Production Of Triple-transgenic Mice Expressing Human HT,DAF And CD59 Gene And The Studies Of These Transgenes In Xenograft Rejection

Our approach is based on the likelihood that a multifaceted, integrated approach will be necessary to prolong xenograft survival to the point of clinical acceptance. Transgenic mice were produced by microinjection of transgene constructs for the human [alpha]l,2-fucosyltransferase (HT), decay accelerating factor (DAF) and cluster of differentiation 59 (CD59). The aim of the paper was to investigate the effect of expressing human HT gene in vivo on α -Gal antigen and the role of human HT, DAF and CD59 in overcoming hypercute rejection (HAR) and acute vascular rejection (AVR).SECTION IConstruction of Recombinant Human Decay accelerating Factor Gene Objective To construct an recombinant human decay accelerating factor gene using UI enhancer for high-performance expression in xenotransplantation. Methods Cutting the pSP73 and pBluescript II SK~+ plasmid with ClaI/EcoRI restriction endonucleases, and obtaining the recombinant pBluescript II SK~+ plasmid which containing UI enhancer. Then cutting the pGEM-7zf-DAF and recombinant pBluescript II SK~+ plasmid with XbaI/BamHI restriction endonucleases, and obtaining the new recombinant pBluescript II SK~+ plasmid which containing UI enhancer and DAF cDNA and ICAM-2 promoter. The plasmids pick-uped from positive transformed germs were identified by the restriction endonucleases and polymerase chain reaction (PCR). Results Obtaining special fragments by different restriction endonucleases. Further, amplifing and obtaining 330 bp and 321 bp special fragments from the positive plasmid by PCR. These results accord with completely the demands of the designs. Conclusion The recombinant human decay accelerating factor gene using UI enhancer for high-performance expression inxenotransplantation is constructed successfully.scetion nGeneration of transgenic mice expressing human HT/CD59 and DAF Objective To generate transgenic mice expressing human HT/CD59 and DAF gene. Methods The HT, DAF and CD59 transgene constructs were cutted from their vectors by using NotI/PvuI, XbaI/ClaI and BglII/SmaI restriction endonucleases respectively. The HT transgene construct mixed with the CD59 transgene contruct were co-injected into cytonucleus of fertilized mouse ova, and the DAF transgene construct was injected into cytonucleus of fertilized mouse ova. Results Co-injection of the HT/CD59 transgene constructs resulted in 132 liveborn mice and 89 transgenic mice expressing human DAF were born. Conclusion The primary generation of transgenic mice expressing human HT/CD59 and DAF gene has been generated successfully.section IIIScreening of transgenic mice expressing human HT/CD59 and DAF Objective To screen the primary transgenic mice expressing human HT/CD59 and DAF. Methods Genomic DNA was isolated from all of liveborn mice. At first, integration of the foreign genes was screened by PCR. Then Southern-blot analysis was done to make further confirmation on the results. Expression of the foreign genes on peripheral blood mononuclear cells (PBMCs) of transgenic mice was detected by Flow Cytometry (FCM). RT-PCR analysis was used to detect the level of human HT, DAF and CD59 mRNA expression in the heart, liver, kidney and lung of transgenic mice. An immunocytochemical survey was performed to examine the distribution of human H antigen in the tissues from transgenic mice. Results Integration rate of human HT, DAF and CD59 gene were 11.4% (15/132), 22.5% (20/89) and 14.5% (19/132) respectively, comprising five HT/CD59 double transgenics. Expression rate of human HT, DAF and CD59 gene were 4.5% (4/89),7.6% (10/132) and 6.1% (8/132) respectively, and four of the five double transgenics expressed human HT and CD59 gene. Expression of human HT, DAF and CD59 mRNA could be detected in the heart, liver, kidney and lung of transgenic mice. The immunocytochemistry and FCM studies showed high-level expression of H antigen and with reduction of a -Gal antigen in mice expressing human HT gene. Conclusion The primary generation of transgenic mice expressing human HT/CD59 and DAF gene have been generated successfully.SECTION IVScreening of expression of transgenes in the offspring from transgenic mice expressing human HT, CD59 and DAF Objective To explore the rule of heredity of foreign genes in transgenic mice and to generate transgenic mice co-expressing human HT/CD59/DAF gene for further experiments. Methods The HT/CD59 transgenic mice were bred with the DAF transgenic mice to generate Fl and F2 generation of transgenic mice. Screening was performed as described above. Results Among seventeen Fl generation transgenic mice, co-expression of human HT/DAI^ HT/CD59s HT/CD59/DAF were two, two and one respectively. Among eight F2 generation transgenic mice, co-expression of human HT/D^ HT/CD59^ HT/CD59/DAF were two, three and zero respectively. Conclusion Transgenic mice co-expressing human HT/CD59/DAF gene has been generated successfully. The level of expression of transgenes is different in offsprings of transgenic mice.SECTION VResearch of the transgenic mice expressing human HT, CD59 and DAF to avoid xenograft rejectionObjective To investigate the ability of transgenic mice expressing human HT, DAF and CD59 to avoid HAR, and to explore the mechanism of transgenic mice to inhibitAVR. Methods Transgenic mice were divided into five groups, group I: normal mice;group II: transgenic mice expressing human HT gene;groupIII: transgenic mice co-expressing human HT/CD59 gene;groupIV: transgenic mice co-expressing human HT/DAF gene;group V: transgenic mice co-expressing human HT/CD59/ DAF gene. The mice's hearts were perfused ex vivo with 12% human plasma by using a modified Langendoff apparatus, and the effect on cardiac function was determined. Paraffin sections of perfused hearts were stained to examine deposition of the IgM, IgQ C3c and C9, activation of NF- k B, expression of ICAM-1 and E-Selectin on the vascular endothelium. Results Compared to hearts of group I, during perfusion with 12% human plasma, the hearts of group II were protected with work maintained at 27% of the maximum level during a period of 60 min. Compared to hearts of group I and group II, the hearts of groupIII and groupIV and group V were further protected. Tissue sections from normal hearts showed deposition of the IgM, IgQ C3c and C9, activation of NF- k B, expression of ICAM-1 and E-Selectin on the vascular endothelium. Tissue sections from the triple-transgenic heart only showed deposition of the IgG Conclusions The present study suggests that the expression of human HT gene partly protected mouse hearts from HAR. The transgenic mice expressing multiple molecules address different humoral components of xeograft rejection to overcome HAR and may partly inhibit AVR by inhibiting activation of NF- k B.