Difference between revisions of "Part:BBa K1633006"

 
 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1633006 short</partinfo>
 
<partinfo>BBa_K1633006 short</partinfo>
Line 18: Line 17:
 
<partinfo>BBa_K1633006 parameters</partinfo>
 
<partinfo>BBa_K1633006 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
==== 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-4 finally functions just as a backup.
 +
 +
[[File:NJU-China-parts-fig 14.png|300px]]
 +
 +
Figure 2. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-4 plasmid.

Latest revision as of 19:31, 18 September 2015

MOR siRNA-4 (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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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-4 finally functions just as a backup.

NJU-China-parts-fig 14.png

Figure 2. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-4 plasmid.