Expression Of Imprinted Genes In Early Embryonic Development

Only very few of these genes in mammalian genome are known as imprinted genes(may be no higher than 5%),but the few genes play an important role in fetal growth and behavior development.In early mammalian embryonic development,imprinted genes have two reprogramming ways: one is the process of zygotes developing into early embryos,and another one is the process of primitive germ cells developing into mature gametes.In these two reprogramming processes,it can cause many diseases if the reprogramming is unusual,and these diseases are associated with abnormal expression caused by genomic imprinting disorders.Therefore,it's crucial to conduct researches on the regulation of early embryonic imprinted genes expression.In the previous early embryonic imprinted genes studies,expression of imprinted genes only limited to using RNA-FISH at cellular level,which is difficult and only detected one gene at a time.Nowadays,with the development of biotechnology,we realized the single-cell detection technology at molecular level for the first time in 2007.There has been studied out four kinds of different single-cell detection technologies so far,respectively are: scRNA-Seq 3'transcriptome analysis,RNA–Seq single-cell tagged reverse transcription(STRT),CEL-Seq and Smart-Seq.The first technique has high accuracy,but only can detect the 3'end of the mRNA.Other three techniques are capable of analyzing whole mRNA,but still with several short comings.To be specific,STRT technology easily leads to fragments in reverse transcription process;CEL-Seq technology has preferences with large amount of amplification and cost highly;Smart-Seq technology holds the disadvantage of instability,and the transcriptome of low level expression may be lost.In this experimental study,we just need to detect the 3'-UTR region of early embryo.We eventually choosed scRNA-Seq 3'transcriptome technology to analysis early embryonic imprinted genesthroughout the comparison of four technologies.Through testing the imprinted genes expression at single cell level,it can provide more data to research early embryonic development in mammals,and also provide theoretical basis for solving early embryonic development programming problem.This experiment firstly use bioinformatics to find out the imprinted genes of father and mother in imprinted genes professional website(Geneimprint website),and summary the expression source,SNP loci,differences between mouse strains,chromosome location and properties of these genes.Then design primers by using the Beacon Designer software for the corresponding mRNA sequences of these imprinted genes in the NCBI database.On the basis of this,collect the embryos of ten periods(zygote,early 2-cell stage,mid-2 cell stage,late 2-cell stage,4-cell stage,8-cell stage,16-cell stage,early blastocyst,mid blastocyst and late blastocyst),and use scRNA-Seq 3'transcriptome analysis technology to extract and amplify single cell,finally the amplified products were purified by using magnetic beads.After this step,two housekeeping gene(Ddx3x,Pdha1)were used to detect the expression of the purified c DNA respectively,subsequently,choose the samples which have been expressed to be the RT-qPCR templates and analysis them.The study shows that the method of RNA extraction to the expression of imprinted gene is feasible.Imprinted gene expression has spatial and temporal-specific expression,the density of embryo sample is different in different periods,so as to gene expression,and this result of which has been proved in the previous literature.Due to the unstableness of housekeeping gene m Gapdh expression,it should not be regarded as the internal genes of imprinted genes expression.There laid the foundation for the further study of reprogramming in early embryonic development.