Team:NJU China/Project/Brain

From 2013.igem.org

(Difference between revisions)
 
(67 intermediate revisions not shown)
Line 1: Line 1:
-
<html lang="en">
+
<!DOCTYPE html>
-
    <head>
+
<html lang="en" class="no-js">
-
        <meta charset="UTF-8" />
+
<head>
-
        <meta http-equiv="X-UA-Compatible" content="IE=edge,chrome=1">  
+
<meta charset="UTF-8" />
-
         <meta name="viewport" content="width=device-width, initial-scale=1.0">  
+
<meta http-equiv="X-UA-Compatible" content="IE=edge,chrome=1">  
-
<style type="text/css">
+
         <meta http-equiv="Content-Type" content="text/html; charset=utf-8">
 +
<meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
<title>NJU_China</title>
 +
<meta name="description" content="Circular Navigation Styles - Building a Circular Navigation with CSS Transforms | Codrops " />
 +
<meta name="keywords" content="css transforms, circular navigation, round navigation, circular menu, tutorial" />
 +
<meta name="author" content="Sara Soueidan for Codrops" />
 +
<link rel="shortcut icon" href="../favicon.ico">
 +
<script src="http://cdn.jquerytools.org/1.2.5/full/jquery.tools.min.js?foo"></script>
 +
 
 +
<link rel="stylesheet" type="text/css" href="http://cs.wellesley.edu/~hcilab/iGEM2012/css/Team.css">
 +
<STYLE type="text/css">
/* hiding the top section*/
/* hiding the top section*/
body{position:absolute; top:0px; width:100%; height:1480px;}
body{position:absolute; top:0px; width:100%; height:1480px;}
Line 59: Line 69:
     float:left;
     float:left;
     white-space: nowrap;
     white-space: nowrap;
-
     top:-3px;
+
     top:-6px;
     width: 490px;
     width: 490px;
     z-index: 5000;
     z-index: 5000;
     font-family: sans-serif;
     font-family: sans-serif;
-
     font-size: 55%;
+
     font-size: 95%;
     line-height: 1em;
     line-height: 1em;
     z-index:99;
     z-index:99;
Line 131: Line 141:
/* end menu (edit, page ...) */
/* end menu (edit, page ...) */
-
/* CSS reset */
+
 
-
body,div,dl,dt,dd,ul,ol,li,h1,h2,h3,h4,h5,h6,pre,form,fieldset,input,textarea,p,blockquote,th,td {  
+
/* HEADER STYLES: banner, navbar, etc. */
-
margin:0;
+
 
-
padding:0;
+
ul#nav {
 +
width:1800px;
 +
margin:40px 0 0 30px;
 +
position:relative;
}
}
-
html,body {
+
 
 +
#nav li {
 +
 
 +
color: #FFFFFF;
 +
background-color:none;
 +
margin: 0 85px 0 0;
 +
float: left;
 +
position: relative;
 +
list-style: none;
 +
text-transform:uppercase;
 +
}
 +
#nav li:last-child { margin:0; }
 +
 
 +
 
 +
/* dropdown */
 +
#nav li:hover > ul {
 +
/*display: block;*/
 +
opacity:1;
margin:0;
margin:0;
-
padding:0;
+
background-color: none;
 +
z-index:0;
}
}
-
table {
+
 
-
border-collapse:collapse;
+
#nav ul li {
-
border-spacing:0;
+
float: none;
 +
margin: 0;
 +
padding: 0;
}
}
-
fieldset,img {  
+
 
-
border:0;
+
#nav ul a {
 +
font-weight: normal;
 +
/*text-shadow: 0 1px 0 #fff;*/
}
}
-
input{
+
 
-
border:1px solid #b0b0b0;
+
 
-
padding:3px 5px 4px;
+
</STYLE>
-
color:#979797;
+
 
-
width:190px;
+
<link href='http://fonts.googleapis.com/css?family=Source+Sans+Pro:400,600' rel='stylesheet' type='text/css'>
 +
 
 +
 
 +
<style type="text/css">
 +
@import url(http://fonts.googleapis.com/css?family=Lato:300,400,700);
 +
@font-face {
 +
font-family: 'codropsicons';
 +
src:url('../fonts/codropsicons/codropsicons.eot');
 +
src:url('../fonts/codropsicons/codropsicons.eot?#iefix') format('embedded-opentype'),
 +
url('../fonts/codropsicons/codropsicons.woff') format('woff'),
 +
url('../fonts/codropsicons/codropsicons.ttf') format('truetype'),
 +
url('../fonts/codropsicons/codropsicons.svg#codropsicons') format('svg');
 +
font-weight: normal;
 +
font-style: normal;
}
}
-
address,caption,cite,code,dfn,th,var {
+
 
-
font-style:normal;
+
.container > header,
-
font-weight:normal;
+
.codrops-top,
 +
.container > section {
 +
font-family: 'Lato', Arial, sans-serif;
}
}
-
ol,ul {
+
 
-
list-style:none;
+
.container > header {
 +
margin: 0 auto;
 +
padding: 2em;
 +
text-align: center;
 +
background: rgba(0,0,0,0.01);
}
}
-
caption,th {
+
 
-
text-align:left;
+
.container > header h1 {
 +
font-size: 2.625em;
 +
line-height: 1.3;
 +
margin: 0;
 +
font-weight: 300;
}
}
-
h1,h2,h3,h4,h5,h6 {
+
 
-
font-size:100%;
+
.container > header span {
-
font-weight:normal;
+
display: block;
 +
font-size: 60%;
 +
opacity: 0.7;
 +
padding: 0 0 0.6em 0.1em;
}
}
-
q:before,q:after {
+
 
-
content:'';
+
/* To Navigation Style */
 +
.codrops-top {
 +
background: #f06060;
 +
background: rgba(255, 255, 255, 0.1);
 +
text-transform: uppercase;
 +
width: 100%;
 +
font-size: 0.69em;
 +
line-height: 2.2;
}
}
-
abbr,acronym { border:0;
+
 
