The Studies Of Transgenic Mice Tissue-specifically Expressing Human DAF,CD59 On The Vascular Endothelium For Xenotransplantation

Using transgenic approaches to express human CRP in animals is believed to be an effective way of overcoming the HAR. Transgenic animals ubiquitously expressing human CRP have been constructed and proved to be afforded significant protection from human serum-mediated attack. However a general expression of the transgene may be incompatible with normal physiology, or even lethal to the host. To eliminate the side-effects of ectopic expression, we constructed the transgenic mice tissue-specifically expressing hDAF, CD59 on the vascular endothelium by co-injection of hCD59 and hDAF expression constructs driven by the human intercellular adhesion molecule 2 (ICAM-2) promoter. The purpose of the present study was to produce transgenic mice tissue-specifically expressing hDAF, CD59 on the vascular endothelium, to investigate whether expression of hDAF, CD59 can provide further protection against human complement-mediated attack, and to explore the rules of endothelial-specific expression and of the heredity in transgenic animals.SECTION I Generation of transgenic mice tissue-specificallyexpressing human DAF, CD59 on the vascular endotheliumObjective To generate transgenic mice tissue-specifically expressing hDAF. hCD59 on the vascular endothelium. Method Plasmid DNAs constructed in our lab were prepared by alkali lysis and purified with polyethylene glycol (PEG). The analysis of restriction map show that two gene were well-constructed and suitable for transgenic animal experiment. The hDAF and hCD59 expressioncassettes were excised from their vectors by using Xba I / BamH I /Sea I and Bgl II /Sma I respectively, then separated, purified, mixed in EDTA buffer at approximately 5 u g/ml each and stored at low temperature. The DAF cassette (3.7kb) comprises human ICAM-2 promoter, hDAFcDNA (including the first hDAF intron) and the SV40 splice/polyA. The CD59 cassette (2.2kb) contains human ICAM-2 promoter, the first hCD59 intron, hCD59cDNA and the BGH polyA. The purified DAF and CD59 constructs were co-injected into the male pronucleus of fertilized mouse ova by standard techniques. After cultured for 1 h in BMOC-3+10%FCS, the morphologically excellent injected Embryos were surgically transferred into pseudopregnant foster mothers, and pregnancy allowed to proceed until term. Results 80 superovulated females were made, and 2100 fertilized eggs were collected. Among the 1700 fertilized eggs with clear pronuclear, 1300 health eggs survived the microinjection. They were transferred into the oviducts of 50 psedopregnant recipient mothers. 30 females were pregnant and 135 mice were born. Conclusion The primary generation of transgenic mice have been generated successfully.section IIScreening of transgenic mice tissue-specificallyexpressing human DAF, CD59 on the vascular endotheliumObjective To screen the primary transgenic mice tissue-specifically expressing h DAF, hCD59 on the vascular endothelium. Method Genomic DNA was isolated from all of newborn mice. At first, the foreign genes were screened by PCR using human ICAM-2 promoter DNA as the template. Then Southern blot analysis was done to make further confirmation on the results. Total RNA was isolated from the peripheral blood leukocytes (PBLs) of transgene-integrated mice. RT-PCR wasdone to test hDAF or hCD59 gene expression at mRNA levels. Cells from the PBLs were incubated with the PE-conjugated mouse anti-human DAF mAb or the FITC-conjugated mouse anti-human CD59 mAb, both at 1:100 dilution. Stained cells were analyzed by FACS analysis to verify the existence of the transgenes at protein level. An immunocytochemical survey was performed to examine the distribution of transgenes in the tissues from transgenic mice. Results PCR indicated there were 55 mice containing the human ICAM-2 promoter DNA. Thirty five (35/55) were transgenic by Southern blot analysis, comprising five double transgenics and thirty single transgenics (21 DAF, 9CD59). Among these transgene-integrated mice, expression of hDAF or hCD59 was detected in five single transgenic...