Latest revision as of 19:30, 18 September 2015

humanpractice

Parts

1.Bba_K1633004

MOR siRNA-2 (siRNA for mouse Mu opioid receptor)

INTRODUCTION

This part is an artificially designed RNA strand. It serves as an element of the Team NJU-CHINA RNAi module. We use it as the siRNA medicine to downregulate the expression of Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free software accessible online. This tool can find the best siRNA sequences on target gene MOR to insure the maximum gene-specificity and silencing efficacy. This tool also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. Then we synthesize the shRNA sequences with the help of a DNA synthesis company. We totally got four such shRNA plasmids. Because MOR siRNA-2 plasmid can efficiently knock down MOR expression and show best interference efficiency, it is selected as the primary siRNA medicine.

Figure 1. The sequence of MOR siRNA-2.

USAGE AND BIOLOGY

We package MOR siRNA into exosomes by transfecting HEK293 cells with 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.

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

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 80 fmol/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 3. 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 4. 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 5. 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 6. RVG exosome-delivered siRNA specifically enters Neuro2A cells and reduce MOR expression. Left panel: 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. Right panel: 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.

The effects of siRNA delivered by RVG exosomes on morphine-induced CPP

MOR and its signaling pathway are known to be involved in the dependence and relapse of opioids such as morphine and heroin. Importantly, relapse always disrupts the process of opioid withdrawal. Subsequently, we focus on investigating the effect of exosomal siRNA of MOR on opioid relapse. We evaluate the consequences of MOR knockdown by exosomal siRNA in the animals by conducting the morphine-induced conditioned place preference (CPP) test, a mouse model for morphine wanting/liking behaviors. In the CPP paradigm, mice learned to associate the rewarding effect of morphine with a drug-paired environment. The CPP test was designed to mimick the process of relapse of morphine. Before conditioning, the mice showed a preference for visiting black chamber. Then, morphine dependence was developed when mice were place-conditioned by intraperitoneal injection with morphine in the white chamber on even-numbered days (on days 2, 4, 6, 8 and 10) and with saline in the black chamber on odd-numbered days (on days 3, 5, 7, 9 and 11). On day 12, CPP test 1 was conducted by allowing the mice to freely visit the morphine-paired white chamber or saline-paired black chambers. As expected, mice showed a significant preference in visiting the morphine-paired white chamber, suggesting the development of morphine dependence. Then, morphine treatment was removed for several days. On day 26, CPP test 2 was conducted and mice spent less time in the morphine-paired white chamber than the saline-paired black chamber, suggesting the disappearance of morphine dependence. Then, mice were intravenously injected with saline or with siRNAs loaded in normal exosome or RVG exosome once every two days for a total of four times, and CPP test 3 was performed on day 32. Mice maintained their natural preference for the black chamber, suggesting that MOR siRNA had no effect on the behavior of the mice. Finally, mice were relapsed on morphine on day 33, and CPP test 4 was performed the next day. Interestingly, the mice treated with RVG exosome-delivered siRNAs maintained their natural preference for the black chamber, while the mice treated with saline or with siRNAs loaded in normal exosome show preference to morphine-paired white chamber, suggesting that the MOR siRNAs delivered by RVG exosome restrain drug addiction when the mice were re-exposed to morphine.

Figure 7. The effects of siRNA delivered by RVG exosomes on morphine-induced CPP. The upper panel is represented by the value of the time mice stay in morphine-paired white chamber minus the time mice stay in saline-paired black chamber. The lower panel is the representives of the heatmap of the mouse mobility.

The effects of siRNA delivered by RVG exosomes on MOR expression in vivo

After the CPP test, mice were sacrificed, and total RNA and protein were extracted from mouse brain to evaluate the expression levels of MOR in vivo. Both MOR protein and mRNA levels were reduced in the mice treated with RVG exosome-delivered siRNA. In contrast, siRNAs delivered by unmodified exosome could not reduce MOR mRNA and protein levels in mouse brain. Thus, these results clearly demonstrate that exosomes with RVG modification passed through the BBB and delivered MOR siRNA into the central nervous system to regulate MOR expression, while natural exosomes without the RVG modification were not capable of delivering siRNA into the central nervous system or regulating target gene expression.

Figure 8. RVG exosomes can transfer MOR siRNA through the BBB and reduce MOR expression levels in vivo. Left panel: Western blot analysis of MOR protein levels in the brains of mice following injection with saline or with MOR siRNA loaded in normal exosomes or RVG exosomes. Right panel: qRT-PCR analysis of MOR mRNA levels in the brains of mice following injection with saline or with MOR siRNA loaded in normal exosomes or RVG exosomes.

2.Bba_K1633008

nSMase2 (coding sequence to express nSMase 2 protein)

INTRODUCTION

This part is a codon-optimized nSMase 2 gene CDS. We express this gene in HEK293 cell to increase the amount of exosomes.

USAGE AND BIOLOGY

Neutral sphingomyelinase 2 (nSMase 2) is a key regulatory enzyme generating ceramide from sphingomyelin and can actively induce exosome secretion from cells and trigger cellular export of small RNAs. The original sequence of nSMase 2 from Homo Sapiens is from NCBI. NCBI Gene ID is 55512. The sequence codon of this part was optimized. We ordered the sequence from a DNA synthesis company. When this part is inserted into pcDNA 3.1 vector and transfected into mammalian cells, it can express nSMase 2 gene in mammalian cells and stimulate the secretion of exosomal siRNAs from cells.

CHARACTERIZATION

Stimulation of exosome and exosomal siRNA secretion by introduction of nSMase 2

Extracellular vesicles (EVs) are generated through biogenetic mechanisms involving neutral sphingomyelinase (nsMase).

Because nSMase2 can stimulate both exosome production and siRNA loading to exosomes, we selected nSMase2 as a “molecular pump” to accelerate the amounts of exosomes released by cells and to promote the generation of exosomal siRNAs. A plasmid expressing nSMase2 was constructed and transfected into HEK293 cells to stimulate the secretion of exosomes and exosomal siRNA from HEK293 cells. As anticipated, both exosomes and exosomal siRNA secreted by HEK293 cells were increased after overexpressing nSMase2 in HEK293 cells.

Figure 9. (A) Total amounts of exosomes (shown as total protein) secreted by HEK293 cells with or without the introduction of nSMase2. (B) Quantitative RT-PCR analysis of siRNA levels in exosomes secreted by HEK293 cells with or without the introduction of nSMase2.

