Research On The Mechanisms Of Embryonic Lethality In Vps4b Knock-out Mice

Background and ObjectiveVacuolar protein sorting 4B(VPS4B),which is associated with the lysosomal degradation pathway,intracellular protein trafficking,virus budding,and regulating the different stages of mitosis and cell division.Our research group has previously identified that VPS4B as one of the pathogenic genes related to Dentin dysplasia type I(DD-I).Besides,through the preliminary study we illuminated the role of VPS4B gene in tooth development.Thus,in this study we constructed a Vps4b knock-out mouse model originally in order to investigate the effects of Vps4b gene on tooth development and related regulatory pathways.However,in the process of mouse breeding and reproduction,we found that the Vps4b-/-ones were lethal in embryonic stage;consequently we decided to explore the mechanism of embryonic lethality so as to laid the foundation for further understanding the function of Vps4b gene in biological growth and development.Materials and MethodsThe spatiotemporal expression of Vps4b gene in mice was detected by RT-PCR,qRT-PCR and immunohistochemistry.Vps4b gene knock-out mouse model was constructed by CRISPR-Cas9 system and mouse genotypes were identified by PCR and Sanger sequencing.And then,mouse transcripts were verified through RT-PCR?TA cloning and Sanger sequencing;Sequence Manipulation Suite bio-soft analysis was used for changes in protein prediction;moreover,the expression of both was validated further by qRT-PCR and immunohistochemisty assays.The body weight growth curve of the postnatal mice was drawn,and lethal time of Vps4b-/-mice embryos was determined by Vps4b+/-mice fixed-point mating.Besides,embryonic lethal morphology of Vps4b-/-mice was observed with visual microscopy and HE staining.What's more,the wild-type eukaryotic expression vector of VPS4B and VPS4B-siRNA were transfected into HEK-293T cells,and then the cell proliferation of over-expression and knockdown group was tested by CCK-8 assay,and cell apoptosis and cell cycle of knockdown group were examined by flow cytometry.Furthermore,the expression of cell apoptosis?cell cycle and endocytosis related target genes in knockdown group and E12.5 days' embryos was determined by qRT-PCR.Res?ltsThe results of RT-PCR,qRT-PCR and immunohistochemistry showed that Vps4b gene was widely and relatively stablely expressed in different tissues and stages.Sanger sequencing indicated successful construction of the Vps4b+/-gene knock-out mouse model.RT-PCR?TA cloning and Sanger sequencing showed a new transcript exhibited in Vps4b+/-mice,and by SMS bio-soft analysis,there was a new truncated protein predicted of them.Additionally,significantly reduced transcription and protein levels in Vps4b gene were seen by qRT-PCR and immunohistochemistry among the Vps4b+/-mice.What's more,the body weight growth curve of the postnatal mice showed that no significant difference in body weight between the Vps4b+/+ and Vps4b+/-mice.However,in the process of Vps4b+/-mice fixed-point mating,the Vps4b-/-mice were identified as die in E9.5 day.Besides,visual microscopy showed that Vps4b-/-embryos after E9.5 esixted serious developmental delay and gradually absorbed by their maters.HE staining indicated E12.5 Vps4b-/-embryos' placental developmental abnormalities,decreased blood vessels and other defects.CCK-8 assay showed that the cell proliferation was markedly promoted or inhibited by over-expression or knockdown of VPS4B in HEK-293T cells.Flow cytometry indicated that significantly increased early apoptosis rate was seen in knockdown group,in aDD-Ition,cells were arrested at G0/G1 phase and decreased at S phase.Moreover,qRT-PCR assay showed that the expression of mRNA levels among cell apoptosis?cell cycle and endocytosis related target genes was significantly altered in knockdown group and E12.5 embryos.ConclusionKnockout Vps4b gene results in the embryonic lethality of Vps4b-/-mice at E9.5.Signal transduction disorders of cell endocytosis is preliminarily clarified as the cause of embryonic death.We indicate that Vps4b gene is very essential for early embryonic development in mice,which providing a theoretical basis and technical support for the prevention and gene therapy of congenital malformations.