The Establishment Of Stable CREB Gene Knock-out Cell Lines With CRISPR/Cas9 Techniques And The Regulatory Effects Of CREB On APP Gene Expression

Objective Amyloid precursor protein is a multifunctional transmembrane protein which could promote the differentiation of neuron and the synaptogenesis.Under pathological condition,the Amyloid precursor protein was abnormally cleavage and produced a 42 amino acids polypeptide fragment named amyloid peptide which deposit in the brain tissues and caused the inevitable damages to neuron and causes the decline of memory,so it has been considered as a main hypothesis for the generation of Alzheimer's disease.Alzheimer's diseases is a common neurodegenerative disease and seriously affects the daily life of the senior people.One of remarkable pathological changes in Alzheimer's disease is the abundant amyloid peptide deposit in the brain tissues.c AMP-response element binding protein,CREB,is an important nuclear transcriptional factor.There are researches demonstrated that the content of CREB decreased in the brain of the patients with Alzheimer's disease and the content of APP increased dramatically.However,he regulatory effects of CREB on APP is still undefined.Based on this point,we employed CRISPR/Cas9 techniques to knock out the CREB gene in mouse hippocampus cell line and established the stable CREB-gene knocked out cell line to observe the regulatory effects of CREB on APP gene expression and try to investigate the detailed mechanism of this regulation of CREB.Methods1.To design 3 different sg RNA sequences and synthesize the oligonucleotides corresponded to the sg RNA sequences to construct the CRISPA/Cas9 gene knock-out plasmids,p X459-CREB.2.To evaluate the activities of the constructed plasmids;the constructed plasmids were transfected into HT22 cells and the whole genomic DNA were extracted,short chain PCR sequencing analysis and long chain PCR products with T7E1 enzyme cutting analysis were performed to evaluated the activities of the constructed plasmids.3.To establish the stable cell lines with p X459-CREB plasmids,western blot assay and q PCR were employed to check the effects of CREB gene knock-out,those with best gene knock-out cell lines were used for the further experiments.4.The protein expression levels of CREB and APP were assessed with western blot assay.5.The expression levels of other factors in the gene transduction pathway,such as GSK-3?,p300,p52 and IKB?were checked with western blot assay.Results1.The results of enzyme cutting and DNA sequencing demonstrated that the CREB sg RNA1,2 and 3 were successfully inserted into p X459 plasmid.The sg RNA sequences and the ORF were correct and consistent with the design.2.sg RNA-1 sequence was located in a DNA sequence repeated area and the plasmid p X459-CREB1 activity could not evaluated with PCR analysis.After being transfected into HT22 cells,p X459-CREB2 and 3 demonstrated the base mutation and part DNA sequence missing when analyzed DNA sequencing and T7E1 cutting.These results demonstrated that p X459-CREB2 and 3 is active.3.Plasmids p X459-CREB1,2 and 3 were transfected into HT22 cells and selected with puromycin to establish the stable cell lines,the efficiency of CREB gene knock-out in 3cell lines were evaluated with q PCR and Western blot,the results demonstrated that all the cell lines have different knock-out efficiency and the knock-out rate were all above 70%,the CREB-2 and 3 have the best knock-out efficiency and reached to 90%,this cell line were chosen for the coming experiments.4.The results of western blot demonstrated that when CREB gene was knocked out,the APP expression level in the cells increased remarkably,when CREB gene was overexpressed in the Neuro-2 cells,CREB expression levels increased but APP expression levels decreased.5.The western blot results also demonstrated that when CREB gene was knocked out,the expression levels of GSK-3?,p300 and p52 in the cells increased but IKB-?decreased.Conclusion1.Successfully constructed CREB gene knock-out plasmids p X459-CREB1,2 and 3,and all the activities of plasmid were evaluated.2.Established the stable cell line with CREB gene knock out.3.CREB gene knocked out could increase APP gene expression,the mechanism of this regulation could be regulated through GSK-3?,p300,p52 and IKB-?.