We then performed nanoparticle tracking analysis (NTA) to have a more precise determination of the quantity and size of secreted exosomes. The use of Nanosight enabled quantification and size determination of the EV, as nanoparticles can be automatically tracked and sized based on Brownian motion and the diffusion coefficient. Because exosomes are more homogenous and generally smaller than most EVs with a diameter size ranging from 40 to 120 nm, the percentage of nanoparticles whose size ranges from 40 to 120 nm could be a good indicator of total amount of exosomes. The shift of peak of size distribution of EV from 170 nm to 120 nm and the significant raise of the peak indicated the increase of relative level of exosomes in secreted EVs after overexpression nSMase2 in HEK293 cells. From what we have discussed above, the improvement of manufacturing exosomes by overexpressing nSMase2 is proved to be feasible and effective.

Figure 10. Characterization of secreted exosomes after overexpression of nSMase2 in HEK293 cells. (A and B) Representative screen shots of the NTA videos for EV from HEK293 cells under normal condition (left) or after transfection with nSMase2 plasmid (right). (C and D) Size and intensity of EV from HEK293 cells under normal condition (left) or after transfection with nSMase2 plasmid (right). (E) Concentration of different particle sizes of exosomes with (red line) or without (blue line) transfection with nSMase2 plasmid. The peak of size distribution of EV shifted from 170 nm to 120 nm after transfection with nsMase2 plasmid, indicating the increase in quantity of secreted exosomes.

3.Bba_K1633003

MOR siRNA-1 (siRNA for mouse Mu opioid receptor)

INTRODUCTION

This part is an artificially designed RNA strand. It serves as an element of the Team NJU-CHINA RNAi module. We use it as the siRNA medicine to downregulate the expression of Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free software accessible online. This tool can find the best siRNA sequences on target gene MOR to insure the maximum gene-specificity and silencing efficacy. This tool also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. Then we synthesize the shRNA sequences with the help of a DNA synthesis company. We totally got four such shRNA plasmids. Although MOR siRNA-1 plasmid can efficiently knock down MOR expression, it does not show best interference efficiency and therefore serve as a backup.

Figure 11. The sequence of MOR siRNA-1.

USAGE AND BIOLOGY

This part is a shRNA designed to target and degrade MOR mRNA. When this shRNA sequence is cut by restriction enzyme and then integrated into mammalian vector, this shRNA can play a RNAi function in mammalian cell lines. When the shRNA vector of MOR is transfected into mammalian cells, the shRNA hairpin structure is cleaved by Dicer into siRNA of MOR and loaded into the RISC. The siRNA-RISC complex targets at MOR mRNA under the guide of siRNA sequence and cleave the MOR mRNA.

CHARACTERIZATION

Interference efficiency of MOR siRNA-1 plasmid

To ensure the interference efficiency, MOR siRNA-1 plasmid was transfected into the mouse neuroblastoma cell line Neuro2A. Efficient knockdown of MOR by MOR siRNA-1 in Neuro2A cells is observed.

Figure 12. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-1 plasmid.

4.Bba_K1633005

MOR siRNA-3 (siRNA for mouse Mu opioid receptor)

INTRODUCTION

This part is an artificially designed RNA strand. It serves as an element of the Team NJU-CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free software accessible online. This tool can find the best siRNA sequences on target gene MOR to insure the maximum gene-specificity and silencing efficacy. This tool also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. Then we synthesize the shRNA sequences with the help of a DNA synthesis company. We totally got four such shRNA plasmids. Although MOR siRNA-3 plasmid can efficiently knock down MOR expression, it does not show best interference efficiency and therefore serve as a backup.

Figure 13. The sequence of MOR siRNA-3.

USAGE AND BIOLOGY

This part is a shRNA designed to target and degrade MOR mRNA. When this shRNA sequence is cut by restriction enzyme and then integrated into mammalian vector, this shRNA can play a RNAi function in mammalian cell lines. When the shRNA vector of MOR is transfected into mammalian cells, the shRNA hairpin structure is cleaved by Dicer into siRNA of MOR and loaded into the RISC. The siRNA-RISC complex targets at MOR mRNA under the guide of siRNA sequence and cleave the MOR mRNA.

CHARACTERIZATION

Interference efficiency of MOR siRNA-3 plasmid

To ensure the interference efficiency, MOR siRNA-3 plasmid was, transfected into the mouse neuroblastoma cell line Neuro2A. Efficient knockdown of MOR by MOR siRNA-3 in Neuro2A cells is observed.

Figure 14. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-3 plasmid.

4.Bba_K1633006

MOR siRNA-4 (siRNA for mouse Mu opioid receptor)

INTRODUCTION

This part is an artificially designed RNA strand. It serves as an element of the Team NJU-CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free software accessible online. This tool can find the best siRNA sequences on target gene MOR to insure the maximum gene-specificity and silencing efficacy. This tool also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. Then we synthesize the shRNA sequences with the help of a DNA synthesis company. We totally got four such shRNA plasmids. Although MOR siRNA-4 plasmid can efficiently knock down MOR expression, it does not show best interference efficiency and therefore serve as a backup.

Figure 15. The sequence of MOR siRNA-4.

USAGE AND BIOLOGY

This part is a shRNA designed to target and degrade MOR mRNA. When this shRNA sequence is cut by restriction enzyme and then integrated into mammalian vector, this shRNA can play a RNAi function in mammalian cell lines. When the shRNA vector of MOR is transfected into mammalian cells, the shRNA hairpin structure is cleaved by Dicer into siRNA of MOR and loaded into the RISC. The siRNA-RISC complex targets at MOR mRNA under the guide of siRNA sequence and cleave the MOR mRNA.

CHARACTERIZATION

Interference efficiency of MOR siRNA-4 plasmid

To ensure the interference efficiency, MOR siRNA-4 plasmid was, transfected into the mouse neuroblastoma cell line Neuro2A. Efficient knockdown of MOR by MOR siRNA-4 in Neuro2A cells is observed.

Figure 16. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-4 plasmid.

6.Bba_K1633007

GFP siRNA

This part is artificial designed to target and downregulate GFP protein. 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 GFP is transfected into mammalian cells, the shRNA hairpin structure is cleaved by Dicer into siRNA of GFP and loaded into the RISC. The siRNA-RISC complex targets at GFP mRNA under the guide of siRNA sequence and cleave the GFP mRNA.

USAGE AND BIOLOGY

This part is artificial designed to target and downregulate GFP protein in GFP- transgenic mice or GFP-overexpressed cells.

CHARACTERIZATION

To determine whether siRNA delivered via RVG exosomes can pass through the BBB and regulate endogenous gene expression, we packaged siRNA against green fluorescent protein (GFP) into RVG exosomes and injected them into GFP-transgenic mice through the tail vein. Then, the GFP levels in various tissues were determined by measuring fluorescence emission using a fluorescence microscope. Compared with control mice, injection of the RVG exosomes loaded with GFP siRNA dramatically reduced GFP levels in different parts of the brain of GFP-transgenic mice. In contrast, unmodified exosomes loaded with GFP siRNA did not induce obvious GFP silencing in mouse brain. On the other hand, while unmodified exosomes loaded with GFP siRNA had significant effect on GFP levels in lung, liver and spleen of GFP-transgenic mice, RVG exosomes loaded with GFP siRNA only induced a slight but non-significant GFP silencing in these tissues. The results successfully demonstrate that exosome-packaged siRNA can be delivered to various tissues and thus silence endogenous gene expression. The results also indicate that RVG peptide on the surface of exosome has some selectivity for neuronal tissues, which may simultaneously prevent siRNA from spreading to non-neuronal tissues.

