Identification Of The Interaction Between DND1 And GRN

Background: Dnd1 gene plays an important role in the development of male mouse reproductive cells and testicular germ cell tumors, but its mechanism of action and its relationship with human diseases are still unknown. In previous work, we have screened out for DND1 interacting protein by yeast two hybrid technique, they are GRN, FLAD1, RNF31,EFEMP1. The Grn gene plays an important role in the regulation of cell growth, damage repair and tumorigenesis. Because the yeast two hybrid screening system may have false positive results, we have used the co-immunoprecipitation, GST pull-down assay and colocalization technique to verify the interaction between DND1 and GRN, which will lay the foundation for further elucidation of the biological functions of Dnd1.Objective: The objective of this research is to identify the interaction between DND1 and GRN by co-immunoprecipitation, GST pull-down and colocalization techniques..Methods:(1) The gene Dnd1 was amplified by PCR. The cloned gene was digested by restrictive endonuclease and subcloned into eulcaryotic experssion vecters pCDNA3.1-myc-his, and pemRFP-C1, and protokaryotic expression vecter pET32 a. Dnd1 had been cloned into pCDNA3.1-myc-his, pemRFP-C1 and pET32 a, and the recombinant plasmid was transduced into TOP10. The positive clones were identified by PCR and sequencing.(2)The gene Grn was amplified by PCR from THP-1 cDNA. The cloned gene was digested by restrictive endonuclease and subcloned into eulcaryotic experssion vecter pemGFP-C1 and protokaryotic expression vecter pGEX-4T-2. Grn was cloned into pemGFP-C1 and pGEX-4T-2, and the recombinant plasmid was transduced into TOP10. The positive clones were identified by PCR andsequencing.(3) By colocalization of intracellular, we observed DND1 and GRN colocalization situation in HEK-293 T cells.(4) In order to verify the interaction of DND1 and GRN in mammalian cells, we used co-immunoprecipitation.(5) By GST pull-down assay, we verify the direct interaction of DND1 and GRN in vitro.Results:(1) PCR and nucleotide sequencing verified that the constructed Dnd1 vectors(pET32a-dnd1, pCDNA3.1-myc-his-dnd1,pemRFP-C1-dnd1) and the Grn vectors(p GEX-4T-2-grn,pemGFP-C1-grn) were corrent.(2) The intracelluar colocalization results showed that in HEK-293 T cells DND1 was expressed both in the cytoplasm and the nucleus, and that GRN was expressed in the nucleus.Colocalization of DND1 and GRN was found in the cytoplasm.(3)Co-immunoprecipitation results showed that DND1 interacted with GRN in HEK-293 T cells.(4) GST pull-down assay results showed that DND1 interacted with GRN in vitro.These observations lay the foundation for the further study of the signaling pathways and the biological function of Dnd1 gene.