Generation Of HIF-1 Alpha Overexpression Transgenic Mice By Pronuclear Microinjection And Its Detection

HIF-1?(hypoxia inducible factor 1 alpha)is the subunit of HIF-1 heterologous dimer structure.HIF-1 is PAS(PER-ARNT-SIM)members of the family of transcription factors,and it mainly through combined with target genes and regulate the transcription of target genes.Its role is largely mediated the body hypoxia ischemia adaptive response,etc.HIF-1 alpha subunit is the activity of HIF-1 subunits,it determines the HIF-1 transcription factor activity and function.Vascular endothelial growth factor(VEGF)is one of the members of platelet derived growth factor family,it has been proved to be promote the follicular development and the role of the hair growth,but also prove to be one of target genes of HIF-1 alpha.This experiment using CISPR/Cas9 gene targeting system integrating HIF-1 alpha to Rosa26 site of murine genome to investigate HEF-1 alpha effects on hair follicle development and so on.This study gets the transgenic mice through pronuclear micro injection technology.At first,we identify the positive mouse through PCR,then test the expression levels of HIF-1? and VEGF by using the methods of Real-time PCR and Western Blot in positive mice skin tissue.1.The construction of HIF-1? over expression vector and function testTo study the relationship between HIF-1? and hair growth means we need study the relationship between HIF-1? and VEGF.We need to verify whether over expression of HIF-1? really causes changes in target gene VEGF and eNOS.And we need to verify whether HIF-1? works in the signaling pathway of HIF-1?/VEGF/VEGFR2.In this study,we first cloned the HIF-1? gene from the mouse genome and successfully constructed the overexpression vector.The vector was successfully transferred into mouse fetal fibroblasts(MEF)by electroporation,then Real-time PCR was performed on the transgenic MEF with non-transgenic MEF as the control group.The expression level of HIF-1? mRNA in the transgenic MEF was 17.91 times higher than that in the control group,the expression level of VEGF mRNA was 15.49 times of that of the control group,the expression level of VEGFR2 mRNA was 6.13 times of that of the control group,the expression of eNOS mRNA was the control Group 2 times;Then according to the Western Blot test,the expression of HIF-1?protein in the MEF was 2.41 times higher than that in the control group,and the protein expression of VEGF was 1.79 times of that of the control group by Western Blot.These results suggest that overexpression of HIF-1? does cause upregulation of VEGF and eNOS,and also upregulates VEGFR2.2.Construction of HIF-1? gene target vectorIn this study,we used CISPR/Cas9 gene targeting system for gene editing,and this system includes three vectors:Cas9 vector,sgRNA vector and HIF-1?homologous recombinant vector.In this study,the target site was selected as the first intron of Rosa26 at 1096bp.And as the laboratory has achieved insert the target gene at this site,Cas9 vector and sgRNA vector directly use the carrier which stored in the laboratory.Then,HIF-1? homologous recombination targeting vector was successfully constructed by amplifying the upstream homologous arm,the human K14 promoter,and the target gene HIF-1? with appropriate cleavage site.The vector was identified by a series of enzyme digestion and then sequenced.The homology of the vector reached 100%,so it was found that the targeting vector was constructed successfully.3.The cellular level relevant detectionIn this study,the Cas9 vector,sgRNA vector and HIF-1? homologous recombinant vector were introduced into MEF by electroporation,and the cell genome was extracted after 48 hours.In this experiment,two pairs of primers were screened:one pair was used to detect whether the target gene HIF-1? was successfully integrated into the cell genome,that is,the randomized primers were detected;the other pair was used to detect whether the target gene was inserted into the target site as expected.Using the transfected cell genome as template,we used the above two pairs of primers for PCR reaction.The PCR reaction product conforms to the expected size and the PCR products were sequenced and sequenced correctly.Two pairs of primers above can be used for subsequent transgenic mouse testing.4.Production and identification of HIF-1? transgenic miceIn this study,transgenic mice were prepared by pronuclear micro injection.The method mainly includes the preparation of the gene for injection,the acquisition of pronucleus stage embryos,the pronuclear microinjection and the embryo transfer.In this study,we obtained 56 transgenic mice.And two random integration positive mice was identified by PCR.Did not get the targeted integration positive mouse.These two positive mice were the good model for the study of the relationship between HIF-1? gene and hair follicle development.5.Detection of expression levels of HIF-1? transgenic miceIn this study,the expression levels of HIF-1? were detected in the skin tissues of two randomized positive mice 661 and 664.In this study,the same batch of non-transgenic mice 682 as control mice.By real-time PCR detection,the expression level of HIF-1? mRNA in 661 mouse was 7.75 times higher than that in control mice,and the expression level of VEGF mRNA was 29.62 times of that of control mice;And the expression level of HIF-1? mRNA in 664 mouse was 3.41 times higher than that in control mice,and the expression level of VEGF mRNA was 2.91 times of that of control mice.And then by Western Blot for protein level expression detection,the expression level of HIF-1? protein in 661 mouse was 2.02 times that of control mice,and the expression level of VEGF protein was 1.72 times of that of control mice;The expression level of HIF-1? protein in 664 mouse was 1.84 times higher than that in control mice,and the expression level of VEGF protein was 1.54 times of that of control mice.