Figure 17. Fluorescence confocal microscopy photographs showing sections from different tissues of GFP-transgenic mice. GFP-transgenic mice were intravenously injected with saline (control) or with GFP siRNA loaded in normal exosomes (siRNA-exosome) or RVG exosomes (siRNA-RVG exosome).

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-<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-model.html" style="font-weight:bold;font-family:幼圆;color:black">Delivery model</a></li>
+<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-model.html" style="font-weight:bold;font-family:幼圆;color:black">Delivery Module</a></li>
-<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/RNAi" style="font-weight:bold;font-family:幼圆;color:black">RNAi model</a></li>
+<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/RNAi" style="font-weight:bold;font-family:幼圆;color:black">RNAi Module</a></li>
-<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/signaling" style="font-weight:bold;font-family:幼圆;color:black">Signaling</a></li>
+<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/signaling" style="font-weight:bold;font-family:幼圆;color:black">Signaling Module</a></li>
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 </div>
-<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-human-practice.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Human Practice</a></li>
+<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/Practices" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Human Practice</a></li>
 <li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-parts.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Parts</a></li>
 <li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-team.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Team</a></li>
 <li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-attribution.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Attribution</a></li>
-<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/Collaborations" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Colaborations</a></li>
+<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/Collaborations" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Collaborations</a></li>
 <li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-safty.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Safety</a></li>
 <div style="line-height:250%;margin-left:10%" id="main2" onClick="document.all.child2.style.display=(document.all.child2.style.display =='none')?'':'none'" > <a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Notebook </div>
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      <TD width="73%" bgColor=#FFFFFF style="vertical-align:top;padding-left:80px;padding-right:80px;padding-top:50px;padding-bottom:50px;word-wrap:break-word;">
-										<h1> <font color=#FF0000>  1.Bba_K1633003 </font> </h1>
-	<B> MOR siRNA -1(siRNA for mouse Mu opioid receptor) </B>
+	<h1>  Parts  </h1>
-	<h2> INTRODUCTION </h2>
-This part is an artificially designed RNA strand. It serves as an element of the Team
+	<h1> <font color=#FF0000>  1.Bba_K1633004 </font> </h1>
-NJU CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
-Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
-software accessible online. This tool can find the best siRNA sequences on target gene
-MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
-designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
-the plasmid. Then we order them through a DNA synthesis company.  We got four such RNA.
-MOR siRNA-1 is a backup.
-<br><br>
-<!-- 插入第一张图 --> <img src="https://static.igem.org/mediawiki/2015/2/2a/NJU-China-parts-
-parts1.png">	<br><br>
-Figure 1. The sequence of MOR siRNA-1.
-<br><br>
-	<h2> USAGE AND BIOLOGY </h2>
-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.
-<br><br>
-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-1 finally functions just as a backup.
-<br><br>
-<!-- 插入第二张图 -->	<img src="https://static.igem.org/mediawiki/2015/e/e8/NJU-China-
-parts-fig_2.png"style="width:500px">	<br><br>
-Figure 2. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-1
-plasmids.
-<br><br>
-	<h1> <font color=#FF0000>  2.Bba_K1633004 </font> </h1>
-	<B> MOR siRNA -2(siRNA for mouse Mu opioid receptor) </B>
+	<B> MOR siRNA-2 (siRNA for mouse Mu opioid receptor) </B>
 	<h2> <B> INTRODUCTION </B> </h2>
@@ Line 314: / Line 242: @@
 This part is an artificially designed RNA strand. It serves as an element of the Team
-NJU CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
+NJU-CHINA RNAi module. We use it as the siRNA medicine to downregulate the expression of
 Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
@@ Line 324: / Line 252: @@
 designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
-the plasmid. Then we order them through a DNA synthesis company.
+the plasmid. Then we synthesize the shRNA sequences with the help of a DNA synthesis
+company. We totally got four such shRNA plasmids. Because MOR siRNA-2 plasmid can
+efficiently knock down MOR expression and show best interference efficiency, it is
+selected as the primary siRNA medicine.
 <br><br>
-<!-- 插入第三张图 -->	<img src="https://static.igem.org/mediawiki/2015/3/34/NJU-China-
+<!-- 插入第三张图 -->	<img src="https://static.igem.org/mediawiki/2015/5/5c/NJU-China-
-parts-parts3.png">	<br><br>
+parts-parts2.png" style="width:500px">	<br><br>
-Figure 3. The sequence of MOR siRNA-2.
+Figure 1. The sequence of MOR siRNA-2.
 <br><br>
@@ Line 339: / Line 273: @@
 	<h2> <B> USAGE AND BIOLOGY </B> </h2>
-We package MOR siRNA into exosomes by transfecting HEK293 cells with a plasmid
+We package MOR siRNA into exosomes by transfecting HEK293 cells with a Lamp2b-RVG
-expressing MOR siRNA and then collect siRNA-encapsulated exosomes. When inject the
+plasmid and the MOR siRNA-2 plasmid and then collect siRNA-encapsulated exosomes. When
-modified exosomes into the bloodstream, exosome will specifically recognize
+inject the modified exosomes into the bloodstream, exosome will specifically recognize
 acetylcholine receptors and fuse with neurons under the direction of the RVG peptide.
@@ Line 359: / Line 293: @@
 <br><br>
-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
+	<h2> <B> CHARACTERIZATION </B> </h2>
-cell line Neuro2A. Efficient knockdown of MOR in Neuro2A cells is observed, and the
+          <B> Interference efficiency of MOR siRNA-2 plasmid </B>
-sequence with the best interfering effect was selected for further study. Not showing
+<br><br>
+To ensure the interference efficiency, MOR siRNA-2 plasmid was transfected into the mouse
-the best efficiency, MOR finally functions just as a backup. Showing the best
+neuroblastoma cell line Neuro2A. Efficient knockdown of MOR by MOR siRNA-2 in Neuro2A
-efficiency, MOR siRNA-2 is used for further study.
+cells is observed.
 <br><br>
@@ Line 375: / Line 310: @@
 <!-- 插入第四张图 -->	<img src="https://static.igem.org/mediawiki/2015/4/4a/NJU-China-
-parts-fig_4.png" style="width:500px">	<br><br>
+parts-fig_4.png" style="width:350px">	<br><br>
-Figure 4. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-1
+Figure 2. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-2
 plasmid.
@@ Line 384: / Line 319: @@
-	<h2> <B> CHARACTERIZATION </B> <h2>
+	<h3> <B> Package of MOR siRNA into exosomes </B> </h3>
-`	<h3> <B> 3.1 Package of MOR siRNA into exosomes </B> </h3>
 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
+assay. The MOR siRNA concentration in exosomes was calculated to be approximately 80
