Analysis Of Structure And Function Of Porcine PNAS-4 And Construction Of Targeting Vector For Knock-out α1,3-GT Gene

Part I Analysis of structure and function of porcine PNAS-4 Being involved in the whole lifecycle of organism, apoptosis, which is ubiquitous and also normal of cell death, is known to play an important role during organism development, survival and maintain normal physiological function especially in embryonic development, cell proliferation, differentiation, formation of apparatus. In different developmental stages, the skeletal muscle involves a great number of differential expression genes which are regulating proliferation and apoptosis for certain cells in the developmental process.In our previous work, a novel gene with differential expression in different tissues and different developmental stages was isolated by screening from a full-length cDNA library constructed with the 55-day-old pig fetus skeletal muscle. In order to study the structure and function of this gene from nucleic acid and protein level, several methods including molecular biology, cytobiology as well as bioinformatics was employed in experiment. And the main results are as follows:1. Obtaining the Full-length cDNA of porcine PNAS-4The full-length 4133bp cDNA sequence of porcine PNAS-4 (GenBank accession number: DQ435075) was obtained by PCR and 5'RACE.2. Functional elements located in the cDNAA functional element called Internal Ribosome Entry Site (IRES) was found in the 5' UTR. Two boxes named Brd-Box and GY-Box located in the 3' UTR of the porcine gene containing two consensus polyadenylation signals (AATAAA) located upstream of the poly (A) stretch. And the Brd-Box plays an important role in negative regulation for the post- transcription via its influence on the stability and translation efficiency.3. Regulation factors in the promoter of human homologous geneA lot of binding site of transcription factors such as Sp1 and AP-1 were discovered nearby the CpG island after analysis of network regulation for the promoter of human clorf121 which is homologous to porcine PNAS-4, indicating the transcription efficiency will be higher than others. In addition, there are many binding sites of apoptosis repressor in the human promoter. This promoter is GC rich and lacks a TATA box as is typical for promoters of ubiquitously expressed "housekeeping genes".4. Characterization of predicted protein sequences The conceptual translation product of the porcine PNAS-4 transcript encodes 194 amino acids residues, which is a non-secretary protein without signal peptide, or transmembrane regions. Three-dimensional simulation structure was carried out according to the Putative conserved domain related to Eukaryotie protein of unknown function (DUF862), which comprises three possible protein binding domains and four amino acids residues exposing outside. Comparison to seven amino acids sequences of different species reported in GenBank, the porcine putative protein has a high level identity (96-98%) in mammals, and furthermore, it shares 74% and 45% similarity with Xenopus laevis and Arabidopsis thaliana, respectively.5. Detection of Polymorphisms and analysis of linkage disequilibriaThe complete second intron comprising of 3.2kb in length (GenBank accession no. DQ406743) was obtained according to the human clorf121 genome structure. After analysis of four mutation site located on the second intron and 3'UTR respectively using PCR-RFLP and DHPLC methods, the allele frequency of the four SNPs displays variable in different pig breeds. And the four mutation sites appear linkage disequilibria in single population though they are approximately 21Kb apart from each other. There are significant associations between the genotype of PNAS-4 with phenotypes of Percentage of Leaf Fat, Internal Fat Rate, Carcass Length, Rib Numbers, Loin-muscle Area, Ratio of M vs S in Ham and Hemoglobin Concentration (P＜0.05).6. Temporal and spatial express patternReal-time Q-PCR was performed to analyze tissue-specific expression of porcine PNAS-4 transcript using the housekeeping geneβ-actin as endogenous control. Relative mRNA expression of PNAS-4 was high in the lymph as well as in skeletal muscle, and with weakest signal observed in kidney and brain. In addition, the higher mRNA expression was found in cancerization cell than the normal kidney tissue. For temporal expression analysis, the porcine PNAS-4 appears down-regulated pattern during embryonic development while as up-regulated pattern after