Study On Growth And Development Index And Related Genes Of Expression Of Meg8-DMR Gene Editing Mice

The mouse Dlk1-Dio3 imprinted gene cluster is an important imprinting cluster,which exists at the end of chromosome 12 and accounts for about 1 Mb of the genomic region.It includes the paternal expression protein encoding genes Dlk1,Rtl1 and Dio3,a variety of maternal expression non-coding RNA genes,in addition to a large number of miRNAs and snoRNAs.This imprinted gene cluster plays an important role in mouse embryonic development,cell reprogramming and the occurrence of hereditary diseases.If the abnormality of the imprinted genes in the region causes growth and development disorder,or even death,the study of this region is very important.Currently,three major differentially methylated regions have been identified in the Dlk1-Dio3 imprinted gene cluster:IG-DMR,Gtl2-DMR and Dlk1-DMR,all of these are paternal methylation.The preliminary findings of this laboratory are now named as Meg8-DMR,the first methylation region of the parental methylation in th region which may have important imprinting regulatory functions.Based on the CRISPR/Cas9 gene editing technology,the Meg8-DMR knockout mice model is used to investigate the effect of Meg8-DMR on the imprinted genes in the region.Firstly,the Meg8-DMR knockout homozygous(Meg8-DMR^-/-),heterozygote(Meg8-DMR^+/-or Meg8-DMR^-/+)and wild-type(Meg8-DMR^+/+)mice embryo phenotype data are collected from the previous group.The analysis finds that knockout of Meg8-DMR has no significant effect on mice embryo morphology,body weight and average number of births compare to wild-type mice,but it can result in a certain proportion of fetal death,Meg8-DMR single-equivalent knockout mouse self-crossing results in a low wild-type ratio?13.98%?in offspring.Secondly,through semi-quantitative and quantitative to detect the effect of Meg8-DMR knockout on the expression of related imprinted genes in the middle and last stages of the mice development,E12.5 embryos and E15.5 tissues reveals that there is no significant effect in the region of the expression of Meg8-DMR knockout genes,and there is no statistical difference.But in Meg8-DMR^-/+and Meg8-DMR^-/-mice,the expression of Irm is significantly up regulated.Finally,SNP sequencing is used to detect the effect of Meg8-DMR knockout on the imprinting pattern of the major imprinted genes in the region.The results shows that the deletion of Meg8-DMR can not change the imprinting patterns of the major imprinting genes Dlk1,Gtl2 and Rian in the region.In summary,the deletion of Meg8-DMR has no significant effect on the morphological weight of mouse embryos,but it can cause a certain proportion of fetal death.At the molecular level,the knockout of Meg8-DMR does not affect the imprinting pattern of the major imprinted genes in the region,nor does it significantly affect the expression of imprinted genes in the region.Only the Irm locates in Rian is in Meg8-DMR^-/+and in Meg8-DMR^-/-mice the expression level is significantly up-regulated.It is suggested that Meg8-DMR may regulate the expression of adjacent genes only as a regulatory region of Rian.This study can help to understand the biological function of Meg8-DMR during embryonic development,also help to further elucidate the transcriptional regulation mechanism of Meg8-DMR.