 +
.codrops-top a {
 +
text-decoration: none;
 +
padding: 0 1em;
 +
letter-spacing: 0.1em;
 +
color: #fff;
 +
display: inline-block;
}
}
-
/* General Demo Style */
+
.codrops-top a:hover {
-
body{
+
background: rgba(255,255,255,0.95);
-
    background: #f7f7f7 url(../images/old_wall.jpg) repeat top left;
+
color: #777;
-
    color: rgba(0,0,0,0.8);
+
-
    font-family: 'Kelly Slab','Myriad Pro', Arial, sans-serif;
+
-
    font-size: 15px;
+
}
}
-
a{
+
 
-
    color: #fff;
+
.codrops-top span.right {
-
    text-decoration: none;
+
float: right;
}
}
-
.container{
+
 
-
    width: 100%;
+
.codrops-top span.right a {
-
    position: relative;
+
float: left;
 +
display: block;
}
}
-
.clr{
+
 
-
    clear: both;
+
.codrops-icon:before {
 +
font-family: 'codropsicons';
 +
margin: 0 4px;
 +
speak: none;
 +
font-style: normal;
 +
font-weight: normal;
 +
font-variant: normal;
 +
text-transform: none;
 +
line-height: 1;
 +
-webkit-font-smoothing: antialiased;
}
}
-
h1{
+
 
-
    font-size: 24px;
+
.codrops-icon-drop:before {
-
    position:relative;
+
content: "\e001";
-
    font-weight: 400;
+
-
    padding: 30px 5px 10px 5px;
+
-
margin: 0px 15px;
+
-
color: rgba(153,157,181,0.6);
+
-
text-shadow: 0px 1px 1px #fff;
+
-
border-bottom: 1px solid rgba(17,17,22,0.1);
+
}
}
-
/* Header Style */
+
 
-
.header{
+
.codrops-icon-prev:before {
-
    font-family:'Arial Narrow', Arial, sans-serif;
+
content: "\e004";
-
    line-height: 24px;
+
-
    font-size: 11px;
+
-
    background: #000;
+
-
    opacity: 0.9;
+
-
    text-transform: uppercase;
+
-
    z-index: 9999;
+
-
    position: relative;
+
-
    -moz-box-shadow: 1px 0px 2px #000;
+
-
    -webkit-box-shadow: 1px 0px 2px #000;
+
-
    box-shadow: 1px 0px 2px #000;
+
}
}
-
.header a{
+
 
-
    padding: 0px 10px;
+
/* Demo Buttons Style */
-
    letter-spacing: 1px;
+
.codrops-demos {
-
    color: #ddd;
+
padding-top: 1em;
-
    display: block;
+
font-size: 0.9em;
-
    float: left;
+
}
}
-
.header a:hover{
+
 
-
    color: #fff;
+
.codrops-demos a {
 +
text-decoration: none;
 +
outline: none;
 +
display: inline-block;
 +
margin: 0.5em;
 +
padding: 0.7em 1.1em;
 +
border: 3px solid #fff;
 +
color: #fff;
 +
font-weight: 700;
}
}
-
.header span.right{
+
 
-
    float: right;
+
.codrops-demos a:hover,
 +
.codrops-demos a.current-demo,
 +
.codrops-demos a.current-demo:hover {
 +
opacity: 0.6;
}
}
-
.header span.right a{
+
 
-
    float: none;
+
.container > section {
-
    display: inline;
+
padding: 2em;
 +
font-size: 1.4em;
 +
max-width: 50em;
 +
margin: 0 auto;
}
}
-
.demos{
+
 
-
    position:relative;
+
.container > section p {
-
    z-index: 1000;
+
padding: 0.8em 0;
-
    text-align:center;
+
text-align: justify;
-
float: right;
+
opacity: 0.5;
-
padding: 10px;
+
font-weight: 300;
 +
line-height: 1.4;
}
}
-
.demos a{
+
 
-
    display: inline-block;
+
@media screen and (max-width: 25em) {
-
    padding: 4px 6px;
+
 
-
    background: #000;
+
.codrops-icon span {
-
    color: #fff;
+
display: none;
-
    text-transform: uppercase;
+
}
-
font-family: Arial;
+
 
-
    font-size: 11px;
+
-
-webkit-font-smoothing:antialiased;
+
-
    box-shadow: 1px 1px 4px rgba(0,0,0,0.2);
+
-
    -webkit-transition: all 0.2s linear;
+
-
    -moz-transition: all 0.2s linear;
+
-
    -o-transition: all 0.2s linear;
+
-
    -ms-transition: all 0.2s linear;
+
-
    transition: all 0.2s linear;
+
-
}
+
-
.demos a:hover{
+
-
    color: #fff;
+
-
    background: #c90047;
+
-
}
+
-
.demos a.current-demo,
+
-
.demos a.current-demo:hover{
+
-
    font-weight: bold;
+
-
    background: #c90047;
+
-
    color: #fff;
+
-
cursor: default;
+
}
}
</style>
</style>
<style type="text/css">
<style type="text/css">
-
h2.ss-subtitle{
+
* {
-
    padding: 10px 10px 40px;
+
-
    font-size: 52px;
+
-
    text-transform: uppercase;
+
-
    color: rgba(0,0,0,0.8);
+
position: relative;
position: relative;
-
text-shadow: 1px 1px 2px rgba(0,0,0,0.2);
+
-moz-box-sizing: border-box;
 +
box-sizing: border-box;
 +
margin: 0;
 +
padding: 0;
 +
list-style: none;
}
}
-
h2.ss-subtitle:before{
+
 
-
width: 4px;
+
html,
-
height: 40px;
+
body {
-
background: rgba(17,17,22,0.8);
+
height: 100%;
-
content: '';
+
-
position: absolute;
+
-
right: 75%;
+
-
margin-right: -4px;
+
-
bottom: -4px;
+
-
-moz-border-radius: 2px 2px 0px 0px;
+
-
-webkit-border-radius: 2px 2px 0px 0px;
+
-
border-radius: 2px 2px 0px 0px;
+
}
}
-
h2.ss-subtitle:after{
+
 
-
width: 25%;
+
body {
-
height: 0px;
+
background: #b4e5ff;
-
border-bottom: 4px dotted rgba(17,17,22,0.8);
+
color: #fff;
-
content: '';
+
-
position: absolute;
+
-
right: 50%;
+
-
margin-right: -1px;
+
-
bottom: -4px;
+
}
}
-
.ss-links{
+
 
