| The chronic shortage of human organs, tissues and cells for transplantation has inspired research on the possibility of using animal donor tissue instead. Transplantation over a species barrier is associated with rejections which are difficult to control. Vascular endothelium is the most immediate barrier between the xenogeneic donor organ and host immune and nonimmune defense systems. Thus, these cells are the prime targets for such genetic modifications.Transgenic pigs have been made that express human complement regulatory proteins such as CDS9 and DAF, which allow these pigs to withstand, at least partly, the attack of human complement following natural antibody binding and complement activation. However, specific high-level expression of transgenes throughout the vascular tree in adult animals has proved difficult to achieve, perhaps because of the inherent heterogeneity of endothelium. In all these cases promoters with activity in a wide range of tissues were used. In cases in which a general expression of the thansgene may be incompatible with normal physiology, or even lethal to the host, one would want to restrict the expression of the transgene to the cell type of interest, e.g. endothelial cells(ECs). Hence, selecting the strong endothelial-specific promoters become one of most important parts in the research of xenotransplantation. The promoter of intercellular adhesion molecule-2 (ICAM-2) has been a endothelial cell-specific promoter studied currently and could drive transgen to expresse high-level in vivo and in vitro. Moreover, many studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograftsurvival and improving the prospect of clinical xenotransplantation.According to the above, we want to establish the transgenic pigs expressing two human complement regulator}' proteins that can prevent rejection .The upstream job of the pigs is to construct the effective specific vectors expressing CD59 andDAF gene. In present study, we cloned the promoter sequence of ICAM-2 fromthe human blood genome, and the fragments of intronl of CD59 and DAF genesrespectively. ICAM-2 promoter acted as a promoter, and the fragment of intron 1acting an enhancer, the expression vectors of CD59 and DAF gene wereconstructed respectively. Using the method of liposomes transfection, the vectorswere transferred into pig aorta endothelial cells and the expression were measuredby flow cytometer and PT-PCR. Finaly, the endothelial cells expressing resistantDAF proteins were incubated with human serum, and the function of thetransfeutants resisting lysis from human serum were measured.l.The construction of the expression vectors of CD59 gene using ICAM-2promoterMethods Produced ICAM-2 promotor fragment and CD59-intronl fragment byPCR from the human blood genome, then inserted these fragments into apcDNA3-CD59 eukaryotic expression vector. Digested this recombinant plasmidwith the special restriction endonucleases (for example.EcoR I /HindLU) . TheICAM-2 promoter and CD59-intronl fragments was identified by PCR, and wassequencd.Results Productions by digestion accord with the design. The two DNAfragment sequences. ICAM-2 promoter and CD59-intronl fragments, are thesame as the frames of the gene bank.Conclusions The specific expression vector of CD59 gene was constructedsuccessfully.2. The reconstruction of the specific expression vectors of DAF geneMethods Cut the pGEM-7Zf -DAF plasmid with restriction endonucleases,andobtaining the recombinant human DAF gene which containing ICAM-2 promotorfragment, DAFcDNA and polyA. In the same way, obtained the pcDNASfragment expression vector. Above two DNA fragments performed recombinantreaction and the production was transformed into reception germs. Theplasmids pick-uped from positive transformed germs were identified by the...
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