Biomod/2014/OhioMOD/results: Difference between revisions

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<h2>6. Conclusion</h2>
<h2>6. Conclusion</h2>
<h2>7. Future Work</h2>
<h2>7. Future Work</h2>
<br><a href="http://openwetware.org/index.php?title=Biomod/2014/OhioMOD/results&action=edit">Edit Results</a><br><br>
<br><a href="http://openwetware.org/index.php?title=Biomod/2014/OhioMOD/results&action=edit">Edit Project and Results</a><br><br>
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Revision as of 21:11, 22 October 2014

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     <li ><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD">Home</a></li>
     <li><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD/project">Background</a></li>

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<h1>Project and Results</h1> <h2>Novel microRNA antisense therapeutic delivery using DNA origami nanostructures</h2>


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<A HREF="#scroll1">1. Structures</A> </br> <A HREF="#scroll2">2. miR 21 Sequestering</A> </br><A HREF="#scroll3">3. Cellular Uptaking</A> </br> <A HREF="#scroll4">4. PTEN Expression</A> </br><ul id="list2"> <A HREF="#scroll4.1">4.1 Gene Expression</A></ul> <ul id="list2"> <A HREF="#scroll4.2">4.2 Protein Expression</A></ul> <A HREF="#scroll5">5. Cellular Viability</A> <A NAME="scroll1"></A></br> <A HREF="#scroll6">6. Conclusion</A> </br> <A HREF="#scroll7">7. Future Work</A>


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<h2>1. Structures</h2> <h2>2. miR 21 Sequestering</h2> <div id="CellularUptaking"> <h2>3. Cellular Uptaking</h2> </br><strong>Hypothesis:</strong> Our structures utilize the endolysosomal pathway to infiltrate cells. </br></br>Once the structures were characterized and functionalized with miR-21 complementary overhangs, the next step was to test whether they would be successfully uptaken into cells. One possible mode of uptake would be through endocytosis, where the structures would be enclosed in an endosome and allowed into the cell. To test this theory, structures labeled with a fluorescent intercalating dye (TOPRO3) were incubated with cells whose lysosomes were labeled with Lysotracker Green. After a 4 hour incubation period, the cells were imaged with a fluorescent microscope to try and observe colocalization between the signals originating from the structures and the lysosomes.

</br></br>As Figure XXX shows, fluorescence from the Lysotracker (shown in green) and fluorescence from TOPRO3 (shown in red) occupy the same coordinates within a cell, suggesting that the structures and endosomes occupy the same space within the cell. In addition to suggesting a possible mechanism for uptake, the Lysotracker also aids in affirming the position of the structures within the cell. Since these images are two-dimensional, without the Lysotracker, there would be no way to determine whether the signal from the structures originated from within the cell, or from above or below the cell. Co-localizing with Lysotracker affixes the signal’s position in the z-plane as well as the x and y planes.

</br></br>Hypothesis: The difference in size and aspect ratio of the structures will affect how they are uptaken. One advantage of generating structures with very different aspect ratios was the ability to test which structures were more easily uptaken by cells. Even though both structures meet the ideal dimensional parameters for cell uptake (Randy’s pyramid citation), the different geometries of the elongated branch versus the compact Block O may have an impact on cell uptake efficiency. While microscopy images do not necessarily provide quantitative data on the uptaking efficiency of structures, some preliminary assessments may be made nonetheless. Firstly, more instances of co-localization could be observed in samples containing branch structures relative to samples containing Block O structures. Perhaps more intriguingly, in the samples containing branch, all instances of fluorescence in the 640 nm channel occurred either within viable cells or within dead cell debris. However, in the Block O samples, a few instances of fluorescence were found outside of cells, as shown in Figure XXX.

</br></br>The fluorescence signal, with an intensity comparable to the intensity of the TOPRO3 dye, originated at the very edge of the cell, on the cell membrane. This could indicate that, while branch structures are quickly uptaken once they come in contact with cells, the Block O structures experience a delay in uptake, and thus tend to congregate outside the cell. While this introduces an interesting possibility in terms of the mechanism of uptake, more experiments must be conducted, both using qualitative methods such as imaging, and quantitative methods such as qPCR, to form a conclusion on this matter. </div><!--end of cellular uptake-->


<h2>4. PTEN Expression</h2> <h3>4.1 Gene Expression</h3> <h3>4.2 Protein Expression</h3> <h2>5. Cellular Viability</h2> <h2>6. Conclusion</h2> <h2>7. Future Work</h2> <br><a href="http://openwetware.org/index.php?title=Biomod/2014/OhioMOD/results&action=edit">Edit Project and Results</a><br><br> </div>


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