-pmol/μg. The results showed that MOR siRNA can be successfully packaged into exosomes,
+fmol/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
@@ Line 402: / Line 335: @@
 <!-- 插入第五张图 -->	<img src="https://static.igem.org/mediawiki/2015/7/72/NJU-China-
-parts-fig_5.png" style="width:500px">	<br><br>
+parts-fig_5.png" style="width:250px">	<br><br>
-Figure 5. The concentration of MOR siRNA in unmodified or RVG-modified exosomes.
+Figure 3. The concentration of MOR siRNA in unmodified or RVG-modified exosomes.
 <br><br>
-	<h3> <B> 3.2 TEM photographs of exosomes carrying MOR siRNA inside and RVG on
+	<h3> <B> TEM photographs of exosomes carrying MOR siRNA inside and RVG on
 membranes </B> </h3>
@@ Line 429: / Line 362: @@
 <!-- 插入第六张图 -->	<img src="https://static.igem.org/mediawiki/2015/f/f9/NJU-China-
-parts-fig6.jpg" style="width:500px">	<br><br>
+parts-fig6.jpg" style="width:250px">	<br><br>
-Figure 6. TEM photographs of the exosomes with outside RVG modification and inside siRNA
+Figure 4. TEM photographs of the exosomes with outside RVG modification and inside siRNA
 loading.
@@ Line 437: / Line 370: @@
 <br><br>
-	<h3> <B> 3.3 RVG exosomes specifically deliver MOR siRNA into neuronal cells
+	<h3> <B> RVG exosomes specifically deliver MOR siRNA into neuronal cells
 </B> </h3>
@@ Line 465: / Line 398: @@
 <!-- 插入第七张图 -->	<img src="https://static.igem.org/mediawiki/2015/7/78/NJU-China-
-parts-fig_7.png" style="width:400px;height:300px">	<br><br>
+parts-fig_7.png" style="width:350px">	<br><br>
-Figure 7. Quantitative RT-PCR analysis of MOR siRNA concentration in Neuro2A and C2C12
+Figure 5. 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
@@ Line 495: / Line 428: @@
 MOR siRNA into the neuronal cells to reduce MOR expression levels.
-<br><br>
 <table>
@@ Line 503: / Line 434: @@
 <TD>	<! -- 插入第八张图 -->	<img src="https://static.igem.org/mediawiki/2015/a/a9/NJU-
-China-parts-fig8.jpg" style="width:400px;height:300px"> </TD>	<br><br>
+China-parts-fig8.jpg" style="width:400px"> </TD>	<br><br>
 <TD>	<!-- 插入第八张图 -->	<img src="https://static.igem.org/mediawiki/2015/4/49/NJU-
-China-parts-fig8B.png" style="width:400px;height:300px"> </TD>	<br><br>
+China-parts-fig8B.png" style="width:250px"> </TD>	<br><br>
 </TR>
@@ Line 513: / Line 444: @@
 </table>
-Figure 8. RVG exosome-delivered siRNA specifically enters Neuro2A cells and reduce MOR
+Figure 6. RVG exosome-delivered siRNA specifically enters Neuro2A cells and reduce MOR
-expression. (A) Western blot analysis of MOR protein levels in untreated control Neuro2A
+expression. Left panel: 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)
+cells or cells treated with MOR siRNA loaded in normal exosomes or RVG exosomes. Right panel:
 qRT-PCR analysis of MOR mRNA levels in untreated control Neuro2A cells or cells treated
@@ Line 526: / Line 457: @@
-	<h3> <B> 3.5 The effects of siRNA delivered by RVG exosomes on morphine-induced
+	<h3> <B> The effects of siRNA delivered by RVG exosomes on morphine-induced
 CPP </B> </h3>
@@ Line 544: / Line 475: @@
 mice learned to associate the rewarding effect of morphine with a drug-paired
-environment. The CPP test was designed to mimick the process of relapse of morphine
+environment. The CPP test was designed to mimick the process of relapse of morphine.
-(Fig. 5A). Before conditioning, the mice showed a preference for visiting black chamber.
+Before conditioning, the mice showed a preference for visiting black chamber.
 Then, morphine dependence was developed when mice were place-conditioned by
@@ Line 589: / Line 520: @@
-<!-- 插入第九张图 -->	<img src="https://static.igem.org/mediawiki/2015/e/ee/NJU-China-
+<table>
-parts-fig9.png" style="width:400px;height:300px">	<br><br>
-Figure 9. The effects of siRNA delivered by RVG exosomes on morphine-induced CPP. A flow
-chart depicting the experimental design is shown. The panel is represented by the value
+<TR>
-of the time mice stay in morphine-paired white chamber minus the time mice stay in
-saline-paired black chamber.
+<TD>
+<!-- 插入第九张图 -->
+<img src="https://static.igem.org/mediawiki/2015/e/ee/NJU-China-
+parts-fig9.png"
+style="width:700px">	<br><br>
+</TR>
+</TD>
+<TR>
+<TD>
+<!-- 插入第九张图 -->	<img src="https://static.igem.org/mediawiki/2015/f/f1/NJU-China-
+parts-CPP.jpg" style="width:700px">	<br><br> <!-- 插入CPP -->
+</TR>
+</TD>
+</table>
+Figure 7. The effects of siRNA delivered by RVG exosomes on morphine-induced CPP.  The
+upper panel is represented by the value of the time mice stay in morphine-paired white
+chamber minus the time mice stay in saline-paired black chamber. The lower panel is the
+representives of the heatmap of the mouse mobility.
 <br><br>
-	h3> <B> 3.6 The effects of siRNA delivered by RVG exosomes on MOR expression in
+	<h3> <B> The effects of siRNA delivered by RVG exosomes on MOR expression in
 vivo </B> </h3>
@@ Line 624: / Line 600: @@
 expression.
-<br><br>
 <table>
@@ Line 632: / Line 606: @@
 	<!-- 插入第十张图 -->	<img src="https://static.igem.org/mediawiki/2015/5/55/NJU-
-China-parts-fig10.jpg" style="width:400px;height:300px">	 </TD>
+China-parts-fig10.jpg" style="width:400px">	 </TD>
 <TD>	<!-- 插入第十张图 -->	<img src="https://static.igem.org/mediawiki/2015/0/0d/NJU-
-China-parts-fig10B.png" style="width:400px;height:300px">	</TD>	<br><br>
+China-parts-fig10B.png" style="width:250px">	</TD>	<br><br>
 </TR>
 </table>
-Figure 10. RVG exosomes can transfer MOR siRNA through the BBB and reduce MOR expression
+Figure 8. RVG exosomes can transfer MOR siRNA through the BBB and reduce MOR expression
-levels in vivo. (A) Western blot analysis of MOR protein levels in the brains of mice
+levels in vivo. Left panel: Western blot analysis of MOR protein levels in the brains of mice
 following injection with saline or with MOR siRNA loaded in normal exosomes or RVG
-exosomes. (B) qRT-PCR analysis of MOR mRNA levels in the brains of mice following
+exosomes. Right panel: qRT-PCR analysis of MOR mRNA levels in the brains of mice following
 injection with saline or with MOR siRNA loaded in normal exosomes or RVG exosomes.
@@ Line 653: / Line 627: @@
 <br><br>
-		<h1> <font color=#FF0000>  3.Bba_K1633005 </font> </h1>
+		<h1> <font color=#FF0000>  2.Bba_K1633008 </font> </h1>
-		<B> MOR siRNA-3(siRNA for mouse Mu opioid receptor) </B>
+		<B> nSMase2 (coding sequence to express nSMase 2 protein) </B>
-		<h2> <B> INTRODUCTION </B> </h2>
-This part is an artificially designed RNA strand. It serves as an element of the Team
+		<h2> <B> INTRODUCTION </B> </h2>
-NJU CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
+This part is a codon-optimized nSMase 2 gene CDS. We express this gene in HEK293 cell
-Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
+to increase the amount of exosomes.
-software accessible online. This tool can find the best siRNA sequences on target gene
+<br><br>
-MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
+		<h2> <B> USAGE AND BIOLOGY </B> </h2>
-designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
+Neutral sphingomyelinase 2 (nSMase 2) is a key regulatory enzyme generating ceramide
-the plasmid. Then we order them through a DNA synthesis company.  We got four such RNA.
+from sphingomyelin and can actively induce exosome secretion from cells and trigger
-MOR siRNA-3 is a backup.
+cellular export of small RNAs. The original sequence of nSMase 2 from Homo Sapiens is
-<br><br>
+from NCBI. NCBI Gene ID is 55512. The sequence codon of this part was optimized. We
-<!-- 插入第十一张图 -->	 <img src="https://static.igem.org/mediawiki/2015/3/34/NJU-China-