-
position: fixed;
+
.component {
-
left: 10px;
+
position: relative;
-
top: 166px;
+
margin-bottom: 3em;
-
width: 40%;
+
height: 15em;
-
z-index: 100;
+
background: rgba(0,0,0,0.05);
 +
font-family: 'Lato', Arial, sans-serif;
}
}
-
.ss-links a{
+
 
-
background: rgba(0,0,0,0.2);
+
@media only screen and (max-width: 620px) {
-
font-size: 16px;
+
.no-csstransforms li {
-
width: 40px;
+
width: 4em;
-
height: 40px;
+
height: 4em;
-
line-height: 40px;
+
line-height: 4em;
-
margin: 5px;
+
}
-
float: left;
+
-
border-radius: 50%;
+
-
display: block;
+
-
text-align: center;
+
-
-webkit-transition: background 0.2s linear;
+
-
-moz-transition: background 0.2s linear;
+
-
-o-transition: background 0.2s linear;
+
-
-ms-transition: background 0.2s linear;
+
-
transition: background 0.2s linear;
+
}
}
-
.ss-links a:hover{
+
 
-
background: rgba(0,0,0,0.4);
+
@media only screen and (max-width: 500px) {
 +
.no-ccstransforms .cn-wrapper {
 +
padding: .5em;
 +
}
 +
 
 +
.no-csstransforms .cn-wrapper li {
 +
width: 4em;
 +
height: 4em;
 +
font-size: .9em;
 +
line-height: 4em;
 +
}
}
}
-
.ss-container{
+
 
-
    width: 100%;
+
@media only screen and (max-width: 480px) {
-
    position: relative;
+
.csstransforms .cn-wrapper {
-
    text-align: left;
+
font-size: .68em;
-
    float: left;
+
}
-
overflow: hidden;
+
 
-
padding-bottom: 500px;
+
.cn-button {
 +
font-size: 1em;
 +
}
}
}
-
.ss-container h2{
+
 
-
    font-size: 40px;
+
@media only screen and (max-width:420px) {
-
    text-transform: uppercase;
+
.no-csstransforms .cn-wrapper li {
-
    color: rgba(78,84,123,0.2);
+
width: 100%;
-
text-shadow: 0px 1px 1px #fff;
+
height: 3em;
-
padding: 20px 0px;
+
line-height: 3em;
-
}
+
}
-
.ss-container:before{
+
-
    position: absolute;
+
-
    width: 4px;
+
-
    background: rgba(17,17,22,0.8);
+
-
    top: 0px;
+
-
    left: 50%;
+
-
margin-left: -2px;
+
-
    content: '';
+
-
    height: 100%;
+
-
}
+
-
.ss-row{
+
-
    width: 100%;
+
-
    clear: both;
+
-
    float: left;
+
-
    position: relative;
+
-
    padding: 30px 0;
+
-
}
+
-
.ss-left, .ss-right{
+
-
    float: left;
+
-
    width: 48%;
+
-
    position: relative;
+
-
}
+
-
.ss-right{
+
-
    padding-left: 2%;
+
-
}
+
-
.ss-left{
+
-
    text-align: right;
+
-
    float: left;
+
-
    padding-right: 2%;
+
-
}
+
-
.ss-circle{
+
-
    border-radius: 50%;
+
-
    overflow: hidden;
+
-
    display: block;
+
-
    text-indent: -9000px;
+
-
    text-align: left;
+
-
    -webkit-box-shadow:
+
-
0px 2px 5px rgba(0,0,0,0.7) inset,
+
-
0px 0px 0px 12px rgba(61,64,85,0.3);
+
-
    -moz-box-shadow:  
+
-
0px 2px 5px rgba(0,0,0,0.7) inset,
+
-
0px 0px 0px 12px rgba(61,64,85,0.3);
+
-
    box-shadow:
+
-
0px 2px 5px rgba(0,0,0,0.7) inset,
+
-
0px 0px 0px 12px rgba(61,64,85,0.3);
+
-
background-size: cover;
+
-
background-color: #f0f0f0;
+
-
background-repeat: no-repeat;
+
-
background-position: center center;
+
-
position: static;
+
-
}
+
-
.ss-small .ss-circle{
+
-
width: 100px;
+
-
height: 100px;
+
-
}
+
-
.ss-medium .ss-circle{
+
-
width: 200px;
+
-
height: 200px;
+
-
}
+
-
.ss-large .ss-circle{
+
-
width: 300px;
+
-
height: 300px;
+
-
}
+
-
.ss-circle-deco:before{
+
-
width: 29%;
+
-
height: 0px;
+
-
border-bottom: 5px dotted #ddd;
+
-
border-bottom: 5px dotted rgba(17, 17, 22, 0.3);
+
-
-webkit-box-shadow: 0px 1px 1px #fff;
+
-
-moz-box-shadow: 0px 1px 1px #fff;
+
-
box-shadow: 0px 1px 1px #fff;
+
-
position: absolute;
+
-
top: 50%;
+
-
content: '';
+
-
margin-top: -3px;
+
-
}
+
-
.ss-circle-deco:after{
+
-
width: 0px;
+
-
height: 0px;
+
-
border-top: 10px solid transparent;
+
-
    border-bottom: 10px solid transparent;
+
-
content: '';
+
-
position: absolute;
+
-
top: 50%;
+
-
margin-top: -10px;
+
-
}
+
-
.ss-left .ss-circle-deco:before{
+
-
    right: 2%; 
+
-
}
+
-
.ss-right .ss-circle-deco:before{
+
-
    left: 2%; 
+
-
}
+
-
.ss-left .ss-circle-deco:after{
+
-
right: 0;
+
-
border-right: 10px solid rgba(17,17,22,0.8);
+
-
}
+
-
.ss-right .ss-circle-deco:after{
+
-
left: 0;
+
-
border-left: 10px solid rgba(17,17,22,0.8);
+
-
}
+
-
.ss-left .ss-circle{
+
-
    float: right;
+
-
    margin-right: 30%;
+
-
}
+
-
.ss-right .ss-circle{
+
-
    float: left;
+
-
    margin-left: 30%;
+
-
}
+
-
.ss-container h3{
+
-
    margin-top: 34px;
+
-
padding: 10px 15px;
+
-
background: rgba(26, 27, 33, 0.6);
+
-
text-shadow: 1px 1px 1px rgba(26, 27, 33, 0.8)
+
-
}
+
-
.ss-container .ss-medium h3{
+
-
margin-top: 82px;
+
-
}
+
-
.ss-container .ss-large h3{
+
-
margin-top: 133px;
+
-
}
+
-
.ss-container .ss-left h3{
+
-
border-right: 5px solid rgba(164,166,181,0.8);
+
-
}
+
-
.ss-container .ss-right h3{
+
-
border-left: 5px solid rgba(164,166,181,0.8);
+
-
}
+
-
.ss-container h3 span{
+
-
    color: rgba(255,255,255,0.8);
+
-
    font-size: 13px;
+
-
    display: block;
+
-
    padding-bottom: 5px;
+
-
}
+
-
.ss-container h3 a{
+
-
    font-size: 28px;
+
-
    color: rgba(255,255,255,0.9);
+
-
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</script>
 
-
    </head>
 
-
    <body>
 
-
        <p>&nbsp;</p>
 
-
        <div class="container">
 
-
            <h2 class="ss-subtitle">RESULTS</h2>
+
</head>
-
<div id="ss-links" class="ss-links">
+
<body>
-
<a href="#Brain">Brain</a>
+
<div style="float:left; position:absolute; margin-top:-30px; margin-left:-50px; z-index:1; display:block; ">
-
<a href="#Liver">Liver</a>
+
</br></br>
-
<a href="#T cell">T cell</a>
+
<a href="https://2013.igem.org"><img src="https://static.igem.org/mediawiki/2013/8/80/NJU-Miniminiminilogocolored.png" ></a>
 +
</div>
 +
<div class="container">
 +
<!--Start NavBar-->
 +
<ul id="nav">  
 +
    <li><a href="https://2013.igem.org/Team:NJU_China">Home</a></li>
 +
 +
    <li><a href="https://2013.igem.org/Team:NJU_China/Project">Project</a>  
 +
        <ul>
 +
  <li><a href="https://2013.igem.org/Team:NJU_China/Project">Overview</a></li>
 +
          <li><a href="https://2013.igem.org/Team:NJU_China/Project">Chassis</a></li>
 +
          <li><a href="https://2013.igem.org/Team:NJU_China/Project">Targeting Module</a></li>
 +
          <li><a href="https://2013.igem.org/Team:NJU_China/Project">Killing Module</a></li>
 +
          <li><a href="https://2013.igem.org/Team:NJU_China/Project">Achievement</a></li>
 +
        </ul>
 +
    </li>
 +
    <li><a href="https://2013.igem.org/Team:NJU_China/Modeling">Modeling</a>
 +
    </li>
 +
 +
    <li><a href="https://2013.igem.org/Team:NJU_China/Human Practice">Human Practice</a>
 +
        <ul>
 +
  <li><a href="https://2013.igem.org/Team:NJU_China/Human Practice">Free debate</a></li>
 +
          <li><a href="https://2013.igem.org/Team:NJU_China/Human Practice">Reach the unreached</a></li>
 +
          <li><a href="https://2013.igem.org/Team:NJU_China/Human Practice">Workshop</a></li>
 +
          <li><a href="https://2013.igem.org/Team:NJU_China/Human Practice">Collaboration</a></li>
 +
        </ul>
 +
    </li>
-
</div>
+
    <li><a href="https://2013.igem.org/Team:NJU_China/Wet lab">Wet lab</a>
-
            <div id="ss-container" class="ss-container">
+
        <ul>  
-
                <div class="ss-row">
+
  <li><a href="https://2013.igem.org/Team:NJU_China/Protocol">Protocol</a></li>  
-
                    <div class="ss-left">
+
          <li><a href="https://2013.igem.org/Team:NJU_China/Notebook">Lab notes</a></li>  
-
                        <h2 id="Brain">Brain</h2>
+
          <li><a href="https://2013.igem.org/Team:NJU_China/Data Page">Data Page</a></li>  
-
                    </div>
+
          <li><a href="https://2013.igem.org/Team:NJU_China/Parts">Parts</a></li>  
-
                    <div class="ss-right">
+
        </ul>
-
                        <h2>2013</h2>
+
    </li>  
-
                    </div>
+
   
-
                </div>
+
    <li><a href="https://2013.igem.org/Team:NJU_China/Safety">Safety</a>
-
                <div class="ss-row ss-medium">
+
        <ul>  
-
                    <div class="ss-left">
+
  <li><a href="https://2013.igem.org/Team:NJU_China/Safety">Safety form</a></li>  
-
<a>Liver:</a>
+
          <li><a href="https://2013.igem.org/Team:NJU_China/Virus-related_safety">Virus-related safety</a></li>  
-
<span>For liver targeting, we need to first find a protein specifically recognize hepatic cells. Since Heptitis B virus can infect hepatic cells distinctively, and from recent study[1], we knew that HBV recognizes the hepatic cells via the interaction between the pre-S1 of the HBV envelop protein and NTCP receptor of the hepatic cells. We tried to engineer the pre-S1 from HBV envelope protein to the lamp 2b.</BR>
+
        </ul>
-
Therefore we cloned the pre-S1 into lamp 2b, and we choose pcDNA 3.1(+) as our backbone.</span>
+
    </li>  
-
                         
+
-
                        </h3>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h3>
+
-
<a>Results for liver targeting:</a>
+
-
<span>To produce the exosomes that have pre-S1 on their surface for liver targeting, we first transfected the exosome-producing cells, HEK 293T cells, with the plasmid encoding the fusion protein of lamp 2b and pre-S1 peptide.</span>
+
-
                         
