Study Of Biological Features Of Bdh2 Knockout Mouse Embryonic Stem Cells

3-hydroxybutyrate dehydrogenase-2(Bdh2),a short-chain dehydrogenase,catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore,playing a key role in iron homeostasis,energy metabolism and apoptosis.However,the function of Bdh2 in embryonic stem cells(ESCs)remains unknown.Our lab has identified a unique pluripotent embryonic stem cell type,ASC(advanced embryonic stem cell),which is highly potent.For example,a single ASC contributes to all the derivatives of the early postimplantation epiblast:the embryo,germline and the extraembryonic mesoderm of the yolk sac.To gain insights into the role of Bdh2 in pluripotency and cell fate decisions of mouse ESCs,we generated Bdh2 homozygous knockout cell lines by CRISPR/Cas9 genome editing technology.In here,we used ASCs and traditional ground-state ESCs to carry on knockout experiment,and further explore the effect of Bdh2 deficiency on ESCs.This study divides into four parts,and the main findings are shown as follows:Part one:The establishment and characterization of Bdh2 homozygous knockout embryonic stem cell lines ASCs and ESCsCRISPR/Cas9 system was applied to obtain a total of seven ASCs with homozygous deletion of Bdh2 gene(Bdh2^-/-),one ASCs with single allele deletion of Bdh2 gene(Bdh2^+/-)and two ESCs with homozygous deletion of Bdh2 gene(Bdh2^-/-).And the expression of BDH2 protein were not detected in all the knockout cell lines,indicating that BDH2 function has been lost.The establishment rate of Bdh2 gene deficient ASCs and ESCs were 8.2%and 2%,respectively.Furthermore,through a series of test including morphology observation,karyotype identification,AP staining,cell growth curves drawing,cell cycle analysis,the expression of pluripotency and differentiation gene,we demonstrated that the Bdh2 gene was dispensable for the maintaining of embryonic stem cell pluripotency;Bdh2 gene may affect the cell proliferation rate by affecting the cell cycle distribution of ESCs;Notably,the m RNA expression,even the protein level of endoderm gene Gata4,Gata6 and Sox17 were significantly up-regulated in Bdh2 gene deficient ASCs and ESCs.In contrast,the expression of mesoderm genes Brachyury(T),Evx1 and trophectoderm gene Cdx2 was significantly decreased.These data suggest that Bdh2 gene is involved in the proper control of germ layer specific transcripts for cell fate decision.Part two:Molecular features of Bdh2 gene deficient ASCsTo gain further molecular insights of Bdh2 gene deficient ASCs,we analyzed global gene expression profiles using RNA sequencing(RNA-Seq).Comparative transcriptome analysis demonstrated that Bdh2 deficiency enables ASCs to have a unique molecular feature.Firstly,after screening of differentially expressed genes,we found that the expression of endoderm markers Gata4,Gata6,Sox7,Sox17 and Pdgfra were significantly increased in Bdh2 deficient ASCs.Meanwhile,Gene ontology(GO)analysis and gene sets enrichment analysis(GSEA)all revealed enrichment for such biological processes as stem cell fate decisions,endoderm formation and development.The RNA-Seq data showed high concordance with the RT-q PCR results.Secondly,we first time systematically analyzed the effect of Bdh2 deficiency on the metabolic processes of embryonic stem cells at the transcriptome level.We found that the down-regulated genes in Bdh2 gene deficient ASCs were significantly enriched in several metabolic processes,which involved in four kinds of macromolecular substance metabolism:amino acid metabolism,carbohydrate metabolism,lipid metabolism and nucleotide metabolism.In here,our preliminary analysis data has shown that Bdh2gene deficiency affected the metabolic processes in the embryonic stem cells.Part three:Bdh2 deficiency influence on the DNA methylation of embryonic stem cellsIn this part of the experiment,we examined the expression of several methyltransferases at the transcription and protein level in the Bdh2 gene deficient ASCs.And the results showed that Bdh2 deficiency inhibit the expression of methyltransferases DNMT3A,DNMT3B and DNMT1 in Bdh2 gene deficient ASCs.At the same time,Bdh2 deficiency also causes the increase of intracellular oxidative stress and activity of m TORC1 signaling pathway.The specific mechanism of Bdh2gene regulating DNA methylation,intracellular oxidative stress and m TORC1signaling in embryonic stem cells still needs to be further explored.Part four:Bdh2 deficiency influence on the differentiation potential of embryonic stem cells in vivo and in vitroIn this part,embryonic body(EB)formation and primitive endoderm differentiation in vitro were investigated,the results showed that Bdh2 gene deficiency promoted the differentiation of embryonic stem cells towards endoderm.In addition,the phenotypic differences in EB sphere between Bdh2 gene deficient cells and control cells were detected,including the differences in the diameter,the number and the structure characteristics of EB sphere.The differences were speculated to be related to the anti-apoptotic function of Bdh2 gene.Finally,we examined the identity and developmental potential of Bdh2 gene deficient ASCs in vivo by producing the chimeric embryos and mice.The results showed that Bdh2 gene deficient ASCs were able to contribute to embryo germline lineages and yolk sac,and a full-term chimeric pup was obtained.In conclusion,we propose that the bi-allelic Bdh2 gene deficient ASC and ESC lines would be a valuable laboratory resource to study the role of Bdh2 in the development,as well as a worthwhile tool for researching stem cell earliest fate decision and DNA methylation.