<p>The prepared pDAS12124-preedited plasmid serves as a positive control to validate the success of the experiment. A technical control with the pZMB938 plasmid confirms successful transfection of the cells. In the main part of the experiment, pDAS12489-2in1 and pCMV-PE2 are co-transfected. Successful transfection is visualised by GFP signals.</p>
<H4text="Conclusion"/>
<divclassName="figure-wrapper">
<figure>
<imgsrc=""style={{height:"10%",width:"auto"}}/>
<figcaption><b>Figure 1</b> XXXX.</figcaption>
</figure>
</div>
<H5text="Conclusion"/>
<p>The microscopy data validates our proof of concept. Compared to our internal positive control, pDAS12124-preedited (see Figure X), less cells co-transfected with pDAS12489 and pCMV-PE2 (see Figure X) showed fluorescence. Contrary to our expectations, the technical transfection control with pZMB938 showed lower transfection efficiency. All negative controls showed no fluorescence.</p>
<H4text="Proof-of-concept 2"/>
<H5text="Workflow"/>
<p>To optimise transfection efficiency, different dilutions and concentrations of DNA were used to find the best transfection conditions.</p>
<H4text="Conclusion"/>
<H5text="Conclusion"/>
<p>All 4 different transfection conditions were done with pZMB938 and showed good results, but best result were done when lipofectamine 2000 was diluted 1:10 and 1000 ng DNA was transfected.</p>
<H4text="Proof-of-concept 3"/>
<H5text="Workflow"/>
<p>Transfection with Lipofectamine 3000 was performed because of the probably better performance and transfection rate. The prepared pDAS12124-preedited plasmid serves as a positive control to validate the success of the experiment. A technical control with the pZMB938 plasmid confirmed successful transfection of the cells as before. In the main part of the experiment, pDAS12489-2in1 and pCMV-PE2 were co-transfected. Successful transfection and prime editing was detected by GFP signals.</p>
<H4text="Conclusion"/>
<H5text="Conclusion"/>
<p>Internal control and technical control showed higher transfection efficiency then in previous experiments, therefore transfection with lipofectamine 3000 seems to be more efficient than transfection with lipofectamine 2000. The fluorescence of pDAS12189+pCMV-PE2 was still quite low. All negative controls are showed no fluorescence.</p>
<H4text="Proof-of-concept 4"/>
<H5text="Workflow"/>
<p>Again a preliminary test with the technical positive control was conducted potentially optimize our transfection protocol and to train the handling.</p>
<H4text="Conclusion"/>
<H5text="Conclusion"/>
<p>The results of the test were not as good as expected. Nearly no transfection efficiency was visible. This could be due to too old hek293 cells</p>
<H4text="Proof-of-concept 5"/>
<H5text="Workflow"/>
<p>HEK cells were thawed and another prelimary test was conducted. In this test two different transfection agents were used (Lipofectamine 3000 & CaCl2) to check which one is better suited for our experiments. The literature uses lipofectamine 3000 but CaCl2 transfection is much cheaper.</p>
<H4text="Conclusion"/>
<H5text="Conclusion"/>
<p>Both transfections are working out well but the efficiency of the lipofectamine transfection was much higher.</p>
<H4text="Proof-of-concept 6"/>
<H5text="Workflow"/>
<p>One last time the transfection of pDAS12189+pCMV-PE2 was conducted. Although our proof-of-concept already showed successful editing the first time, we repeated the experiment to get better transfection efficiency.</p>
<H4text="Conclusion"/>
<H5text="Conclusion"/>
<p>The transfection efficiency was much better. Our proof-of-concept was working correctly. The reporter system pDAS12189 only led to production of a fluorescent signal when co transfected with a prime editing complex as pCMV-PE2.</p>
