Construction of recombinant lentiviral vector containing shRNA against mouse Fndc5 gene in order to transduct of mouse embryonic stem cells

Objective: RNA interference (RNAi) is considered as a potential approach for knocking down of target genes and their functional assessment. Lentiviral vectors serve as an efficient tool for transducing of foreign genes in a wide variety of mammalian cells. Fibronectin type I domain-containing 5 (Fndc5) is a glycosylated membrane protein which is shown that its transcript levels to be increased during the neural and cardiac differentiation of mouse embryonic stem cells (mESCs). Here, we reported the efficacy of Fndc5 gene silencing in mESCs using lentiviral vectors expressing shRNA.
Methods: Two distinct shRNA sequences targeting Fndc5 coding sequence (CDS) and one scramble shRNA sequence as a negative control were designed and commercially synthesized. Synthetic shRNA oligonucleotides were cloned into a lentiviral inducible vector after annealing downstream of the tetracycline inducible H1 promoter. The recombinant lentiviral vector was packaged in HEK293T cells, then mESCs were transduced by lentiviral particles. Expression of shRNA in transduced cell lines was induced by Doxycycline treatment for 48h.
Results: Evaluation of transcript levels of Fndc5 by real-time PCR showed a significant decrease in transduced cells by a mixture of two shRNAs.
Conclusion: Taken together, lentiviral-mediated RNA interference targeting Fndc5 gene could be considered as an efficient tool to silence the expression of the gene in transduced cell line to study the function of Fndc5.