Identification,Functionalanalysis And Application Of Mouse Kiss1 Gene Promoter

Kiss1 encodes the neuropeptide Kisspeptin,which is a key regulator of the hypothalamus-pituitary-gonadal(HPG)axis,regulates the release of gonadotropin releasing hormone(Gn RH)and and plays an important role in sexual development.The regulation mechanism of Kiss1 expression has always been a focus of neuroendocrinology research.Promoters regulate gene expression at the transcriptional level,which directly determine the spatio-temporal specificity of downstream gene expression.Therefore,the resolution of Kiss1 promoter is of great significance for studying the expression regulation mechanism of Kiss1.The development of gene-specific promoters also plays an important role in the construction of transgenic animals and the optimization of genetic engineering tools(CRISPR-Cas9 and Cre-lox P system).The purpose of this study is to analyze the sequence and regulatory patterns of the Kiss1 promoter and to explore its potential application in genetic engineering tools.Methods: First,build a series of recombinant luciferase reporter vectors with 5’-truncated Kiss1 promoter.The results of promoter activity analysis in different cell lines showed that the three Kiss1 promoters with high activity in GT1-7 cells had no activity or very low activity in N6 cells,indicating that Kiss1 promoters had certain cell specificity.We screened out the promoter p2(-1089～+18)with the highest transcriptional activity for further research and application.We analyzed the transcription factor(TF)binding of the p2(-1089～+18)sequence.A green fluorescent(EGFP)reporter vector driven by p2(-1089～+18)was constructed and transfected into GT1-7 cells and treated with estrogen(estradiol,E2).The changes of reporter gene expression were detected by flow cytometry and q RT-PCR to verify the promoter’s response to estrogen signal.Then,the application potential of the promoter in the following three genetic engineering tools was explored.First,the pLL3.7-p2(-1089～+18)-EGFP was further packaged into lentivirus to infect primary hypothalamic neurons to achieve fluorescent labeling of Kiss1 neurons.The Cas9 expression vector driven by p2(-1089～+18)was constructed and co-transfected with sg RNA expression vector.T7E1 assay was used to verify the shear efficiency of CRISPR-Cas9 system.The Cre expression vector driven by p2(-1089～+18)was constructed and co-transfected with Ai9 vector.The recombinant efficiency of Cre-lox P system was verified by observing the red fluorescence protein expression and PCR method.Results: In this study,six recombinant luciferase reporter vectors with 5’-truncated Kiss1 promoter were successfully constructed for the identification of the transcriptional activity regions of Kiss1 promoter.DLRA in different cell lines(GT1-7,N6,293T)all showed that the promoter p2(-1089～+18)had the highest transcriptional activity,so it was used as the main research object in the future.TF binding analysis of p2(-1089～+18)revealed that there were two estrogen receptor(ER)binding sites(-421 and-502)on the promoter——the ER response element(ERE).The analysis results were verified by flow cytometry and q RT-PCR,and the results showed that the expression level of the downstream reporter gene of p2(-1089～+18)was significantly increased after E2 stimulation(P<0.05),indicating that the promoter was able to respond to exogenous estrogen signals and presented positive regulation.About the promoter application part: First of all,the primary hypothalamic neurons were successfully isolated and cultured in vitro,and pLL3.7-p2(-1089～+18)-EGFP was packaged into lentivirus to infect primary hypothalamic neurons.Although fluorescence labeling of neurons was not observed under the microscope,q RT-PCR results showed that EGFP was significantly overexpressed in cells,which was about2000 times higher than that in the control group(P<0.01).The Cas9 expression vector driven by p2(-1089～+18)was successfully constructed and proved to be able to shear the target sites to a certain extent under the guidance of sg RNA.The Cre expression vector driven by p2(-1089～+18)was successfully constructed.The expression of the downstream RFP reporter gene caused by lox P site recombination was detected by q RT-PCR(P<0.01),which was more than 8 times that of the negative control,thus the function of Cre recombinase was proved.Conclusion: In this study,the transcriptional activity regions of mouse Kiss1 promoter were identified,and Kiss1 promoter with high activity was screened out,and the application potential of this promoter in genetic engineering tools was explored,providing ideas and improvement directions for subsequent studies.In addition,the sequence analysis and hormone response analysis of the promoter not only contributed to revealing the regulation of the transcription of Kiss1 gene,but also provided important reference for the optimization of the subsequent promoter activity and specificity.