| Osteogenesis imperfecta (01) is a connective tissue disorder characterized by a variable degree of bone fragility, often accompanied by short stature, blue sclera, hearing loss, dentinogenesis imperfecta, and hyperlaxity of ligaments and skin. The prevalence of OI is estimated to be 1/10,000-1/15,000 worldwide.Type I collagen is the major components of fibrillar collagens, which constitute a quarter of the total protein weight in mammals. It mainly exists in bone, periosteum, skin, tendon, ligament, blood vessels, sclera and cornea tissues. Defects in the continuous triple helix structure of type I collagen lead to OI. Most of the human OI cases are caused by mutations in the COL1A1 or COL1A2 genes, which encode the proal and proa2 chains of collagen I, respectively.To determine the genetic defect of OI in a family, we applied a PCR-DHPLC-Sequencing method and identified a missense mutation in exon 36 of COL1A1 gene, a G to A transition at position 2461(2461 G>A), resulting in a Gly to Ser substitution at codon 821 (G821S). Polymorphism screening ruled out the possibility of polymorphism of this mutation. Hence, this mutation probably led to OI. Dppa2 (Developmental pluripotency-associated gene 2) has been identified recently as a gene that is specially expressed in pluripotent cells and some carcinoma tissues, involving in maintaining the pluripotency and self-renewal of embryonic stem cells (ESCs). Suppressing its expression induces the differentiation of ESCs. K1f4 plays a significant role in the process of reprogramming somatic cells to induced pluripotent stem cells (iPSCs), and its function of regulation in the process of embryonic development has caused a widespread concern. Previous study has shown that Dppa2 may participate in the regulatory network, which regulates the pluripotency and self-renewal of ESCs, as a target gene of Oct4. In this study, applying bioinformatics analysis, using mouse embryonic stem cells (mESCs) as materials, we explored how the changes of K1f4's expression influenced on the expression of Dppa2.Through bioinformatics analysis, we found four K1f4's transcriptional factor binding sites in the region of Dppa2's promoter, indicating that Dppa2 may be a target gene of Klf4 involved in the maintenance of ESCs' undifferentiated state and self-renewal.We firstly obtained the open reading frame (ORF) of Klf4 using the cDNA of mESCs as template by PCR, and then cloned it into the eukaryotic expression vector pCMV-myc. Its successful construction was confirmed by PCR and DNA sequencing. Then we used PLKO.1 vector to construct the RNA interference (RNAi) vector of Klf4, and its successful construction was proved by DNA sequencing.To identify the interference effect of the RNAi vector, we used 293T cells (no expression of mouse K1f4 gene) as material. We separately single-transfected pCMV-myc-K1f4, and co-transfected these two vectors, to 293T cells, while using untreated 293T cells as control. Demonstrated by RT-PCR, Realtime PCR and Western Blot, high expression of Klf4 could only be detected in the 293T cells, which was single-transfected by pCMV-myc-Klf4 vector, but could neither be detected in the co-transfected 293T cells nor in the untreated 293T cells. This result showed that both of the vectors we constructed could play roles in cells.Finally, we respectively transfected the over-expression vector of K1f4 and the RNAi vector of K1f4 to mESCs, and then determined the change of Dppa2's expression. Results showed that the expression of Dppa2 experienced a corresponding down-regulation after transfecting the RNAi vector of Klf4 into mESCs, and it also showed a corresponding up-regulation after transfecting the over-expression vector of K1f4 into mESCs. This study initially confirmed the change of Klf4's expression had influence on Dppa2's expression. Chip and EMS A are still needed to confirm that the combination does exist in vivo and vitro.
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