BBa_K4840009 (RNAi-Ready pSIREN-RetroQ-ShRNA-2)

Construction Design

RNAi-Ready pSIREN-RetroQ-ShRNA-2 (BBa_K4840009) is composed of RNAi-Ready pSIREN-RetroQ (BBa_K4840004) and ShRNA-1 (BBa_K4840006). The glycoprotein-Osteoglycin (OGN) was selected, and a knockout plasmid of OGN was constructed. The plasmid vector RNAi-ReadypSIREN-RetroQ was selected for successful transfection into cells. Two interference sequences (ShRNA-2) and a negative control sequence (ShRNA-N) were designed. Restriction endonuclease BamHI and EcoRI sites were designed at both ends of the sequence. RNAi-ReadypSIREN-RetroQ and ShRNA-2, ShRNA-N were ligated using T4 ligase, respectively named RNAi-ReadypSIREN-RetroQ-ShRNA-2 and RNAi-Ready pSIREN-RetroQ-ShRNA-N (Figure 1 AB).

Figure 1: The map of RNAi-ReadypSIREN-RetroQ-ShRNA-2; RNAi-Ready pSIREN-RetroQ-ShRNA-N

Engineering Principle

In this study, we constructed a cell model of chondrocyte matrix metabolism disorder induced by the inflammatory factor IL-1β. We used the intervention method of gene silencing to clarify the regulation of OGN expression in chondrocytes by inflammatory factors and determine the effect of gene silencing OGN on IL-1β-regulated chondrocyte matrix metabolism.

Experimental Approach

We designed and synthesized the interference sequences of OGN gene for the construction of OGN gene silencing plasmids. We performed experiments to explore whether OGN is involved in the initiation and progression of OA. The forward and reverse sequences of ShRNA were connected by annealing. The obtained DNA fragments were inserted into the RNAi-Ready pSIREN-RetroQ vector. E. coli strains transformed with constructed plasmids were spread onto LB solid medium plates containing corresponding antibiotics and cultured at 37℃ overnight (Figure 2).

Figure 2: Monoclonal colonies of E. coli transformed with OGN gene silencing plasmids (A) RNAi-Ready pSIREN-RetroQ-ShRNA-2; (B) RNAi-Ready pSIREN-RetroQ-ShRNA-N

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 3A). At the same time, we sent the positive monoclonal antibody to the company for sequencing. Figure 3C showed that the gene sequence was correct and there was no mutation. It proves that RNAi-Ready pSIREN-RetroQ-ShRNA-2 and RNAi-Ready pSIREN-RetroQ-ShRNA-N plasmids were successfully constructed.

Figure 3: Electrophoretic gel (A) and Sequencing (B.C) Verification of RNAi-Ready pSIREN-RetroQ plasmid

Characterization/Measurement

As shown in Figure 4 and Figure 5, the mRNA and protein levels of OGN were significantly decreased in IL-1β group and OGN-Sh-2 group compared with those of the Control group or OGN-Sh-N group. In addition, the results of qRT-PCR revealed that the decrease of OGN expression can induce the disruption of matrix metabolism in chondrocytes, indicative of the regulatory role of OGN in OA.

Figure 4: RNAi-Ready pSIREN-RetroQ-ShRNA-1 and 2 plasmids inhibited the expression of OGN in chondrocytes (A) qRT-PCR results; (B) western blot results

Figure 5: The decrease of OGN expression induced matrix metabolism disorder in chondrocytes

Reference

Leong I. Osteoglycin-linking bone and energy homeostasis. Nat Rev Endocrinol. 2018 Jul;14(7):379.
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.
Nulali J, Zhan M, Zhang K, et al. Osteoglycin: An ECM Factor Regulating Fibrosis and Tumorigenesis. Biomolecules. 2022 Nov 11;12(11):1674.

Sequence and Features

Assembly Compatibility: