Construction Of A Rat Model Of MeCP2 Knockout And Study Of Target Genes By MeCP2

Rett syndrome(RTT)is an X-linked neurological disorder.MeCP2(Methyl-CpG-binding protein 2)mutations have been considered as the cause of RTT.At present,the mechanism of MeCP2-protein mutations resulting in RTT is not very clear.The phenotype of the existing MeCP2 knockout mice does not match the symptoms of RTT patients.In this study,we constructed the MeCP2 knockout rat model and investigated the differences between the MeCP2-null and the wild type rats at transcription level.By using the chromatin immunoprecipitation sequencing(ChIP-Seq)technique,we identified the target genes regulated by MeCP2 in rat brain.Our purpose is to explore the pathogenesis of RTT and provide theoretical support for clinical diagnosis and treatment.Part I:Generation of MeCP2 knockout rat model To set up an animal model mimicking Rett syndrome,the vector targeting to MeCP2 exon2was constructed by CRISPR/Cas9 technology.The Cas9 mRNA and sgRNA mixtures were microinjected into fertilized ova to obtain the MeCP2 knockout rat.The off-target effect of MeCP2-null rat was evaluated.Methods:The vector of MeCP2-sgRNA was constructed.Cas9 mRNA and sgRNA were transcribed in vitro and both were microinjected into fertilized ova.The ova were transplanted into the uteruses of pseudopregnant rats.MeCP2 gene from the new born rats was sequenced and the MeCP2 protein was checked by Western blot.The possible off-target sites were predicted by software and the sites were sequenced.The off-target effect was detected.Results:MeCP2 knockout rat model was successfully set up.Comparing the MeCP2sequencing results of MeCP2 knockout rats with wild type rats,two kinds of MeCP2-null rats with different genotypes were obtained.Western blotting was performed to validate that both types were positive.In both positive knockout rats,no off-target sites were detected.Conclusion:In this study,the MeCP2-null rat model was set up.The model was validated at both gene and protein levels.It provides an excellent animal model for study of the pathogenesis of RTT syndrome.Part?:Phenotype and behavioral analysis of MeCP2 knockout rat In this study,we made further efforts to analyze behavior of MeCP2-null rat.The body weight and phenotype of the knockout rats were compared with the wild type rats.We observed the behavioral performance of the MeCP2-null rats and compared it with some symptoms of Rett syndrome patients.Through this animal model,the effects of MeCP2deficiency in rat's brain was deeply explored.Methods:The weight and phenotype of the new born rats were analyzed.Open field test and step-through test were carried out for taking insight into the anxiety and cognitive ability of the MeCP2-null rat.The data were collected for SPSS software analysis.Results:The weight of MeCP2-null rats declined dramatically.The movement in the central region increased in the open field test,indicating that the rats had tendency of anxiety and the error frequency was significantly increased for the knockout rats compared with that of normal rats in the step-though experiment,which indicated the cognition of the MeCP2-null rats was flawed.Conclusion:The results of this study confirmed that MeCP2 play an important role in Rett syndrome and the behavioral performance of MeCP2-null rats was similar to that of the Rett syndrome patients.Part?:ChIP-Seq combined with RNA-Seq to search for the target genes of MeCP2MeCP2 plays an important role in the process of neural development in rats.Loss of MeCP2 function can lead to the dysregulation of downstream genes' expression.For identifying the genes regulated by MeCP2,we investigated the transcriptome changes between knockout rats and wild type rats and meanwhile we used the ChIP-Seq technique to identify the genes regulated by MeCP2.That will help to further understand the biological function of MeCP2 protein,so as to give new insight into the pathogenesis of Rett syndrome.Methods:Using RNA-Seq technology,the changes of RNA expression level between MeCP2 knockout rat and wild type rat were analyzed.Combined with the unique antibody technique of ChIP-Seq,we try to identify the downstream genes regulated by MeCP2.Results:Through the analysis of the ChIP-Seq and RNA-Seq data,the genes regulated by MeCP2 were different in different regions of rat brain.It is associated with different functions for different regions in rat brain.In this study,we found MeCP2 was a genome-wide binding protein.MeCP2 protein prefers to bind to long genes in different areas of rat brain(cerebral cortex,cerebellum,and hippocampus)and most of these binding sites were in introns of the genes.Conclusion:We identified the key genes regulated by MeCP2 and their DNA binding motif.Our data proved that the functions of MeCP2 protein were different in different brain regions.The data also suggest that the MeCP2 protein may regulate downstream genes in the Rett syndrome.