+
-
                        </h3>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row">
+
    <li><a href="https://2013.igem.org/Team:NJU_China/Team">Team</a></li>
-
                    <div class="ss-left">
+
-
                        <h2 id="Liver">Liver</h2>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h2>2013</h2>
+
-
                    </div>
+
-
                </div>
+
-
                <div class="ss-row ss-medium">
+
-
                    <div class="ss-rihgt">
+
-
                        <a class="ss-circle ss-circle-2">Week2 (14th-18th, April)</a>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-small">
+
-
                    <div class="ss-left">
+
-
                        <h3>
+
-
<a>siRNA screening (Failed)</a>
+
-
<span>14th: We extracted plasmids and cultured the 293t cells.</span>
+
-
<span>15th: We transfected plasmids into 293t cells.</span>
+
-
<span>16th: We collected cells and preserved it in Trizol.</span>
+
-
<span>17th: We extracted RNA, then did RT-PCR with the RNA.</span>
+
-
<span>18th: We did qPCR with the cDNA we got on 17th April.</span>
+
-
<span>21th: We reexamined the concentration of RNA, and redid qPCR.</span>
+
-
                        </h3>
+
-
                    </div>
+
-
                    <div class="ss-row ss-medium">
+
-
                    <div class="ss-left">
+
-
                        <a class="ss-circle ss-circle-3">Week3 (27th, April – 1st, May)</a>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-small">
+
-
                    <div class="ss-right">
+
-
                        <h3>
+
-
<a> siRNA screening (Failed)</a>
+
-
<span>27th: We cultured the 293t cells in a 12-well plate.</span>
+
-
<span>28th: We transfected 293t cells.</span>
+
-
<span>29th: We collected cells and extracted RNA from it.</span>
+
-
<span>30th: We did RT-qPCR with the RNA we extracted on 29th April.</span>
+
-
<span>1st: We did qPCR with the cDNA we got on 30th April.</span>
+
-
                        </h3>
+
-
                    </div>
+
-
                   
+
-
<div class="ss-row">
+
-
                    <div class="ss-left">
+
-
                        <h2 id="T cell">T cell</h2>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h2>2013</h2>
+
-
                    </div>
+
-
                </div>
+
-
                <div class="ss-row ss-medium">
+
-
                    <div class="ss-left">
+
-
                        <a class="ss-circle ss-circle-4">Week4 (8th-13th, May)</a>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-small">
+
-
                    <div class="ss-left">
+
-
                        <h3>
+
-
<a> siRNA screening (Failed), examination whether siRNA are capsulated into exosomes (Success)</a>
+
-
<span>8th: We extracted plasmids of 3 kinds of siRNA.</span>
+
-
<span>9th: We cultured 293t cells in eight D10 dishes and a 12-well plate.</span>
+
-
<span>10th: We extracted 2 kinds of over-expression plasmids and examined the concentration of it. Then we transfect these plasmids into 293t cells respectively.</span>
+
-
<span>11th: We collected cells and exosomes from 8 D10 dish, and cells from 12-well plate.</span>
+
-
<span>12th: We extracted RNA from cells and exosomes.</span>
+
-
<span>13th: We did RT-PCR and qPCR.</span>
+
-
                        </h3>
+
    <li><a href="https://2013.igem.org/Team:NJU_China/Extras">Extras</a>
-
                    </div>
+
            <ul>  
-
                    <div class="ss-right">
+
  <li><a href="https://igem.org/2013_Judging_Form?id=1180">Judging criteria</a></li>  
-
<a href="http://sc.chinaz.com/" class="ss-circle ss-circle-15">Scrollbar Visibility with jScrollPane</a>
+
          <li><a href="https://2013.igem.org/Team:NJU_China/Attributions">Attribution</a></li>  
-
                    </div>
+
          <li><a href="https://2013.igem.org/Team:NJU_China/Acknowledgement">Acknowledgement</a></li>
-
                </div>
+
        </ul>
-
<div class="ss-row ss-medium">
+
    </li>
-
                    <div class="ss-left">
+
   