+ordered the sequence from a DNA synthesis company. When this part is inserted into pcDNA
-parts-parts3.png">	<br><br>
+.1 vector and transfected into mammalian cells, it can express nSMase 2 gene in
-Figure 11. The sequence of MOR siRNA-2.
+mammalian cells and stimulate the secretion of exosomal siRNAs from cells.
 <br><br>
-		<h2> <B> USAGE AND BIOLOGY </B> </h2>
+		<h2> <B> CHARACTERIZATION </B> </h2>
-We package MOR siRNA into exosomes by transfecting HEK293 cells with a plasmid
+		<h3> Stimulation of exosome and exosomal siRNA secretion by introduction
-expressing MOR siRNA and then collect siRNA-encapsulated exosomes. When inject the
+of nSMase 2 </h3>
-modified exosomes into the bloodstream, exosome will specifically recognize
+Extracellular vesicles (EVs) are generated through biogenetic mechanisms involving
-acetylcholine receptors and fuse with neurons under the direction of the RVG peptide.
+neutral sphingomyelinase (nsMase).
-Once inside neurons, MOR siRNA will degrade MOR mRNA by base-pairing, resulting in sharp
+<br><br>
-decrease of MOR on neuron membrane. As a consequence, MOR reduction and disturbed
+Because nSMase2 can stimulate both exosome production and siRNA loading to exosomes, we
-function will result in the inhabitation of the secretion of GABA and the suppression of
+selected nSMase2 as a “molecular pump” to accelerate the amounts of exosomes released
-the dopaminergic reward pathway, which ultimately have some therapeutic effects on
+by cells and to promote the generation of exosomal siRNAs. A plasmid expressing nSMase2
-opioid dependence.
+was constructed and transfected into HEK293 cells to stimulate the secretion of exosomes
+and exosomal siRNA from HEK293 cells. As anticipated, both exosomes and exosomal siRNA
+secreted by HEK293 cells were increased after overexpressing nSMase2 in HEK293 cells.
 <br><br>
-To ensure the interference efficiency of the MOR siRNA, three siRNA sequences targeting
+<!-- 插入第十六张图 -->		<img src="https://static.igem.org/mediawiki/2015/5/58/NJU-
-different sites of MOR mRNA were designed and transfected into the mouse neuroblastoma
+China-parts-fig16.jpg" style="width:500px">	<br><br>
+Figure 9. (A) Total amounts of exosomes (shown as total protein) secreted by HEK293
-cell line Neuro2A. Efficient knockdown of MOR in Neuro2A cells is observed, and the
+cells with or without the introduction of nSMase2. (B) Quantitative RT-PCR analysis of
-sequence with the best interfering effect was selected for further study. Not showing
+siRNA levels in exosomes secreted by HEK293 cells with or without the introduction of
-the best efficiency, MOR siRNA-3 finally functions just as a backup.
+nSMase2.
 <br><br>
-<!-- 插入第十二张图 -->	<img src="https://static.igem.org/mediawiki/2015/8/83/NJU-China-
+We then performed nanoparticle tracking analysis (NTA) to have a more precise
-parts-fig12.png" style="width:400px;height:300px">	<br><br>
+determination of the quantity and size of secreted exosomes. The use of Nanosight
-Figure 12. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-3
+enabled quantification and size determination of the EV, as nanoparticles can be
-plasmid.
+automatically tracked and sized based on Brownian motion and the diffusion coefficient.
-<br><br>
+Because exosomes are more homogenous and generally smaller than most EVs with a diameter
-		<h1> <font color=#FF0000>  4.Bba_K1633006 </font> </h1>
-		<B> MOR siRNA-4(siRNA for mouse Mu opioid receptor) </B>
+size ranging from 40 to 120 nm, the percentage of nanoparticles whose size ranges from
-		<h2> <B> INTRODUCTION </B> </h2>
+to 120 nm could be a good indicator of total amount of exosomes. The shift of peak of
-This part is an artificially designed RNA strand. It serves as an element of the Team
+size distribution of EV from 170 nm to 120 nm and the significant raise of the peak
-NJU CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
+indicated the increase of relative level of exosomes in secreted EVs after
-Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
+overexpression nSMase2 in HEK293 cells. From what we have discussed above, the
-software accessible online. This tool can find the best siRNA sequences on target gene
+improvement of manufacturing exosomes by overexpressing nSMase2 is proved to be feasible
-MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
+and effective.
-designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
+<br><br>
-the plasmid. Then we order them through a DNA synthesis company.  We got four such RNA.
+<div>
+<div>
+<table>
+  <tr>
+   <div style="margin-right:300px"><td><img
-MOR siRNA-4 is a backup.
+src="https://static.igem.org/mediawiki/2015/9/92/NJU-China-parts-293t-ctrl_.gif"></td></div>
-<br><br>
+<div><td><img src="https://static.igem.org/mediawiki/2015/b/b8/NJU-China-parts-293t-
+nSMase.gif"></td></div>
+  </tr>
+</table>
+</div>
+<img
+src="https://static.igem.org/mediawiki/2015/f/fe/NJU-China-Parts-
+basic_experiment_fig5.jpg"
-<!-- 插入第十三张图 -->		<img src="https://static.igem.org/mediawiki/2015/0/03/NJU-
+style="width:500px">
-China-parts-parts4.png">	<br><br>
-Figure 13. The sequence of MOR siRNA-4.
 <br><br>
-		<h2> <B> USAGE AND BIOLOGY </B> </h2>
+Figure 10. Characterization of secreted exosomes after overexpression of nSMase2 in
-We package MOR siRNA into exosomes by transfecting HEK293 cells with a plasmid
+HEK293 cells. (A and B) Representative screen shots of the NTA videos for EV from HEK293
-expressing MOR siRNA and then collect siRNA-encapsulated exosomes. When inject the
+cells under normal condition (left) or after transfection with nSMase2 plasmid (right).
-modified exosomes into the bloodstream, exosome will specifically recognize
+(C and D) Size and intensity of EV from HEK293 cells under normal condition (left) or
-acetylcholine receptors and fuse with neurons under the direction of the RVG peptide.
+after transfection with nSMase2 plasmid (right). (E) Concentration of different particle
-Once inside neurons, MOR siRNA will degrade MOR mRNA by base-pairing, resulting in sharp
+sizes of exosomes with (red line) or without (blue line) transfection with nSMase2
-decrease of MOR on neuron membrane. As a consequence, MOR reduction and disturbed
+plasmid. The peak of size distribution of EV shifted from 170 nm to 120 nm after
-function will result in the inhabitation of the secretion of GABA and the suppression of
+transfection with nsMase2 plasmid, indicating the increase in quantity of secreted
-the dopaminergic reward pathway, which ultimately have some therapeutic effects on
+exosomes.
-opioid dependence.
-<br><br>
-To ensure the interference efficiency of the MOR siRNA, three siRNA sequences targeting
+	<h1> <font color=#FF0000>  3.Bba_K1633003 </font> </h1>
-different sites of MOR mRNA were designed and transfected into the mouse neuroblastoma
+	<B> MOR siRNA-1 (siRNA for mouse Mu opioid receptor) </B>
+	<h2> INTRODUCTION </h2>
-cell line Neuro2A. Efficient knockdown of MOR in Neuro2A cells is observed, and the
+This part is an artificially designed RNA strand. It serves as an element of the Team
-sequence with the best interfering effect was selected for further study. Not showing
+NJU-CHINA RNAi module. We use it as the siRNA medicine to downregulate the expression of
-the best efficiency, MOR siRNA-4 finally functions just as a backup.
+Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
-<br><br>
+software accessible online. This tool can find the best siRNA sequences on target gene
