Promoter Recognition Of The Autism-associated Gene NRXN2β And Regulation Of Protein Synthesis By The 5'UTR Region Of The MRNA Sequence

Objective: To investigate the effect of the edge effects by multiple cell plate on luciferase reporter gene assay.Methods: Use the conventional methods to culture Human Embryo Kidney cells(HEK293),and transfect the luciferase reporter gene PGL4.13 Z and PRL-TK with the transfection reagent Lipofectamine 2000(Invitrogen)into HEK293,which is seeded in 48 well plate.Luciferase activity was measured with the dual luciferase reporter assay reagent(Promega)and the cell numbers were counted 24 h after transfection.Calculate the relative firefly luciferase activity(Fluc),the relative renilla luciferase activity(Rluc),the ratio of Fluc vs Rluc.Compare those values between corner wells,edge wells and central wells of 48 well plate.Results: we have found that the single relative firefly luciferase activity,the single relative renilla luciferase activity,the ratio of Fluc vs Rluc are different between corner wells,edge wells and central wells of 48 well plate.This difference is not related to cell number in 48 well plate.Pre-incubating plate with newly seeded cells at room temperature(RT),plate stacking patterns(one-by-one),avoiding use of the peripheral wells on plates are useless for reducing edge effect,however pre-filling liquid in alledge wells can abolish edge effects.Conclusion: The results of luciferase reporter assay performed with48 well plate can be affected significantly by edge effects of multiple well plate,which may cause wrong interpretation of the result.Filling edge well with liquid can eliminate edge effect.Objective : To define the promoter region of NRXN2β gene,investigate the effect of m RNA 5’UTR on protein synthesis of NRXN2βgene.Methods : Using bioinformatics to identify TSS and upstream regulational element.Various 5’ upstream DNA fragments of the human NRXN2βgene were inserted into p GL4.10 vector to build the luciferase reporter plasmids of containing different NRXN2β gene promoter regions according to conventional molecular cloning techniques.The NRXN2βreporter plasmids and Renilla luciferase control reporter plasmid were co-transfected into HEK293 cells with the transfection reagent Lipofectamine 2000.Luciferase activity in HEK293 cell was measured with the dual luciferase assay reagent(Promega)24 hours after transfection,and the firefly luciferase activity(Fluc)from NRXN2β reporter plasmid was normalized to Renilla luciferase ativity(Rluc)from control plasmid to reflect the Luciferase activity.To define the promoter region,the Luciferase activity of various NRXN2β luciferase reporter plasmids containing different promoter regions was compared with each other.To identify the regulation of 5’UTR on protein synthesis of NRXN2βgene,the DNA fragment of 5’UTR was inserted after SV40 promoter in the plasmid p GL4 promoter to build the luciferase reporter plasmids of containing different NRXN2β gene 5’UTR regions according to conventional molecular cloning techniques.Results:After successfully constructing a series of NRXN2β promoter luciferase report plasmids containing different size.The longest inserted fragment is 2065 bp,which contains 1661 nucleotides upstream first exon and 404 necleotids downstream of transcriptional start site,named as p4NRXN2β1661/+404E.The results indicated that the cis-acting region of-558/-184 is the promoter region of NRXN2β gene.There is obvious different luciferase activity between plasmid p4NRXN2β-558/+512E and P4NRXN2β-558/-1E,and the region of +1/+512 is the 5’UTR of NRXN2β,which inhibits the protein synthesis of NRXN2β.Conclusion:We have found a promoter region of NRXN2β gene and identified that 5’UTR of NRXN2β can inhibite the protein synthesis of NRXN2β gene.