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Subsequently, MOR siRNA levels were assayed in recipient Neuro2A cells when incubating with RVG exosomes loaded with MOR siRNA. The siRNAs concentrations were barely detected in untreated control cells or in cells treated with RVG exosomes or unmodified exosomes loaded with MOR siRNA. In contrast, a significant amount of siRNAs were detected in Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. As a control, MOR siRNA was also barely detected in C2C12 cells treated with RVG-exosome loaded with MOR siRNA. Taken together, these results clearly demonstrate that the RVG peptide modification on the exosome membrane specifically guides exosomes to target neuronal cells bearing the surface acetylcholine receptor, allowing for the efficient delivery of MOR siRNA into the recipient cells. | Subsequently, MOR siRNA levels were assayed in recipient Neuro2A cells when incubating with RVG exosomes loaded with MOR siRNA. The siRNAs concentrations were barely detected in untreated control cells or in cells treated with RVG exosomes or unmodified exosomes loaded with MOR siRNA. In contrast, a significant amount of siRNAs were detected in Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. As a control, MOR siRNA was also barely detected in C2C12 cells treated with RVG-exosome loaded with MOR siRNA. Taken together, these results clearly demonstrate that the RVG peptide modification on the exosome membrane specifically guides exosomes to target neuronal cells bearing the surface acetylcholine receptor, allowing for the efficient delivery of MOR siRNA into the recipient cells. | ||
| − | [[File:NJU-China-parts-fig 7.png| | + | [[File:NJU-China-parts-fig 7.png|300px]] |
Figure 7. Quantitative RT-PCR analysis of MOR siRNA concentration in Neuro2A and C2C12 cells treated with RVG exosomes (RVG exosome), unmodified exosomes loaded with MOR siRNA (siRNA-exosome) or RVG exosomes loaded with MOR siRNA (siRNA-RVG exosome). | Figure 7. Quantitative RT-PCR analysis of MOR siRNA concentration in Neuro2A and C2C12 cells treated with RVG exosomes (RVG exosome), unmodified exosomes loaded with MOR siRNA (siRNA-exosome) or RVG exosomes loaded with MOR siRNA (siRNA-RVG exosome). | ||
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| + | ====== RVG exosomes loaded with MOR siRNA specifically reduce MOR expression in neuronal cells ====== | ||
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| + | We next evaluate the effect of RVG exosome-delivered siRNA on MOR expression in vitro. MOR expression levels were assayed in Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. Compared with control cells, MOR protein and mRNA levels were dramatically reduced by RVG exosome-delivered siRNA, while no reduction in the MOR protein and mRNA levels were observed by exosomes without the RVG peptide on their surface. The results suggest that the RVG peptide modification on the exosome membrane can specifically guides exosomes to target neuronal cells, allowing for the delivery of MOR siRNA into the neuronal cells to reduce MOR expression levels. | ||
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| + | [[File:NJU-China-parts-fig8.jpg|300px]] [[File:NJU-China-parts-fig8B.png|300px]] | ||
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| + | Figure 8. RVG exosome-delivered siRNA specifically enters Neuro2A cells and reduce MOR expression. (A) Western blot analysis of MOR protein levels in untreated control Neuro2A cells or cells treated with MOR siRNA loaded in normal exosomes or RVG exosomes. (B) qRT-PCR analysis of MOR mRNA levels in untreated control Neuro2A cells or cells treated with MOR siRNA loaded in normal exosomes or RVG exosomes. | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 11:49, 18 September 2015
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Applications of BBa_K1633004
USAGE AND BIOLOGY
We package MOR siRNA into exosomes by transfecting HEK293 cells with a a Lamp2b-RVG plasmid and the MOR siRNA-2 plasmid 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
Interference efficiency of MOR siRNA-2 plasmid
To ensure the interference efficiency,MOR siRNA-2 plasmid was transfected into the mouse neuroblastoma cell line Neuro2A. Efficient knockdown of MOR by MOR siRNA-2 in Neuro2A cells is observed.
Package of MOR siRNA into exosomes
The levels of MOR siRNA in isolated exosomes were assayed by a quantitative RT-PCR assay. The MOR siRNA concentration in exosomes was calculated to be approximately 0.14 pmol/μg. The results showed that MOR siRNA can be successfully packaged into exosomes, no matter the exosomes were modified on the outside membrane with or without RVG peptide.
Figure 5. The concentration of MOR siRNA in unmodified or RVG-modified exosomes.
TEM photographs of exosomes carrying MOR siRNA inside and RVG on membranes
We next characterized the RVG exosomes loaded with MOR siRNA using transmission electron microscopy (TEM). The TEM photographs showed that the exosomes presented normal morphological characteristics after outside modification and siRNA loading, with a diameter of approximately 90 nm and a double-layer membrane surrounded. These characteristics indicate that the exosome properties were not affected by the modifications.
Figure 6. TEM photographs of the exosomes with outside RVG modification and inside siRNA loading.
RVG exosomes specifically deliver MOR siRNA into neuronal cells
Subsequently, MOR siRNA levels were assayed in recipient Neuro2A cells when incubating with RVG exosomes loaded with MOR siRNA. The siRNAs concentrations were barely detected in untreated control cells or in cells treated with RVG exosomes or unmodified exosomes loaded with MOR siRNA. In contrast, a significant amount of siRNAs were detected in Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. As a control, MOR siRNA was also barely detected in C2C12 cells treated with RVG-exosome loaded with MOR siRNA. Taken together, these results clearly demonstrate that the RVG peptide modification on the exosome membrane specifically guides exosomes to target neuronal cells bearing the surface acetylcholine receptor, allowing for the efficient delivery of MOR siRNA into the recipient cells.
Figure 7. Quantitative RT-PCR analysis of MOR siRNA concentration in Neuro2A and C2C12 cells treated with RVG exosomes (RVG exosome), unmodified exosomes loaded with MOR siRNA (siRNA-exosome) or RVG exosomes loaded with MOR siRNA (siRNA-RVG exosome).
RVG exosomes loaded with MOR siRNA specifically reduce MOR expression in neuronal cells
We next evaluate the effect of RVG exosome-delivered siRNA on MOR expression in vitro. MOR expression levels were assayed in Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. Compared with control cells, MOR protein and mRNA levels were dramatically reduced by RVG exosome-delivered siRNA, while no reduction in the MOR protein and mRNA levels were observed by exosomes without the RVG peptide on their surface. The results suggest that the RVG peptide modification on the exosome membrane can specifically guides exosomes to target neuronal cells, allowing for the delivery of MOR siRNA into the neuronal cells to reduce MOR expression levels.
Figure 8. RVG exosome-delivered siRNA specifically enters Neuro2A cells and reduce MOR expression. (A) Western blot analysis of MOR protein levels in untreated control Neuro2A cells or cells treated with MOR siRNA loaded in normal exosomes or RVG exosomes. (B) qRT-PCR analysis of MOR mRNA levels in untreated control Neuro2A cells or cells treated with MOR siRNA loaded in normal exosomes or RVG exosomes.
User Reviews
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