-
                        <a href="http://sc.chinaz.com/" class="ss-circle ss-circle-16">Multi-level Photo Map</a>
+
</ul>
-
                    </div>
+
<!--End NavBar-->
-
                    <div class="ss-right">
+
</br></br></br></br></br>
-
                        <h3>
+
<section>
-
                            <span>September 27, 2013</span>
+
<h1>Brain:</h1>
-
                            <a href="http://sc.chinaz.com/">Multi-level Photo Map</a>
+
<p>
-
                        </h3>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-large">
+
-
                    <div class="ss-left">
+
-
                        <a href="http://sc.chinaz.com/" class="ss-circle ss-circle-17">Responsive Image Gallery with Thumbnail Carousel</a>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h3>
+
-
                            <span>September 20, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Responsive Image Gallery with Thumbnail Carousel</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-small">
+
-
                    <div class="ss-left">
+
-
<h3>
+
-
                            <span>September 12, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Elastislide - A Responsive jQuery Carousel Plugin</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
<a href="http://sc.chinaz.com/" class="ss-circle ss-circle-18">Elastislide - A Responsive jQuery Carousel Plugin</a>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-medium">
+
-
                    <div class="ss-left">
+
-
                        <a href="http://sc.chinaz.com/" class="ss-circle ss-circle-19">Slicebox - A fresh 3D image slider with graceful fallback </a>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h3>
+
-
                            <span>September 5, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Slicebox - A fresh 3D image slider with graceful fallback </a>
+
-
                        </h3>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row">
+
-
                    <div class="ss-left">
+
-
                        <h2 id="august">August</h2>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h2>2013</h2>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-large">
+
-
                    <div class="ss-left">
+
-
<h3>
+
-
                            <span>August 30, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Automatic Image Montage with jQuery</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
<a href="http://sc.chinaz.com/" class="ss-circle ss-circle-20">Automatic Image Montage with jQuery</a>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-medium">
+
-
                    <div class="ss-left">
+
-
                        <a href="http://sc.chinaz.com/" class="ss-circle ss-circle-21">Image Zoom Tour with jQuery</a>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h3>
+
-
                            <span>August 23, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Image Zoom Tour with jQuery</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-small">
+
-
                    <div class="ss-left">
+
-
<h3>
+
-
                            <span>August 16, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Circular Content Carousel with jQuery</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
<a href="http://sc.chinaz.com/" class="ss-circle ss-circle-22">Circular Content Carousel with jQuery</a>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-large">
+
-
                    <div class="ss-left">
+
-
                        <a href="http://sc.chinaz.com/" class="ss-circle ss-circle-23">Portfolio Image Navigation with jQuery</a>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h3>
+
-
                            <span>August 9, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Portfolio Image Navigation with jQuery</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-medium">
+
-
                    <div class="ss-left">
+
-
<h3>
+
-
                            <span>August 4, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Expanding Fullscreen Grid Portfolio</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
<a href="http://sc.chinaz.com/" class="ss-circle ss-circle-24">Expanding Fullscreen Grid Portfolio</a>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row">
+
-
                    <div class="ss-left">
+
-
                        <h2 id="july">July</h2>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h2>2013</h2>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-small">
+
-
                    <div class="ss-left">
+
-
                        <a href="http://sc.chinaz.com/" class="ss-circle ss-circle-25">Content Rotator with jQuery</a>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h3>
+
-
                            <span>July 29, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Content Rotator with jQuery</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-large">
+
-
                    <div class="ss-left">
+
-
<h3>
+
-
                            <span>July 22, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Vertical Sliding Accordion with jQuery</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
<a href="http://sc.chinaz.com" class="ss-circle ss-circle-26">Vertical Sliding Accordion with jQuery</a>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-medium">
+
-
                    <div class="ss-left">
+
-
                        <a href="http://sc.chinaz.com" class="ss-circle ss-circle-27">Animated Text and Icon Menu with jQuery</a>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h3>
+
-
                            <span>July 12, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Animated Text and Icon Menu with jQuery</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-small">
+
-
                    <div class="ss-left">
+
-
<h3>
+
-
                            <span>July 5, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Fullscreen Slideshow with HTML5 Audio and jQuery</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
<a href="http://sc.chinaz.com/" class="ss-circle ss-circle-30">Fullscreen Slideshow with HTML5 Audio and jQuery</a>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-large">
+
-
                    <div class="ss-left">
+
-
                        <a href="http://sc.chinaz.com/" class="ss-circle ss-circle-28">Sliding Background Image Menu with jQuery</a>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h3>
+
-
                            <span>July 3, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Sliding Background Image Menu with jQuery</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row">
+
-
                    <div class="ss-left">
+
-
                        <h2 id="june">June</h2>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
                        <h2>2013</h2>
+
-
                    </div>
+
-
                </div>
+
-
<div class="ss-row ss-small">
+
-
                    <div class="ss-left">
+
-
                      <h3>
+
-
                            <span>June 9, 2013</span>
+
-
                            <a href="http://sc.chinaz.com/">Grid Navigation Effects with jQuery</a>
+
-
                        </h3>
+
-
                    </div>
+
-
                    <div class="ss-right">
+
-
<a href="http://sc.chinaz.com/" class="ss-circle ss-circle-29">Grid Navigation Effects with jQuery</a>
+
-
                    </div>
+
-
                </div>
+
-
            </div>
+
-
        </div>
+
-
        <script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.7.1/jquery.min.js"></script>
+
-
<script language="Javascript">
+
-
/*
+
-
* jQuery Easing v1.3 - http://gsgd.co.uk/sandbox/jquery/easing/
+
-
*
+
-
* Uses the built in easing capabilities added In jQuery 1.1
+
-
* to offer multiple easing options
+
-
*
+
-
* TERMS OF USE - jQuery Easing
+
-
*
+
-
* Open source under the BSD License.
+
-
*
+
-
* Copyright © 2008 George McGinley Smith
+
-
* All rights reserved.
+
-
*
+
-
* Redistribution and use in source and binary forms, with or without modification,
+
-
* are permitted provided that the following conditions are met:
+
-
*
+
-
* Redistributions of source code must retain the above copyright notice, this list of
+
-
* conditions and the following disclaimer.
+
-
* Redistributions in binary form must reproduce the above copyright notice, this list
+
-
* of conditions and the following disclaimer in the documentation and/or other materials
+
-
* provided with the distribution.
+
-
*
+
-
* Neither the name of the author nor the names of contributors may be used to endorse
+
-
* or promote products derived from this software without specific prior written permission.
+
-
*
+
-
* THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS" AND ANY
+
-
* EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF
+
-
* MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE
+
-
*  COPYRIGHT OWNER OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL,
+
-
*  EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE
+
-
*  GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED
+
-
* AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING
+
-
*  NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED
+
-
* OF THE POSSIBILITY OF SUCH DAMAGE.
+
-
*
+
-
*/
+
-
// t: current time, b: begInnIng value, c: change In value, d: duration
+
For brain targeting, we chose to use RVG, which is a short peptide from Rabies Virus, as our targeting protein. RVG can specifically recognize acetylcholine receptor in the central nervous system [2], thus we engineer the RVG peptide into the lamp 2b and we use pcDNA 3.1(+) as our vector.</br>
-
jQuery.easing['jswing'] = jQuery.easing['swing'];
+
To produce the brain-specific RVG exosomes, we first transfected the exosome-producing cells, HEK 293T cells, with the plasmids encoding the fusion protein of lamp 2b and RVG peptide.</br>
 +
Also, we use a siRNA as an indicator to assess the targeting effect of RVG-exosomes and loaded the siRNA into the RVG-modified exosomes by direct transfection of the HEK 293T cells with siRNA probe.
 +
</p></section>
 +
<header>
 +
<h1>
 +
<img src="https://static.igem.org/mediawiki/2013/3/3e/ShibieRVG_small.png">
 +
<img src="https://static.igem.org/mediawiki/2013/a/ae/Pcdna3.1_RVG_small.png">
 +
</h1>
 +
</header>
-
jQuery.extend( jQuery.easing,
+
<section>
-
{
+
<h1>Experimental procedure and Results:</h1>
-
def: 'easeOutQuad',
+
 
-
swing: function (x, t, b, c, d) {
+
<p>1. Quantification of siRNA contained in the exosomes</BR>
-
//alert(jQuery.easing.default);
+
    We first quantify the amount of siRNA encapsulated into the exosomes. siRNA was transfected to the HEK 293T cells(which has already been transfected with RVG-Lamp2b plasmids) before exosome purification. Control group was not transfected with such siRNA.</BR>
-
return jQuery.easing[jQuery.easing.def](x, t, b, c, d);
+
    By quantitative PCR analysis of a series of siRNA with known concentration, we drew a standard curve and then use it to calculate the absolute amount of siRNA in the exosomes. As shown in Fig.1, the amount of siRNA in the negative control is quite low (background) while the siRNA contained in the RVG exosomes transfected with siRNA probe reached as high as 0.8 fmol (RNA)/μg (exosome).</br>
-
},
+
The result demonstrates that siRNAs were successfully encapsulated into RVG exosomes.</p>
-
easeInQuad: function (x, t, b, c, d) {
+
 
