ABSTRACT
Objective:To clone swine leukocyte antigen,class II,DO alpha (SLA-DOA) gene from Banna mini-pig inbred line (BMI) and detect its mRNA expression level in 19 important tissues.Methods:The complete eDNA sequence of SLA-DOA gene was cloned by RT-PCR method from BMI and the mRNA expression pattern in BMI important tissues was examined by semi-quantative RT-PCR method.Nucleotide and protein sequences of SIA-DOA were used to carry out bioinformatics analysis and construct the phylogenetic tree.Results:The eDNA length of BMI SLA-DOA was 1 079 bp,which encoded a protein of 250 amino acids with molecular weight (Mw) 27.81 kD,and isoelectric point (pI) 6.48.Genome structure analysis showed it localized to Sus scrofa chromosomes 7 and consisted of four exons and three introns.Semi-quantitative expression analysis showed that SLA-DOA gene expressed highly in the lymph nodes and stomach;weakly in the heart,skin and duodenum and none in other 14 tissues.Functional bioinformatics analysis indicated that SLA-DOA protein contained one signal poptide,one transmembrane region,three conserved domains,four O-GlcNAc glycosylation sites,18 potential phosphorylation sites and to be located in the cytoplasm with 94.1％ certainty.Phylogenetic analysis demonstrated that BMI (pig) had the closest relationship with cattle.Conclusion:This study have successfully cloned the SLA-DOA gene of Banna mini-pig inbred line,performed bioinformatics analysis and tissue expression profile analysis.It will provide a basis for the studies of BMI xenotransplantation.
ABSTRACT
Objective To establish a method of identification of DKO mouse model of Duchenne muscular dystro-phy, and to assess the dystrophin regeneration after stem cell transplantation.Methods Heterozygous mice were mated and the resulting offspring were used to identify their genotype by SSP-PCR.The plasma creatine kinase level was measured by biochemical analyzer and histological changes in the DKO mice were analyzed using HE staining.Human umbilical cord mesenchymal stem cells were prepared and injected into the DKO mice hindlimb muscle, and dystrophin expression was de-tected by immunofluorescence staining at 2 months after injection.Results Mating of heterozygous mice generated three kinds of genotype offsprings, and 21.2%of the offsprings were identified as DKO genotype (285 bp) .DKO mice showed dystrophic symptoms, their plasma creatine kinase level was as high as 16988.52 ±617.48 IU/L, and significant histologi-cal changes including diverse myocyte sizes, numerous centrally nucleated cells and connective tissue proliferation or in-flammatory cells infiltration.Human dystrophin expression was detected in the DKO mouse hindlimb muscle at two months after injection of human umbilical cord mesenchymal stem cells.Conclusion DKO mouse genotype can be identified by SSP-PCR, and DKO mouse is an ideal animal model for studies of stem cell therapy for Duchenne muscular dystrophy.