-<!-- 插入第十四张图 -->		<img src="https://static.igem.org/mediawiki/2015/e/ef/NJU-
+MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
-China-parts-fig_14.png">	<br><br>
+designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
-Figure 14. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-4
+the plasmid. Then we synthesize the shRNA sequences with the help of a DNA synthesis
-plasmid.
+company. We totally got four such shRNA plasmids. Although MOR siRNA-1 plasmid can
+efficiently knock down MOR expression, it does not show best interference efficiency and
+therefore serve as a backup.
 <br><br>
-		<h1> <font color=#FF0000>  5.Bba_K1633007 </font> </h1>
+<!-- 插入第一张图 --> <img src="https://static.igem.org/mediawiki/2015/2/2a/NJU-China-parts-
-		<h2> <B> USAGE AND BIOLOGY </B> </h2>
+parts1.png" style="width:500px">	<br><br>
-It is artificial designed to target and downregulate GFP protein. This part is a short
+Figure 11. The sequence of MOR siRNA-1.
-hairpin RNA (shRNA) sequence. When this shRNA sequence is cut by restriction enzyme and
+<br><br>
-then integrated into pcDNA 6.2 vector, this shRNA can play a RNAi function in mammalian
+	<h2> USAGE AND BIOLOGY </h2>
-cell lines such as HEK293 cell. When the shRNA vector of GFP is transfected into HEK293
+This part is a shRNA designed to target and degrade MOR mRNA. When this shRNA sequence
-cells, the shRNA hairpin structure is cleaved by Dicer into siRNA of MOR and loaded into
+is cut by restriction enzyme and then integrated into mammalian vector, this shRNA can
-the RISC. The siRNA-RISC complex targets at GFP mRNA under the guide of siRNA sequence
+play a RNAi function in mammalian cell lines. When the shRNA vector of MOR is
-and cleave the GFP mRNA.
+transfected into mammalian cells, the shRNA hairpin structure is cleaved by Dicer into
-<br><br>
+siRNA of MOR and loaded into the RISC. The siRNA-RISC complex targets at MOR mRNA under
-		<h2> <B> CHARACTERIZATION </B> </h2>
+the guide of siRNA sequence and cleave the MOR mRNA.
-To determine whether siRNA delivered via RVG exosomes can pass through the BBB and
+<br><br>
-regulate endogenous gene expression, we packaged siRNA against green fluorescent protein
+	<h2> CHARACTERIZATION </h2>
-(GFP) into RVG exosomes and injected them into GFP-transgenic mice through the tail
+	<h3> Interference efficiency of MOR siRNA-1 plasmid </h3>
-vein. Then, the GFP levels in various tissues were determined by measuring fluorescence
+To ensure the interference efficiency, MOR siRNA-1 plasmid was transfected into the
-emission using a fluorescence microscope. Compared with control mice, injection of the
+mouse neuroblastoma cell line Neuro2A. Efficient knockdown of MOR by MOR siRNA-1 in
-RVG exosomes loaded with GFP siRNA dramatically reduced GFP levels in different parts of
+Neuro2A cells is observed.
-the brain of GFP-transgenic mice. In contrast, unmodified exosomes loaded with GFP siRNA
+<br><br>
-did not induce obvious GFP silencing in mouse brain. On the other hand, while unmodified
+<!-- 插入第二张图 -->	<img src="https://static.igem.org/mediawiki/2015/e/e8/NJU-China-
-exosomes loaded with GFP siRNA had significant effect on GFP levels in lung, liver and
+parts-fig_2.png" style="width:350px">	<br><br>
-spleen of GFP-transgenic mice, RVG exosomes loaded with GFP siRNA only induced a slight
+Figure 12. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-1
-but non-significant GFP silencing in these tissues. The results successfully demonstrate
+plasmid.
-that exosome-packaged siRNA can be delivered to various tissues and thus silence
+<br><br>
-endogenous gene expression. The results also indicate that RVG peptide on the surface of
-exosome has some selectivity for neuronal tissues, which may simultaneously prevent
+		<h1> <font color=#FF0000>  4.Bba_K1633005 </font> </h1>
-siRNA from spreading to non-neuronal tissues.
+		<B> MOR siRNA-3 (siRNA for mouse Mu opioid receptor) </B>
-<br><br>
+		<h2> <B> INTRODUCTION </B> </h2>
-<!-- 插入第十五张图 -->		<img src="https://static.igem.org/mediawiki/2015/4/47/NJU-
+This part is an artificially designed RNA strand. It serves as an element of the Team
-China-PARTS-Figure15.jpg">	<br><br>
+NJU-CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
-Figure 15. Fluorescence confocal microscopy photographs showing sections from different
+Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
-tissues of GFP-transgenic mice. GFP-transgenic mice were intravenously injected with
+software accessible online. This tool can find the best siRNA sequences on target gene
-saline (control) or with GFP siRNA loaded in normal exosomes (siRNA-exosome) or RVG
+MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
-exosomes (siRNA-RVG exosome).
+designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
+the plasmid. Then we synthesize the shRNA sequences with the help of a DNA synthesis
-		<h1> <font color=#FF0000>  6.Bba_K1633008 </font> </h1>
+company. We totally got four such shRNA plasmids. Although MOR siRNA-3 plasmid can
-		<B> nSMase2 (coding sequence to express nSMase 2 protein) </B>
+efficiently knock down MOR expression, it does not show best interference efficiency and
+therefore serve as a backup.
-		<h2> <B> INTRODUCTION </B> </h2>
+<br><br>
-This part is a codon-optimized nSMase 2 gene CDS. We express this gene in HEK 293 cell
+<!-- 插入第十一张图 -->	 <img src="https://static.igem.org/mediawiki/2015/3/34/NJU-China-parts-parts3.png" style="width:500px">	<br><br>
-to increase the amount of exosomes.
+Figure 13. The sequence of MOR siRNA-3.
 <br><br>
 		<h2> <B> USAGE AND BIOLOGY </B> </h2>
-Neutral sphingomyelinase 2 (nSMase 2) is a key regulatory enzyme generating ceramide
+This part is a shRNA designed to target and degrade MOR mRNA. When this shRNA sequence
-from sphingomyelin and can actively induce exosome secretion from cells and trigger
+is cut by restriction enzyme and then integrated into mammalian vector, this shRNA can
-cellular export of small RNAs. The original sequence of nSMase 2 from Homo Sapiens is
+play a RNAi function in mammalian cell lines. When the shRNA vector of MOR is
-from NCBI. NCBI Gene ID is 55512. The sequence codon of this part was optimized. We
+transfected into mammalian cells, the shRNA hairpin structure is cleaved by Dicer into
-ordered the sequence from a DNA synthesis company. When this part is inserted into pcDNA
+siRNA of MOR and loaded into the RISC. The siRNA-RISC complex targets at MOR mRNA under
-.1 vector and transfected into mammalian cells, it can express nSMase 2 gene in
+the guide of siRNA sequence and cleave the MOR mRNA.
-mammalian cells and stimulate the secretion of exosomal siRNAs from cells.
 <br><br>
+			<h2> CHARACTERIZATION </h2>
-		<h2> <B> CHARACTERIZATION </B> </h2>
+	<h3> Interference efficiency of MOR siRNA-3 plasmid </h3>
-		<h3> Stimulation of exosome and exosomal siRNA secretion by introduction
+To ensure the interference efficiency, MOR siRNA-3 plasmid was, transfected into the
-of nSMase 2 </h3>
+mouse neuroblastoma cell line Neuro2A. Efficient knockdown of MOR by MOR siRNA-3 in
-Extracellular vesicles (EVs) are generated through several different and poorly
+Neuro2A cells is observed.
-understood biogenetic mechanisms, of which neutral sphingomyelinase (nsMase) is involved
+<br><br>
-in emission of exosomes [1]. nSMase2 was reported to play the vital role in exosome
+<!-- 插入第十三张图 -->		<img src="https://static.igem.org/mediawiki/2015/8/83/NJU-China-parts-fig12.png" style="width:300px">	<br><br>