-
return c*(t/=d)*t + b;
+
<img width="500px"; height="500px" src="https://static.igem.org/mediawiki/2013/2/20/Njubrain.png">
-
},
+
 
-
easeOutQuad: function (x, t, b, c, d) {
+
<p>Figure.1 Empty exosome is collected from HEK 293 T cells without transfection of siRNA probe while the RVG exosome +siRNA is collected from the HEK 293T cells after transfection of siRNA probe. The amount of siRNA contained in the exosome is measured by qPCR.</p>
-
return -c *(t/=d)*(t-2) + b;
+
 
-
},
+
<p>2.In vitro evidence for the entry of RVG exosomes into the primary cortical neuron</br>
-
easeInOutQuad: function (x, t, b, c, d) {
+
  To confirm the targeting capability of modified exosomes, we co-culture the siRNA-containing RVG exosomes with primary cortical neurons, and then measure the amounts of siRNA probe in the neurons to see if our RVG exosome can successfully get into the neurons. </br>
-
if ((t/=d/2) < 1) return c/2*t*t + b;
+
From Fig.2, we can see that siRNA probe labeled with Alexaflour 555（red ）successfully get into the neurons.</p>
-
return -c/2 * ((--t)*(t-2) - 1) + b;
+
 
-
},
+
<img  src="https://static.igem.org/mediawiki/2013/e/e9/Nju_BRAIN2.png">
-
easeInCubic: function (x, t, b, c, d) {
+
<p>Figure.2 Confocal microscopy image of the internalization of Alexaflour 555（red ）labeled siRNA into  neurons.</p>
-
return c*(t/=d)*t*t + b;
+
 
-
},
+
<p>In qPCR analysis of the relative siRNA probe level in the neurons co-cultured with both empty RVG-exosomes and siRNA containing RVG-exosomes, respectively (Fig.3), almost no siRNA was detected from the neurons co-cultured with 40 μg empty RVG-exosomes, while the siRNA detected in neurons co-cultured with siRNA probe containing RVG-exosomes show both significant and dose-dependent increase.  </p>
-
easeOutCubic: function (x, t, b, c, d) {
+
 
-
return c*((t=t/d-1)*t*t + 1) + b;
+
<img width="500px"; height="500px" src="https://static.igem.org/mediawiki/2013/5/51/Njubrain3.png">
-
},
+
 
-
easeInOutCubic: function (x, t, b, c, d) {
+
<p>Figure.3 The RNA was extracted from the primary cortical neurons co-cultured 24h with 40 μg empty exosome, 20μg siRNA containing RVG-exosomes,40μg siRNA containing RVG-exosomes, respectively. And the RNA extracted was measured by qPCR using probe for the siRNA encapsulated in the exosomes.</p>
-
if ((t/=d/2) < 1) return c/2*t*t*t + b;
+
 
-
return c/2*((t-=2)*t*t + 2) + b;
+
<p>3.In vivo evidence for the entry of RVG-exosomes entry into the brain</br>
-
},
+
    To further investigate whether the RVG-exosome can get into brain, we intravenously injected the empty RVG-exosomes, siRNA containing RVG-exosomes, respectively, into the mice. Then we extracted brain tissue and measured the siRNA level in the cortex and medulla.</p>
-
easeInQuart: function (x, t, b, c, d) {
+
<p>As shown in Fig.4, no siRNA was detected in both cortex and medulla after the injection of empty RVG-exosomes and siRNA containing exosomes, while for the siRNA containing RVG-exosomes, the siRNA detected in the cortex and medulla are significant higher than the others.</p>
-
return c*(t/=d)*t*t*t + b;
+
 
-
},
+
<img width="700px"; height="500px" src="https://static.igem.org/mediawiki/2013/c/ce/Njubrain5.png">
-
easeOutQuart: function (x, t, b, c, d) {
+
 
-
return -c * ((t=t/d-1)*t*t*t - 1) + b;
+
<p>Figure.4 The mice were intravenously injected with 200 μg empty exosomes,siRNA containing exosomes and siRNA containing RVG-exosomes, respectively once a day, and continued for four days. On the fifth day, the mice were killed and their brains were taken out. The RNA from their cortex and medulla were measured using siRNA probe for the siRNA encapsulated in the exosome.</p>
-
},
+
 
-
easeInOutQuart: function (x, t, b, c, d) {
+
<p>4.In vivo evidence for the targeting effect of RVG-exosome</br>
-
if ((t/=d/2) < 1) return c/2*t*t*t*t + b;
+
 
-
return -c/2 * ((t-=2)*t*t*t - 2) + b;
+
The siRNA contained in the exosomes were designed to silence OPRM gene, which encodesμ- opioid receptor in neuron cells. We intravenously injected three groups of mice: control group is injected with PBS (Mock), second group is injected with empty exosome, third group is injected with RVG-exosome which has siRNA in it. Next, by qPCR analysis we determined the relative OPRM mRNA level in mice brain. The result (Fig.5) shows that RVG exosome successfully target to neuron cells and the siRNA brought by the exosome down-regulate the gene significantly. </p>
-
},
+
 
