construct a bovine mammary specific expressionvector for hamn LTF. Combined the PBase mRNA, we obatined stable transgenic BMECs. Toremove the selectable-reporter gene, His-NLS-TAT-Cre recombinant protein generated byprokaryotic expression was transduced into the transgenic BMECs. The findings indicatedthat the PB tranposon system mediated by PBase mRNA can be transposable in BMECs.After deletion of selectable-reporter with His-NLS-TAT-Cre recombinant protein, expressionof LTF is normal in the transgenic selectable-reporter free BMECs. The cloned embryosgenerated by selectable-reporter free BMECs had good developmental potential. Thesecomprehensive information suggested that the selectable-reporter BMECs is higher biosaftyand totipotency. In present study, modifications for PB transpsoncan improved the transpositionefficiency and biosafty in gene transfer. The improved selectable-reporter makes moreconveniently identify and select the positive cells. The transgenic embryos generated by PBtranspson have good developmental potential in vitro. This finding will lay the foundation forproduction of transgenic animals and mammary gland bioreactors.