-biogenesis, the inhibition of which markedly reduced the release and loading capability
+Figure 14. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-3 plasmid.
-of exosome [2].
 <br><br>
-Because nSMase2 can stimulate both exosome production and siRNA loading to exosomes, we
+               <h1> <font color=#FF0000>  4.Bba_K1633006 </font> </h1>
-selected nSMase2 as a “molecular pump” to accelerate the amounts of exosomes released
+		<B> MOR siRNA-4 (siRNA for mouse Mu opioid receptor) </B>
-by cells and to promote the generation of exosomal siRNAs. A plasmid expressing nSMase2
+                <h2> <B> INTRODUCTION </B> </h2>
+<br><br>
-was constructed and transfected into HEK293 cells to stimulate the secretion of exosomes
+This part is an artificially designed RNA strand. It serves as an element of the Team NJU-CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free software accessible online. This tool can find the best siRNA sequences on target gene MOR to insure the maximum gene-specificity and silencing efficacy. This tool also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. Then we synthesize the shRNA sequences with the help of a DNA synthesis company. We totally got four such shRNA plasmids. Although MOR siRNA-4 plasmid can efficiently knock down MOR expression, it does not show best interference efficiency and therefore serve as a backup.
-and exosomal siRNA from HEK293 cells. As anticipated, both exosomes and exosomal siRNA
+<br><br>
-secreted by HEK293 cells were increased after overexpressing nSMase2 in HEK293 cells.
+<img src="https://static.igem.org/mediawiki/2015/0/03/NJU-China-parts-parts4.png" style="width:500px">	<br><br>
+Figure 15. The sequence of MOR siRNA-4.
 <br><br>
-<!-- 插入第十六张图 -->		<img src="https://static.igem.org/mediawiki/2015/5/58/NJU-
-China-parts-fig16.jpg">	<br><br>
+		  <h2> <B> USAGE AND BIOLOGY </B> </h2>
-Figure 16. (A) Total amounts of exosomes (shown as total protein) secreted by HEK293
+This part is a shRNA designed to target and degrade MOR mRNA. When this shRNA sequence is cut by restriction enzyme and then integrated into mammalian vector, this shRNA can play a RNAi function in mammalian cell lines. When the shRNA vector of MOR is transfected into mammalian cells, the shRNA hairpin structure is cleaved by Dicer into siRNA of MOR and loaded into the RISC. The siRNA-RISC complex targets at MOR mRNA under the guide of siRNA sequence and cleave the MOR mRNA.
-cells with or without the introduction of nSMase2. (B) Quantitative RT-PCR analysis of
+<br><br>
-siRNA levels in exosomes secreted by HEK293 cells with or without the introduction of
+                    <h2> CHARACTERIZATION </h2>
+	<h3> Interference efficiency of MOR siRNA-4 plasmid </h3>
+To ensure the interference efficiency, MOR siRNA-4 plasmid was, transfected into the mouse neuroblastoma cell line Neuro2A. Efficient knockdown of MOR by MOR siRNA-4 in Neuro2A cells is observed.
-nSMase2.
 <br><br>
-We then performed nanoparticle tracking analysis (NTA) to have a more precise
+<!-- 插入第十四张图 -->		<img src="https://static.igem.org/mediawiki/2015/e/ef/NJU-
-determination of the quantity and size of secreted exosomes [3]. The use of Nanosight
+China-parts-fig_14.png" style="width:300px">	<br><br>
-enabled quantification and size determination of the EV, as nanoparticles can be
+Figure 16. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-4 plasmid.
-automatically tracked and sized based on Brownian motion and the diffusion coefficient.
+<br><br>
-Because exosomes are more homogenous and generally smaller than most EVs with a diameter
+		<h1> <font color=#FF0000>  6.Bba_K1633007 </font> </h1>
-size ranging from 40 to 120 nm [4], the percentage of nanoparticles whose size ranges
+		<B> GFP siRNA </B> <br><br>
-from 40 to 120 nm could be a good indicator of total amount of exosomes. The shift of
-peak of size distribution of EV from 170 nm to 120 nm and the significant raise of the
+This part is artificial designed to target and downregulate GFP protein. This part is a
-peak indicated the increase of relative level of exosomes in secreted EVs after
+short hairpin RNA (shRNA) sequence. When this shRNA sequence is cut by restriction
-overexpression nSMase2 in HEK293 cells. From what we have discussed above, the
+enzyme and then integrated into pcDNA 6.2 vector, this shRNA can play a RNAi function in
-improvement of manufacturing exosomes by overexpressing nSMase2 is proved to be feasible
+mammalian cell lines such as HEK293 cell. When the shRNA vector of GFP is transfected
-and effective.
+into mammalian cells, the shRNA hairpin structure is cleaved by Dicer into siRNA of GFP
-<br><br>
+and loaded into the RISC. The siRNA-RISC complex targets at GFP mRNA under the guide of
-<!-- 插入第十七张图 -->		<img src="https://static.igem.org/mediawiki/2015/2/2c/NJU-
+siRNA sequence and cleave the GFP mRNA.
-China-parts-fig17.jpg">	<br><br>
+<br><br>
-Figure 17. Characterization of secreted exosomes after overexpression of nSMase2 in
+		<h2> <B> USAGE AND BIOLOGY </B> </h2>
-HEK293 cells. (A and B) Representative screen shots of the NTA videos for EV from HEK293
+This part is artificial designed to target and downregulate GFP protein in GFP-
-cells under normal condition or after transfection with nSMase2 plasmid. (C and D) Size
+transgenic mice or GFP-overexpressed cells.
-and intensity of EV from HEK293 cells under normal condition or after transfection with
+<br><br>
-nSMase2 plasmid. (E) Concentration of different particle sizes of exosomes with (red
+		<h2> <B> CHARACTERIZATION </B> </h2>
-line) or without (blue line) transfection with nSMase2 plasmid. The peak of size
+To determine whether siRNA delivered via RVG exosomes can pass through the BBB and
-distribution of EV shifted from 170 nm to 120 nm after transfection with nsMase2
+regulate endogenous gene expression, we packaged siRNA against green fluorescent protein
-plasmid, indicating the increase in quantity of secreted exosomes.
+(GFP) into RVG exosomes and injected them into GFP-transgenic mice through the tail
+vein. Then, the GFP levels in various tissues were determined by measuring fluorescence
+emission using a fluorescence microscope. Compared with control mice, injection of the
+RVG exosomes loaded with GFP siRNA dramatically reduced GFP levels in different parts of
+the brain of GFP-transgenic mice. In contrast, unmodified exosomes loaded with GFP siRNA
+did not induce obvious GFP silencing in mouse brain. On the other hand, while unmodified
+exosomes loaded with GFP siRNA had significant effect on GFP levels in lung, liver and
+spleen of GFP-transgenic mice, RVG exosomes loaded with GFP siRNA only induced a slight
+but non-significant GFP silencing in these tissues. The results successfully demonstrate
+that exosome-packaged siRNA can be delivered to various tissues and thus silence
+endogenous gene expression. The results also indicate that RVG peptide on the surface of
+exosome has some selectivity for neuronal tissues, which may simultaneously prevent
+siRNA from spreading to non-neuronal tissues.
+<br><br>
+<!-- 插入第十五张图 -->		<img src="https://static.igem.org/mediawiki/2015/4/47/NJU-
+China-PARTS-Figure15.jpg" style="width:700px">	<br><br>
+Figure 17. Fluorescence confocal microscopy photographs showing sections from different
+tissues of GFP-transgenic mice. GFP-transgenic mice were intravenously injected with
+saline (control) or with GFP siRNA loaded in normal exosomes (siRNA-exosome) or RVG
+exosomes (siRNA-RVG exosome).