birth. And in the process of skeletal muscle development, there is no differential expression between indigenous and exotic pig breeds for this novel gone.7. Expression P4-p28c in E. coliA prokaryotic expression plasmids P4-pET28c was Constructed, and transferred into E. coli BL21 (DE3) expression strain. As a result from codon preference, no special signal peptide was found via conventional methods including SDS-PAGE.8. The porcine CGI-146 intracellular distributionThe intracellular distribution of porcine CGI-146 (the product of PNAS-4) in PK-15 cells was analyzed by fluorescence and confocal analysis of transiently transfected with P4-pEGFP-C1. After stained with MitoTracker Red and Hoechst33342, CGI-146-pEGFP fusion proteins were predominantly detected in Golgi apparatus, which was identified using the homo-functional plasma P4-pEGFP-N3 as the complementation.9. Overexpression of the porcine CGI-146Transiently transfection of PK-15 cells with the overexressed plasmid P4-pcDNA3.1 which express 21.4kDa non-tagged protein, the cell shrinkage and the less well defined cell surface was observed in the normal view with normal optical microscope. After the transfected cells were stained with Annexin V and Propidium Iodide, the apoptosis (annexin V-positive/propidium iodide-negative) were observed by the fluorescence microscope with barely vector alone as controls. As a new medical technology born in 20th century, clinical transplantation has become one of the important and powerful treatment methods for end-stage organ failure. There are tens of thousands patients coming to a new life by this medical technology every year in all over the world. However many patients die in the course of waiting because the donor organs are serious insufficiency. Therefore the Xenotransplantation has become to the most appropriate method to solve the urgently problem of medicine.The major obstacle to success is the hyperacute rejection (HAR) in pig-to-human discordant xenotransplantation, which attribute to the presence of the Galactose-α1,3-galactose (αGal) epitopes on the surface of pig cells and tissues. And the Synthesis of theα1,3-Gal epitope is catalyzed by the enzymeα1,3-galactosyltransferase (α1, 3-GT, GGAT1), but humans, apes, and Old World monkeys have lost the corresponding galactosyltransferase activity because only with a non-functional copy of theα1,3-GT gene in the course of evolution and therefore produce a large quantity of natural antibodies to the epitope, which are responsible for hyperacute rejection of porcine organs. Consequently, the genetic knockout of the a1,3-galactosyltransferase locus in pigs would provide permanent and complete protection against the HAR at extant range using gene engineering technology.Gene targeting is an important genetic manipulation technology, which defined as the introduction of site-specific modifications into the genome by homologous recombination, has revolutionarized the field of mouse genetics and allowed the analysis of diverse aspects of gene function in vivo. In present, the cloning of sheep, goat, cattle, and pigs by somatic cell nuclear transfer provides a feasible means of disrupting or deleting theα1,3-GT in mammals other than mice.According to the porcineα1,3-GT fragment obtained from the NCBI, a gene targeting vector, which comprise of positive and negative selection, was used to establish the highly inbred Wuzhishan miniature pig (WZSP) line knocked out theα1,3-GT gene by the method of gene targeting in somatic cells. At the same time, to offer the possible experimental animals used for Xenotransplantation in our country. Using the long-range (LR) PCR method, we have cloned two DNA fragments which are 5.4Kb and 1.6Kb respectively from the highly inbred WZSP genome. The 5.4Kb DNA fragment includes the whole eighth intron of porcineα1,3-GT gene, and the other spans the most part of exon 9, which are cloned to vector for sequencing. And sequencing analysis showed that the two fragment are genomic DNA of the porcineα1,3-GT gene.Because there is a restriction digestion enzyme SalⅠlocated in the former of 5.4Kb DNA fragment, 5.1Kb was amplified for 5'arm of targeting vector using conventional PCR method while the 1.6Kb DNA fragment as the 3'arm. And then, the construction taregeting vector containing 15.2 Kb used for knock-out porcineα1,3-GT gene was completed and conformed by sequencing, in which the neomycin phosphotransferase (neo') and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selection markers.