Piggybac Transposon Mediates Foreign Gene Expression In Porcine Fibroblasts And Cloned Embryos

Transgenic animals have great production and research roles, which they can be employed to detect nature of life, such as study the relationship of gene structure and function, cell development potential, embryo development regulation and tumor formation. They can also be applied to establish animal model to study the pathogenesis and therapeutic methods. Moreover, transgenesis technologies can be used to increase the animal breeding efficiency. Also transgenic animals can be used as the medical and eating protein bioreactor. However, transgenic research encountered with some questions,for example, transgene with plasmid random insertion results in the gene lost and shutdown because of the site effect and methylation in germ line, which will lead to the low transgene efficiency; on the other hand, titre control and and virus vectors auxiliary virus derived from virus vectors limit its preclinical and production apllication. Therefore, it's an important premise to find a suitable transgenic vector. In this study, we employed piggyBac transposon as the transgenic vector to deliver EGFP and follistatin into porcine fetal fibroblast and cloned embryos to identify its relative merit in porcine transgenesis. And all the results are as follows:1 In order to estimate whether there was a difference in plasmid entering cytoplasm process between transposon binary plasmid system and pEGFP-C1 unitary plasmid system, FCM was employed to assess the EGFP transient expression at 12hr,24hr,48hr after transfected with donor/helper and pEGFP-C1. The eGFP positive cell rate for piggyBac group were 6.3%, 12.53% and 8.9% while for pEGFP-C1 group the rates were 6.4%, 16% and 8.3% at this three timing, respectively.2 Cells were rendered G418 selection at 48hr after transfection. Cell colonies were counted on the 7th day. At the premise of same amount of transfected cells, piggyBac transposon system produced 21 cell colonies per 1000 cell, representing a 18-fold increase over transfection with pEGFP-C1 random insertion.3 Genome walking method was employed to amplify the flanking sequences of 5'TRs to identify the piggyBac transposition. The sequencing results proved that the transposon flanking sequences were not homogeneous to the donor plasmid backbone.4 Several of the sequences were identified as a part of sequence on porcine chromosome 2,9,15. And all the junction sites of piggyBac backbone and genome were TTAA tetra-nucleotides. Several of the sequences from genome walking were identified as a part of sequence on porcine chromosome 2,9,15, even though the whole porcine genome sequence was inavaiable to date. 5 Foreign gene copy numbers was exhibited by southern blot, and the result showed that piggyBac transposon induced 2 copies in porcine genome, which was confirmed by three enzymes digesting porcine genome.6 Real-Time PCR was employed to detect the EGFP expression mediated by piggyBac system and random insertion in EGFP positive cells . The results showed that piggyBac mediated 4-folds EGFP mRNA more than random insertion in cells.7 To detect the foreign gene promotor methylation status change during cell passage, we identify the promotor methylation change in different cell passage, and the result showed that there was no changes in foreign gene promotor methylation status.8 Different cell groups were transfected with the same amount of donor plasmid while different amount of helper plasmid to detect the piggyBac transposase OPI in porcine PFF. Ten days after selection, G418-resistant cell colonies were counted under microscope. The resistant cell colonies increased when the ratio of donor and helper was 1:1 to 1:4, but there was a sharp decrease when the ratio of donor and helper was 1:5.9 CCK-8 was employed to assess the effect of transposase on PFF proliferation. OD450 values of cell culture medium was decreased along with the increase of transposase in both donor/helper group and pEGFP-C1/helper group. The OD450 values of donor/helper group were significantly higher than that of pEGFP-C1/helper group regardless of the concentration of transposase, only but the amount of helper was equal with donor or pEGFP-C1.10 EGFP positive cells derived from piggyBac transposition and random insertion were employed as the donor cells to produce nuclear transfer embryos.The cleavage rate, blastocyst rate and blastocyst cell number of constructed embryos derived from piggyBac transposition were 85.27% ,12.34% and 47, respectively, while 86.24%, 16.21% and 42 for embryos derived from pEGFP-C1 random insertion. The cleavage rate and blastocyst rate and blastocyst cell number were 84.62%, 13.84% and 47.5 in the control group, in which the donor cells were non-transfected PFF. There were no statistic differences with the cleavage rate, blastocyst rate and the blastocyst cell numbers among the three groups.11 Real-Time PCR was employed to detect the EGFP expression mediated by piggyBac system and random insertion in NT embryos. The results showed that piggyBac mediated 17-folds eGFP mRNA more than random insertion i in cloned blastocysts.12 In follistatin positive porcine fetal fibroblast, the cells proliferate thinner and longer and clone formed. On the other hand, Real-Time PCR results showed that gene expression of p53 and desmin increased significantly.