| IntroductionTransplantation has become the treatment of choice for end - stage organ failure. Low rates of organ donation, however, have resulted in a shortage of a-vailable organs. Xenotransplantation offers a potential solution to this problem. In recent years, researchers have identified the pig as the most suitable donor of organs for use in humans.Transplantation of unmodified pig organs into primates results in rapid and vigorous graft destruction by an innate immune response known as hyperacute rejection. HAR is characterized by interstitial hemorrhage and thrombosis, and occurs due to complement activation initiated by the deposition of preformed xe-noreactive natual antibodies ( XNA ). XNA, found in humans and old world monkeys, are directed against the a - gal epitope that is present on the cells of lower mammals and New World monkeys. Higher primates lack a - gal due to evolutionary loss of a galactosyl transferase enzyme that accounts for its production.The objectives of this study were to clone the PFGL2 gene and cDNA; and to make a preliminary analysis of it's structure. Also the m RNA transcription level were analyzed after incubation of PAEC with xeno serum. After a pig - baboon kidney xenotransplantation, the pathology changes and FGL2 transcription will be explored and the role of pig FGL2 in the development of acute vascular rejection will be explored.Materials and Methods1. Pig genomic library screening: A bacteriophage porcine genomic library was screened by hybridization with a murine FGL2 probe, which were labeledwith a[32P] dCTP by random primer mehtod. DNA inserts from positive clones were mapped through restriction enzyme digestion, southern blot, and subcon-ing. Relevant regions were sequenced on both strands. 2. Chromsome walking; New probes were designed according to the 3' end of acquired sequence of porcine FGL2 gene. Forward primer3:5'-ATA, TAA,GCG, AGT,GTC,CCC, TGA,TT-3'; reverse primer3: 5'-TIT, TCC, TCA, GTG, ATG, AGT, GAA, CC-3'Forward primer4: 5'-CAA, AAA, TGC, AGC,TTT,CCC, CAT,T-3'; reverse primer4: 5'-GGA, GTG, CAC, TTT,TGA, TCA, CAG, AAA-3'. Genomic DNA was extracted from porcine aortic endothelium cells culture and the PCR product were labelled and used in the rescreening procedure. 3. PCR reaction using porcine genomic DNA as the template: forward primer was designed according to the acquired consensus region of human and pig FGL2 3' sequences while reverse primer was designed from human FGL2 - 3 'end downstream sequence; forward primer 5'-AAT, AGG, ATA,CCA, AAT, GTA, AAT, G-3'; Reverse primer: 5'-TGG, TGT, TCC, TCT, ATT, TGC, CTC, T-3'; Advantage 2 polymerase mix was applied and genomic DNA was used as the template of the PCR reaction. The PCR product was then TA cloned into a plasmid vector and sequenced. 4. Alignment analysis of FGL2 gene within porcine, human and mouse species: Blast soft ware. 5. Fluorescence in situ hibrid-ization analysis of porcine FGL2 gene: The positive clone DNA was labelled by a[32P] dCTP and hybridized with the G2 phase porcine cell to detect the location of this gene in the pig chromsome set. 6. FGL2 gene prediction analysis: Blast and gene scan software were used to analyzed the gene structure of FGL2, including the exon and intron location, transcription starting and terminating sites, potential transcription factors and promoter regions. 7. the coding region was amplified by RT - PCR from intestinal RNA and sequenced on both strands. Forward primer: GCC, ACCG,GAG, AGA,TGA,AG; Reverse primer: GAG, ACC, CAA, GTA, CTT, TAA, GCC, ATA, A; No template RNA and no reverse transcriptase were used as the negative controls and GADPH was used as positive controls. 8. Northern Blot: Total RNA were extracted from fresh porcine heart, liver, lung, kidney, small bowel, pancreas, and spleen tissues, then they wereloaded in Rnase free agarose gel and transferred to a nitro cellulose membrane. CDNA probe was prepared by labelling of 1341 RTPCR product with a[32P] dCTP through random primer method. The membrane was then hybr...
|
PDF Full Text Request