ABSTRACT
To summarize the breeding and identification of Wuzhishan miniature pig models with α-l,3-galactosyltransferase (GGTA1) gene-knockout (GTKO).Methods The breeding and reproduction perform of GTKO Wuzhishan miniature pigs were assessed and the quantity of piglets was counted.The GTKO Wuzhishan miniature pig models with GGTA1gene knockout were validated by polymerase chain reaction(PCR).The αGal phenotype of peripheral blood mononuclear cells (PBMC) in human,wild-type Wuzhishan miniature pigs and GTKO Wuzhishan miniature pigs was detected by fluorescent microscope and flow cytometry.Routine blood test parameters were statistically compared between the GTKO and wild-type Wuzhishan miniature pigs.Results The inheritance of GGTA1 genotype complied with Mendel's law.Flow cytometry detected no fluorescent expression of PBMC in GGTA1-/-pig models,which were consistent with the genotype identification results.The mean piglets of the primiparous GTKO Wuzhishan miniature pigs were (6.8±1.8) and (8.3±2.2) for the multiparous Wuzhishan miniature pigs.No statistical significance was noted in routine blood test parameters between the GTKO and wild-type Wuzhishan miniature pigs (all P＞0.05).Conclusions Stable inheritance and normal reproductive capacity are observed in two generations of Wuzhishan miniature pigs continuously.GTKO Wuzhishan miniature pig is a reliable donor for heterogeneous organ transplantation.
ABSTRACT
Objective This study was aimed to characterize the swine leukocyte antigen( SLA) class I genes of GGTA1 -/ - Wuzhishan minipigs and compare their similarity to human leukocyte antigen( HLA) . It has important implica?tions for understanding the cellular rejection in xenotransplantation. Methods Specimens of ear tissue from six founding GGTA1 -/ - Wuzhishan minipigs were collected, and the SLA class I genes (SLA?1, SLA?3, SLA?2) were amplified by RT?PCR. Purified products were cloned into pEASY?T1 vectors and sequenced, followed by BLAST alignment and using bioin? formatc analysis to characterize the SLA class I genes and compare with the similarity to HLA. Results A total of six al?leles were detected, among them alleles were previously reported (SLA?1?0703,SLA?2?1102, SLA?3?0401, SLA?3?0403), and the other were novel (SLA?1?0401wz01, SLA?2?11wz01). The homology between alleles of SLA class I genes in Wuzhishan minipigs and HLA was from 70?5% to 72?1%. The homology analysis of critical amino acid residues on HLA binding with human CD8 + molecules showed that SLA?1?0401wz01, SLA?1?0703, SLA?2?11wz01, SLA?2?1102 and SLA?3?0401 occurred mutant at amino acid positions 225 and 228 ( T→S,T→M) , whereas the other loci were highly conserved. There was a high homology at amino acid level between SLA?2?11wz01, SLA?2?1102 and HLA class I genes which are NK cell KIRs binding sites. Conclusions The amino acid sequences of SLA class I genes of GGTA1 -/ -Wuzhishan minipigs have a high homology to HLA. From the point of view of cell?mediated xenograft rejection, the amino acid sequences of SLA class I genes of GGTA1 -/ - Wuzhishan minipigs have a high homology to HLA, therefore, Wzhishan minipigs may become a good potential donor for pig?human xenotransplantation.
ABSTRACT
Objective Getting the robust exogenous gene expression vector under the control of porcine insulin promoter, and to lay the foundation for pancreaticβ-cells specific transgene expressing pigs.Method Using porcine insu-lin promoter ( PIP, 1500 bp of the 5′UTR from the porcine INS gene including the first exon and the first intron) to con-struct expression vector, the HindIII restriction site which connected the sequences of PIP and EGFP was designed before ATG, named PIP-HindIII-EGFP.Considering that the different location of restriction site may affect the expression efficien-cy of the transgene, we optimized the expression vector.Firstly the HindIII restriction site was deleted to realize the seam-less connection of PIP and EGFP,the vector was named PIP-EGFP.Also we mutated the 3′intron splicing acceptor site( SA) of the first intron into HindIII restriction site, named as PIP-SA( M)-EGFP.Three different EGFP expression vectors were respectively transfected MIN-6 mouse pancreatic β-cells, pig ear fibroblasts and kidney cells.The transfected cells were cultured for 48 h and harvested for RT-PCR, flow cytometry and Western blot analysis, to analyze and compare the expres-sion efficiency of vectors.Results After transfection,green fluorescence was observed only in MIN-6 mouse pancreaticβ-cells.RT-PCR analysis and product sequencing showed that the three expression vectors did have different stability with in-tron splicing.The PIP-HindIII-EGFP construct and PIP-EGFP vector produced two kinds of mRNA with the first intron spliced and no spliced, indicating the instability of intron splicing.Mutation of the PIP splice site would cause the first in-tron not spliced, while flow cytometry and Western blot displayed that the mutation induced a most efficient expression of the downstream gene.Conclusions A robust and specific β-cells expression vector has been successfully generated by mutating the intron splicing acceptor site of the porcine insulin promoter.It provides the foundation for preparation of pigs with pancreaticβ-cells specifically expressing the transgene.