-
easeInQuint: function (x, t, b, c, d) {
+
<img width="700px"; height="500px" src="https://static.igem.org/mediawiki/2013/4/4c/无标题副本.png">
-
return c*(t/=d)*t*t*t*t + b;
+
-
},
+
-
easeOutQuint: function (x, t, b, c, d) {
+
-
return c*((t=t/d-1)*t*t*t*t + 1) + b;
+
-
},
+
-
easeInOutQuint: function (x, t, b, c, d) {
+
-
if ((t/=d/2) < 1) return c/2*t*t*t*t*t + b;
+
-
return c/2*((t-=2)*t*t*t*t + 2) + b;
+
-
},
+
-
easeInSine: function (x, t, b, c, d) {
+
-
return -c * Math.cos(t/d * (Math.PI/2)) + c + b;
+
-
},
+
-
easeOutSine: function (x, t, b, c, d) {
+
-
return c * Math.sin(t/d * (Math.PI/2)) + b;
+
-
},
+
-
easeInOutSine: function (x, t, b, c, d) {
+
-
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<p>Figure.5 in vivo evidence for targeting effect of RVG-exosome. First group (Mock) was injected with PBS and second group was injected with Empty exosome. The last group was injected with RVG-exosome which contained siRNA that could silence OPRM gene. The result shows that modified exosome successfully crossed blood-brain-barrier, entered neuron cells and thus significantly silenced OPRM gene.</p>
-
*
+
-
* TERMS OF USE - EASING EQUATIONS
+
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*
+
-
* Open source under the BSD License.  
+
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*
+
-
* Copyright © 2001 Robert Penner
+
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* All rights reserved.
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*
+
-
* Redistribution and use in source and binary forms, with or without modification,
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*
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* Redistributions of source code must retain the above copyright notice, this list of
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*
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var $sidescroll = (function() {
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<p>Finally, with all these data, we demonstrated that our modified exosome can not only cross the blood-brain-barrier but also specifically deliver siRNA to target tissue and thus silence specific gene.</p>
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</html>
</html>

Latest revision as of 01:31, 29 October 2013

<!DOCTYPE html> NJU_China








Brain:

For brain targeting, we chose to use RVG, which is a short peptide from Rabies Virus, as our targeting protein. RVG can specifically recognize acetylcholine receptor in the central nervous system [2], thus we engineer the RVG peptide into the lamp 2b and we use pcDNA 3.1(+) as our vector.
To produce the brain-specific RVG exosomes, we first transfected the exosome-producing cells, HEK 293T cells, with the plasmids encoding the fusion protein of lamp 2b and RVG peptide.
Also, we use a siRNA as an indicator to assess the targeting effect of RVG-exosomes and loaded the siRNA into the RVG-modified exosomes by direct transfection of the HEK 293T cells with siRNA probe.

Experimental procedure and Results:

1. Quantification of siRNA contained in the exosomes
We first quantify the amount of siRNA encapsulated into the exosomes. siRNA was transfected to the HEK 293T cells(which has already been transfected with RVG-Lamp2b plasmids) before exosome purification. Control group was not transfected with such siRNA.
By quantitative PCR analysis of a series of siRNA with known concentration, we drew a standard curve and then use it to calculate the absolute amount of siRNA in the exosomes. As shown in Fig.1, the amount of siRNA in the negative control is quite low (background) while the siRNA contained in the RVG exosomes transfected with siRNA probe reached as high as 0.8 fmol (RNA)/μg (exosome).
The result demonstrates that siRNAs were successfully encapsulated into RVG exosomes.

Figure.1 Empty exosome is collected from HEK 293 T cells without transfection of siRNA probe while the RVG exosome +siRNA is collected from the HEK 293T cells after transfection of siRNA probe. The amount of siRNA contained in the exosome is measured by qPCR.

2.In vitro evidence for the entry of RVG exosomes into the primary cortical neuron
To confirm the targeting capability of modified exosomes, we co-culture the siRNA-containing RVG exosomes with primary cortical neurons, and then measure the amounts of siRNA probe in the neurons to see if our RVG exosome can successfully get into the neurons.
From Fig.2, we can see that siRNA probe labeled with Alexaflour 555（red ）successfully get into the neurons.

Figure.2 Confocal microscopy image of the internalization of Alexaflour 555（red ）labeled siRNA into neurons.

In qPCR analysis of the relative siRNA probe level in the neurons co-cultured with both empty RVG-exosomes and siRNA containing RVG-exosomes, respectively (Fig.3), almost no siRNA was detected from the neurons co-cultured with 40 μg empty RVG-exosomes, while the siRNA detected in neurons co-cultured with siRNA probe containing RVG-exosomes show both significant and dose-dependent increase.

Figure.3 The RNA was extracted from the primary cortical neurons co-cultured 24h with 40 μg empty exosome, 20μg siRNA containing RVG-exosomes,40μg siRNA containing RVG-exosomes, respectively. And the RNA extracted was measured by qPCR using probe for the siRNA encapsulated in the exosomes.

3.In vivo evidence for the entry of RVG-exosomes entry into the brain
To further investigate whether the RVG-exosome can get into brain, we intravenously injected the empty RVG-exosomes, siRNA containing RVG-exosomes, respectively, into the mice. Then we extracted brain tissue and measured the siRNA level in the cortex and medulla.

As shown in Fig.4, no siRNA was detected in both cortex and medulla after the injection of empty RVG-exosomes and siRNA containing exosomes, while for the siRNA containing RVG-exosomes, the siRNA detected in the cortex and medulla are significant higher than the others.

Figure.4 The mice were intravenously injected with 200 μg empty exosomes,siRNA containing exosomes and siRNA containing RVG-exosomes, respectively once a day, and continued for four days. On the fifth day, the mice were killed and their brains were taken out. The RNA from their cortex and medulla were measured using siRNA probe for the siRNA encapsulated in the exosome.

4.In vivo evidence for the targeting effect of RVG-exosome
The siRNA contained in the exosomes were designed to silence OPRM gene, which encodesμ- opioid receptor in neuron cells. We intravenously injected three groups of mice: control group is injected with PBS (Mock), second group is injected with empty exosome, third group is injected with RVG-exosome which has siRNA in it. Next, by qPCR analysis we determined the relative OPRM mRNA level in mice brain. The result (Fig.5) shows that RVG exosome successfully target to neuron cells and the siRNA brought by the exosome down-regulate the gene significantly.

Figure.5 in vivo evidence for targeting effect of RVG-exosome. First group (Mock) was injected with PBS and second group was injected with Empty exosome. The last group was injected with RVG-exosome which contained siRNA that could silence OPRM gene. The result shows that modified exosome successfully crossed blood-brain-barrier, entered neuron cells and thus significantly silenced OPRM gene.

Finally, with all these data, we demonstrated that our modified exosome can not only cross the blood-brain-barrier but also specifically deliver siRNA to target tissue and thus silence specific gene.

Retrieved from "http://2013.igem.org/Team:NJU_China/Project/Brain"