Difference between revisions of "NJU-China-parts.html"

Latest revision as of 19:30, 18 September 2015

Parts

1.Bba_K1633004

INTRODUCTION

USAGE AND BIOLOGY

CHARACTERIZATION

Package of MOR siRNA into exosomes

TEM photographs of exosomes carrying MOR siRNA inside and RVG on membranes

RVG exosomes specifically deliver MOR siRNA into neuronal cells

RVG exosomes loaded with MOR siRNA specifically reduce MOR expression in neuronal cells

The effects of siRNA delivered by RVG exosomes on morphine-induced CPP

The effects of siRNA delivered by RVG exosomes on MOR expression in vivo

2.Bba_K1633008

INTRODUCTION

USAGE AND BIOLOGY

CHARACTERIZATION

Stimulation of exosome and exosomal siRNA secretion by introduction of nSMase 2

3.Bba_K1633003

INTRODUCTION

USAGE AND BIOLOGY

CHARACTERIZATION

Interference efficiency of MOR siRNA-1 plasmid

4.Bba_K1633005

INTRODUCTION

USAGE AND BIOLOGY

CHARACTERIZATION

Interference efficiency of MOR siRNA-3 plasmid

4.Bba_K1633006

INTRODUCTION

USAGE AND BIOLOGY

CHARACTERIZATION

Interference efficiency of MOR siRNA-4 plasmid

6.Bba_K1633007

USAGE AND BIOLOGY

CHARACTERIZATION