The Study Of MEIS1 Regulate PAX6 Transcriptional Expression

Many genes that are expressed in different spatiotemporal pattern and play ed themselves function take part in the process of development.Transcription F actors that targets the region of transcriptional regulation,play an important rol e in the development,activity or repress the transcription of gene and regulate the spatiotemporal expression of gene.MEIS1 is a TALE homeodomain transcri ptional factor,have an important effect on embryogenesis.The study has found that the loss of MEIS1 result in a nervous system development of disorder in model animal and demonstrated that MEIS1 is a key transcriptional factor in n eurogenesis.Thus,the research of MEIS1 functions and the mechanism of its t ranscription are a significant in nervous system development.In this study,we utilize CRISPR/Cas9 technology and establish a Dox ind ucible Cas9 expressed cell lines named 293T-iCas9.Using 293T-i Cas9 cell line s,we establish a MEIS1 knockout of 293 T cell line.According to the literatur e report,Meis1 regulates Pax6 transcriptional factor expression in the developm ent of mouse eye.However,Pax6 between human and mouse has different fun ctions.Especially,during human neural ectoderm differentiation PAX6 is human neuroectoderm cell fate determinant.To detect preliminarily whether MEIS1 re gulate human PAX6 gene expression,we first predict the potential binding site of MEIS1 in PAX6 promoter and enhancer region and next construct these luci ferase reporter vectors by corresponding to possible site.Transfecting PAX6 luc iferase reporter vectors into MEIS1 knockout cell lines,we detect the mechanis m that MEIS1 regulate PAX6 expression preliminarily.1.Establishing an inducible Cas9 expression HEK293 T cell linesCRISPR/Cas9 is a novel genome editing technology that can achieve the k onckin or knockout of gene simply and quickly in mammal.But the routine C RISPR/Cas9 expression system that has a weak directly transfection efficiency and a low virus packaging productivity has been limited to popularization and application.In this study,we utilize a tet-on system and construct pTight-Cas9-GFP-Puro that a Dox inducible Cas9 expression vector.Togethering p Tight-Cas9-GFP-Puro and pSIN-rtTA-neo,we transfect them transiently into 293 T cell line,and identify the inducible Cas9 expression system by fluorescence micro sco py and Western Blotting.Packaging pTight-Cas9-GFP-Puro vector and p SIN-rtT A-neo vector into virus respectively,we use the harvest two viruses to affect a293 T cell line synchronously,and screen positive cell by adding puro.Next we active tet-on system to induce Flag-Cas9-P2A-GFP expression by adding D ox and use flow cytometry sorting 293 T cells expressing GFP and reseed them into 96-well plates by single cell.Last,we expand the single clone cell and i dentify an inducible Cas9 expressed 293 T cell line by adding Dox and fluoresc ence microscopy and Western Blotting.These results demonstrate that we establ ish a dox inducible Cas9 expression of 293 T cell line named 293T-iCas9.2.Establishing a MEIS1 knockout 293 T cell line by CRISPR/Cas9MEIS family,the myeloid ectropic viral integration site family,have three members containing MEIS1,MEIS2 and MEIS3 that all belong to TALE home odomain transcriptional factor and has an important function in embryonic deve lopment,Disease occurrence and development.MEIS1 mainly take part in som e biologic processes,for example,nervous system,hematopoietic system and le ukemia.Despite the MEIS1 gene has been studied extensively,but its mechanis m of action is still not entirely clear.Therefore,the knockout of MEIS1 is of great significance to the study of the nervous system development and transcrip tional regulation of events in the process of development.We add 293T-i Cas9 to the Dox cell line,and then transfect the targeting MEIS1 Exon3 of sgMEIS1 expression vector(p LKO5.sg RNA.EFS.t RFP)into the 293T-i Cas9.SURVEYO R assay and Western Blotting showed that sg MEIS1 was effective in guiding Cas9 for genome editing.293T-iCas9 cell lines express GFP and Cas9,while s gMEIS1 expression vector RFP and sgMEIS1,thus GFP and RFP can be used as the marker whether the CRISPR/Cas9 system is introduced successfully.GF P/RFP double positive cells were sorted by flow cytometry,and next to Blottin g Western detection and genotype identification,the results showed that we suc cessfully knocked out the MEIS1 gene in HEK293 T cells.3.The effect of MEIS1 on the transcriptional regulation of human PA X6 promoter and enhancerPax6 is human neuroectoderm decisive transcription factor.To preliminary proven whether MEIS1 has a transcriptional regulation to PAX6,we first predi cted MEIS1 possible binding sites in the human PAX6 promoter and enhancer regions.Next,we cloned the human PAX6 P0 and P1 promoter,and linked th em to the p GL3 luciferase reporter vector.293 T cell express endogenous MEIS1,so we take 293 T as the control group,MEIS1 knockout 293 T as the experi mental group,and luciferase assay detect the role of MEIS1 to PAX6 on the p romoter.Finally,we cloned the fragment of predictive MEIS1 binding sites in the enhancer regions,and these enhancers are connected to P0-p GL3,P1-pGL3 luciferase reporter vector respectively and luciferase assay detect the role of M EIS1 to PAX6 on the enhancer.