Construction And Identification Of PHIF-1α/EGFP-C₂

ObjectiveHypoxia-inducible factor-1(HIF-1) is a nuclear factor to educe activity in anoxic condition.HIF-1 extensively resides in mammal and human histiocyte in anoxic condition.Then it regulates the transcription of more than two hundred kinds of target genes.So it possesses many kinds of biological function and plays important role in process of anoxic diseases.Therefore it is one of key responders to hypoxia.HIF-1 is composed by aαsubunit and aβsubunit.HIF-1αis unique in HIF-1 comparing with other numbers of HIF,regulated by oxygen,plays major role in functional regulation. And HIF-1βis also called aryl hydrocarbon receptor nuclear translocator.It is a subunit of aryl hydrocarbon receptor compound and express continuously in cytoplasm.In normal oxygen saturation HIF-1αis hydrolyzed by ubiquitin-protease hydrolysis compound with the 5 min half life.So it is difficult to be detected.In anoxic condition the degradation ofαsubunit is inhibited.HIF-1αand HIF-1βcombine.The heterodimers transfer into cell nucleus to regulate the transcription of downstream target genes.The functions involve in erythrocytopoiesis,vasoformation,energy metabolism such as nucleoside and amino acids,cell survival,apoptosis,cell migration and drug resistance to the maintenance of cell homeostasis in hypoxia.HIF-1 and its target genes play important roles especially in physiological hypoxia such as embryonic development,stem cell proliferation,differentiation and in many kinds of pathological condition such as tumor development,transfer.The research of HIF-1αis confined because of degradation.To construct the vector of pHIFα/EGFP-C2.The transfected cells express HIF-1αin high level.Methord1.Cell culture:To culture mouse embryonic stem cell(D3) and human renal cell carcinoma cell(A498) in 37℃,5%CO₂ incubator with 10%DMEM(containing 10%FBS,100U/mlmycillin).To refresh the medium per 3 days.To passage in routine method when 80-90%fusion.These two kinds cells are supplied with CoCl₂(200uM) and cultured in 3%O₂ for 4h,6h,16h.And the two kinds cells are cultured in 37℃,5%CO₂ as control groups.2.Gene fragment extracton of HIF-1αcDNA:To extract the mRNA of the cells. To detect the integrity of mRNA in 1%agarose gel electrophoresis.To inverse transcript 500ng total mRNA as templatet into cDNA in Random9 primer.To amplify cDNAby PCR.1%agarose gel electrophoresis.3.Construction amplification vector of pHIF-1α-T:To purify the cDNA and link it with linear T-vector.To transform DH5αcompetent cell.To amplify white colony.To extract plasmid according to instruction.Double enzymes(Sacâ… and Apaâ… ) digest in 37℃for 2h.1%agarose gel electrophoresis.DNA sequencing in M13-47 and RV-M.4.Construction expression vector of pHIF-1α/EGFP:Preparation HIF-1αgene fragment and pEGFP-C2 vector.T4 DNA ligase links pEGFP-C2 vector and HIF-1αcDNA fragment.To transform DH5αcompetent cell.LB agar plate containing Amp (100ug/ml).Select several colonies.To amplify the colonies and extract plasmid. Double enzymes(Sacâ… and Apaâ… ) digest in 37℃for 2h.1%agarose gel electrophoresis.DNA sequencing in PCR primer.Result1.Agarose gel electrophoresis shows RT-PCR production of HIF-1α.The straps only was found in the group of A498 cultured in 3%O₂.The position of straps conformed to the expectation.The transcriptional level of HIF-1αis elevated following the hypoxia existant.The straps was not detected in other groups.2.Amplification vector of pHIF-1α-T after Double enzymes(Sacâ… and Apaâ… ) digestion shows 2.6kb and 1.7kb two straps.DNA sequence was the same to the DNA fragment(NM001530)announed by Genebank,no mutation and no frame shif.3.Expression vector of pHIF-1α/EGFP after Double enzymes(Sacâ… and Apaâ… ) digestion shows 4.7kb and 1.7kb two straps.DNA sequence was the same to the DNA fragment(NM001530) announed by Genebank,no mutation and no frame shif.ConclusionWe constructed the vector of pHIF-1α/EGFP -C₂.pHIF-1α/EGFP -C₂ expresses HIF-1αin cells.The degrdn of HIF-1αafter transcription is solved.And it established a base for researching the biological effect of HIF-1.