Part:BBa_K1942000
A shRNA corresponding DNA sequence for KRAS which could silence the gene
This part is a short hairpin RNA (shRNA) sequence. When this sequence is cut by restriction enzyme and then integrated into pcDNA 6.2 vector, it can play an RNAi function in mammalian cell lines such as A549 cells. When the shRNA vector loading anti-KRAS is transfected into A549 cells, the shRNA hairpin structure will be cleaved into anti-KRAS siRNA by Dicer and then construct the RNA-induced silencing complex (RISC). The siRNA-RISC complex will target at KRAS mRNA under the guide of siRNA sequence and digest it.
USAGE AND BIOLOGY
We packaged KRAS siRNA into exosomes by transfecting HEK293 cells with a plasmid expressing KRAS siRNA and then collected siRNA-encapsulated exosomes. When modified exosomes being intravenously injected, they will specifically recognize integrin receptors and fuse with Lung adenocarcinoma cells under the direction of the iRGD peptide. Once inside cells, KRAS siRNA will bind to KRAS mRNA through base-pairing and digest the mRNA, resulting in sharp decrease of K-ras in lung cancer cells. As a consequence, K-ras protein’s reduction and disturbed function will both result in the inhabitation of the proliferation of cancer cells, which ultimately have some therapeutic effects on lung cancer (non-small cell lung cancer in this case).
CHARACTERIZATION
To ensure the interference efficiency of anti-KRAS siRNA plasmid, we transfected it into human lung adenocarcinoma cell line A549 and then extracted protein from these cells to perform western blot. Significant down-regulation of K-ras can be observed in A549 cells treated with anti-KRAS siRNA, demonstrating that anti-KRAS siRNA has the gene silencing effect on lung cancer cells.
Figure 1. Protein quantitatively analysis of K-ras protein after transfection of KRAS siRNA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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