The Study Of LncRNA Expression Profile During Germ Cell Differentiation In Vitro

Objective:1.Establishment of primordial germ cell-like cells(PGCLCs)differentiation system in vitro.2.Identification the expression of lncRNA during in vitro induced of mouse primordial germ cells.3.Prediction of key lncRNA during induction of mouse primordial germ cells.4.Obtain the E5.5 day ectoderm and E12.5 day primordial germ cells in mice.Methods:First,we simulated the in vivo differentiation of mouse primordial germ cells(PGCs)and established a PGCLCs induction system in vitro using a two-step induction method.We used mouse embryonic stem cells(ESC)to induce flattened epiblast-like cells(Epi LCs)after two days of adherent culture,and then used the appropriate cell number for hanging drop culture.The primordial germ cell-like cells(PGCLCs)were further cultured for 4-5 days.We use the PGCs differentiation-specific marker gene Blimp1-GFP to indicate the success of the in vitro induction phase,and then use CD61 and SSEA1 for flow sorting to obtain a complete PGCLCs population on the fourth day.To obtain the cell samples of each stage,RNA was extracted and inverted into cDNA.The marker genes at each stage were detected by QPCR to prove that the induction of primordial germ cells in vitro was successful.Total RNA contains more than 80% rRNA,so common sequencing results contain large amounts of rRNA data.This affects the lncRNA data content in the sequencing data to a large extent,so we use a DNA probe with rRNA reverse sequence to remove the rRNA from the sample and we did not use Oligo(dT)beads,so we can obtain more numbers and types of lncRNA data than the normal transcriptome sequencing.We used the PE150 double-end sequencing of Illumina platform,and we obtained an average of 30 M data volume per sample.We use Gencode to compare and classify the data.We used the FPKM value to indicate gene expression level,and then we analyzed the transcriptome-level expression of the induced two-stage genes.On the one hand,we verified the authenticity of our data.In addition,we can once again verify our in vitro induction.When judging the difference in gene expression between different samples,we defined that the logarithm of the ratio of FPKM between different samples was greater than two for significant differences.Through comparison between different groups of samples,we finally identified 38 lncRNAs that were highly expressed in PGCLC and low in ESCs.We performed QPCR verification on the top 10 lncRNAs with the most significant difference.The expression of eight lncRNAs in PGCLC was indeed higher than that in ESCs,and the expression of lncRNA-GM29156,GM-39397 and GM-20005 was the most significant difference.Next,we used cat RAPID to predict proteins that interact with these three lncRNAs.We used catRAPID to predict proteins that interact with these three lncRNAs.We have seen that their interacting proteins are involved in gametogenesis and that they are involved in G protein-coupled receptor signaling pathways and cell surface receptor signaling pathways.Finally,we also obtained mouse ectodermal cells at day 5.5 and primordial germ cells at day 12.5.For ease of manipulation and access to a more purified cell population,we used Oct4-GFP transgenic mice.Oct4 is specifically expressed in E5.5 ectoderm cells.We use appropriate glass needles of appropriate thickness to cut the desired ectodermal cells along the edge of GFP.In E12.5 embryos,the primordial germ cells also specifically expressed Oct4,so we stripped the middle kidney and genital ridge under a stereomicroscope,and then performed flow sorting of the primordial germ cells we needed.Results:1.Sucessful establishment of in vitro primordial germ cell-like cells induction system.2.Successful acquisition of lncRNA expression profiles in vitro inducing primordial germ cell.3.Ten kinds of lnc RNAs with the most significant differences in ESC and PGCLC expressions were verified,and predict the proteins interacting with the first three lncRNAs.These lncRNAs were mainly found to be related to the gametogenesis.4.Obtain the E5.5 mouse ectoderm cells and E12.5 primordial germ cells.