Part:BBa_K4840002
pCMV-3Tag-1A-OGN
BBa_K4840002 (pCMV-3Tag-1A-OGN)
Usage and Biology
Osteoglycin (OGN) is a proteoglycan released from bone and muscle that is involved in extracellular matrix metabolism (Tanaka et al., 2012). Recent studies have found that the expression of OGN is significantly reduced in the articular cartilage of patients with OA (Balakrishnan et al., 2014). As the only cell type in the articular cartilage, chondrocytes can express and secrete large amounts of extracellular matrix, including collagen and polysaccharide, which play an important role in the maintenance of cartilage homeostasis (Carballo, Nakagawa, Sekiya, & Rodeo, 2017).
Introduction
We used genetic engineering techniques to construct OGN overexpressed and silencing plasmids and determined the effect of OGN expression change on the matrix metabolism of chondrocytes in order to evaluate the possibility of OGN as a therapeutic target for OA treatment.
Construction Design
BBa_K44840002 (pCMV-3Tag-1A-OGN) is composed of pCMV-3Tag-1A (BBa_K4840000) and OGN (BBa_K4840001). We selected OGN, which is involved in the regulation of matrix metabolism in various tissues. At the same time, the restriction sites EcoRI and NotI were added at both ends of the primer. In order to be successfully transfected into cells, we selected the plasmid vector pCMV-3Tag-1A, digested OGN and plasmid vector pCMV-3Tag-1A, and connected OGN and plasmid vector pCMV-3Tag-1A by T4 ligase. Finally, we constructed the overexpression plasmid of OGN, named pCMV-3Tag-1A-OGN (Figure 1).
Engineering Principle
In this study, we will construct a cell model of chondrocyte matrix metabolism disorder induced by inflammatory factor IL-1β and use the intervention method of gene overexpression to clarify the regulation of OGN expression in chondrocytes by inflammatory factors (Figure 2). To determine the effect of OGN on IL-1β-regulated chondrocyte matrix metabolism.
Experimental Approach
In this work, we first constructed OGN overexpressed plasmid. In brief, DNA fragment of OGN was amplified by using the cDNA of ATDC5 cells and then inserted into the pCMV-3Tag-1A vector. The E. coli strains solution transformed with constructed plasmid were spread onto LB solid medium plate containing corresponding antibiotic and cultured at 37℃ overnight (Figure 3).
We randomly selected several monoclonal colonies from the plate for cultivation and extracted plasmid DNA from the cultured bacterial solution. The oligo DNA in plasmid was identified by using agarose gel electrophoresis (Figure 4A). Our results show that a band of about 5000 bp is present in sample 2 and 8, suggesting that pCMV-3Tag-1A-OGN plasmid was constructed successfully. At the same time, we sent the positive monoclonal antibody to the company for sequencing. Figure 4B showed that the gene sequence was correct and there was no mutation. It proves that pCMV-3Tag-1A-OGN plasmid was successfully constructed.
Characterization/Measurement
1. Identification of ITS-induced ATDC5 cells
In order to explore the function of OGN on the matrix metabolism of chondrocytes, we firstly added ITS to the growth medium to induce the differentiation of ATDC5 cells. Since mature chondrocytes contain a large amount of proteoglycan, which can be stained blue by Alcian Blue, we performed related assays in ITS-induced ATDC5 cells. As shown in Figure 5, our cultured ATDC5 cells have differentiated into chondrocytes.
2. The increase of OGN expression improved matrix metabolism in IL-1β-treated chondrocytes
Next, we established an in vitro research model of OA by using IL-1β as a stimulator on chondrocytes and detected the mRNA and protein levels of OGN in the cells by using qRT-PCR and western blotting. As shown in Figure 6, the mRNA and protein levels of OGN in IL-1β-stimulated chondrocytes were significantly decreased compared with those of the control group, which is consistent with the clinical observation that OGN expression in chondrocytes from osteoarthritis patients is reduced. Moreover, both the mRNA and protein levels of OGN in chondrocytes transfected with previously constructed pCMV-3Tag-1A-OGN plasmid were notably increased compared to the mock group, further indicating the overexpressed plasmid was successfully constructed.
Additionally, we investigated whether OGN participates in the pathogenesis of OA. As shown in Figure 7, mRNA levels of matrix metabolism-related genes such as Aggrecan, Col-2, and Sox9 were downregulated, while MMP3 and MMP13 mRNA levels were upregulated in IL-1β-induced chondrocytes. These results indicated the disruption of matrix metabolism in chondrocytes. Notably, OGN overexpression antagonized such IL-1β-induced effects in chondrocytes. Thus, all results suggest that OGN is a promising therapeutic target for OA.
Reference
- Tanaka K, Matsumoto E, Higashimaki Y, et al. Role of osteoglycin in the linkage between muscle and bone. 2012 Apr 6;287(15):11616-28.
- Balakrishnan, L., Nirujogi, R. S., Ahmad, S., Bhattacharjee, M., Manda, S. S., Renuse, S., . . . Pandey, A. (2014). Proteomic analysis of human osteoarthritis synovial fluid. Clin Proteomics, 11(1), 6. doi:10.1186/1559-0275-11-6
- Carballo, C. B., Nakagawa, Y., Sekiya, I., & Rodeo, S. A. (2017). Basic Science of Articular Cartilage. Clin Sports Med, 36(3), 413-425. doi:10.1016/j.csm.2017.02.001
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 597
Illegal NotI site found at 667 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 763
Illegal XhoI site found at 808 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1460
Illegal NgoMIV site found at 2743
Illegal NgoMIV site found at 3026 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3238
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