| Objective:GLI family zinc finger 3 (Gli3), as an important transcriptional factor in Hedgehog(Hh) signal pathway with conserved sequence, plays an important role in embryonic patterning and Organogenesis. Gli3 mutation is associated with many types of syndromes and malformation of organ. However,the full length of Gli3 coding sequence is not publicly avalable. The major aim of this study is to clone the full-length cDNA sequence of Gli3 gene in landrace pig.Methods:(1)Extraction the RNA of lung from landrace pig and reverse synthesis cDNA; (2) Design the gene specific primers for Gli3 based on the highly conserved sequencesof Gli3 from other species, and clone 5’and 3’sequences by RACE technology to obtain the full-length sequence of Gli3 gene. (3) Prediction of the open reading frame(ORF) of Gli3 gene, the characters of Gli3 protein,N-glycosylation sites, phosphorylation sites, secondary structure,3D structural model and construction of phylogenetic tree by the Gli3protein sequences of pigand other species. (4) Determination the relative mRNA level of Gli3 gene in various tissues including heart, liver, spleen, lung, kidney, brain, skin, muscle and tongue from 110-day old fetals and adult pigs by qRT-PCR.Results:The full-length sequence of Gli3 gene is 6289bp with a 4761 bp coding sequence(CDS), a 291 bp 5’terminal untranslated region(UTR) and a 1237 bp 3’ UTR. Gli3 gene of landrace encodes 1587 amino acid(AA). The molecular weight of the predicted protein is 169kD and its isoelectric point (pI) is about 7.2. There is no N-signal peptide, transmembrane domain and O-glycosylation site, while there are six N-glycosylation sites and many Phosphorylation sites, with four conserved zinc domain in the predicted pig Gli3 protein. The random coil is dominant in the secondary structure. The mRNA of Gli3 gene is abundant in spleen, lung, kidney and brain in 110-day old pig fetalsand adult pigs as detected by qRT-PCR.Conclusion:The the full-length cDNA sequence of Gli3 gene in landrace pig was cloned successfully by RT-PCR and RACE technology, which will provide a basis for further study of molecular functionof Gli3 in landrace pig.Objective:To understand the function of GIP in glucose homeostasis by dissect the phenotype of dominant-negative GIPR transgenic pig model.Methods:Oral glucose tolerant testing(OGTT) was performed on two and five-month old GIPRdn transgenic pigs and age-matchedwild-type pigs, respectively. Pancreas was isolated and measured after oral glucose tolerance test. The expression of Ki67 and insulinin of GIPRdn transgenic and wild-type pigs was analysed by immunohistochemistry.Results:Two-monthold GIPRdn transgenic pigs exhibited elvated significant blood glucose level in 1 hour after as well as increased insulin secretion in 1.5 hour after glucosechallenge compared with control.Similarly,blood glucose of 5-month old QIPRdn transgenic pigs was significantly decreased in 1.5 hour, but insulin secretion was significantly increased 0.5 hour after glucose challenge, implying impaired glucose tolecance in GIPRdn transgenic pigs. Although 2-month-old and 5-month old QIPRdn transgenic pigs showed no difference in glucose tolenance, the insulin secretion was significantly decreased as the transgenic pigs grew. The weight of pancrease, insulin secretion and proliferation of pancreatic cells in GIPRdn transgenic pigswere distinctly reduced compared with controls as indicated by immunohistochemistry analysis.Conclusion:Glucose tolerance and insulin secretionwere impaired in two and five-month old GIPRdn transgenic pigs compared with nontransgenic pigs, which indicated that it could be an ideal large animal model for diabetes study. |
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