Part:BBa_K1633005
MOR siRNA-3 (siRNA for mouse Mu opioid receptor)
This part is a short hairpin RNA (shRNA) sequence. When this shRNA sequence is cut by restriction enzyme and then integrated into pcDNA 6.2 vector, this shRNA can play a RNAi function in mammalian cell lines such as HEK293 cell. When the shRNA vector of MOR is transfected into HEK293 cells, the shRNA hairpin structure is cleaved by Dicer into siRNA of MOR and loaded into the RISC. The siRNA-RISC complex targets at Mus musculus MOR mRNA under the guide of siRNA sequence and cleave the MOR mRNA.
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]
USAGE AND BIOLOGY
We package MOR siRNA into exosomes by transfecting HEK293 cells with a plasmid expressing MOR siRNA and then collect siRNA-encapsulated exosomes. When inject the modified exosomes into the bloodstream, exosome will specifically recognize acetylcholine receptors and fuse with neurons under the direction of the RVG peptide. Once inside neurons, MOR siRNA will degrade MOR mRNA by base-pairing, resulting in sharp decrease of MOR on neuron membrane. As a consequence, MOR reduction and disturbed function will result in the inhabitation of the secretion of GABA and the suppression of the dopaminergic reward pathway, which ultimately have some therapeutic effects on opioid dependence.
CHARACTERIZATION
To ensure the interference efficiency of the MOR siRNA, three siRNA sequences targeting different sites of MOR mRNA were designed and transfected into the mouse neuroblastoma cell line Neuro2A. Efficient knockdown of MOR in Neuro2A cells is observed, and the sequence with the best interfering effect was selected for further study. Not showing the best efficiency, MOR siRNA-3 finally functions just as a backup.
Figure 2. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-3 plasmid.
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