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HEK Cells
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09/07/24 - 30/08/24
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<li>Lea</li>
<li>Vasilis</li>
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<h2>RAD301: Transfection of mRNA in HEK cells</h2>
<p>
<b>Background:</b> eGFP mRNA made by us and control eGFP mRNA was transfected into HEK cells to check the functionality of our synthesized mRNA. In this transfection, RNA is diluted with buffer and formed into liposomes using a transfection reagent, this is mixed with HEK cells and incubated overnight.
It was planned to also transfect LNPs containing eGFP mRNA, but this was postponed due to an experimental error in synthesizing these LNPs.
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<p>19th of September 2024</p>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
</ul>
<h4>Experimental:</h4>
<ulclass="emptylist">
<li>transfection solution was prepared:</li>
<li>
50 ul rNA buffer containing 400ng RNA was mixed with 1ul reagent, mixed and left for 15 min:
<table>
<tr>
<td></td>
<td>mRNA iGEM</td>
<td>3CB mRNA</td>
<td>Mock</td>
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<tr>
<td>RNA</td>
<td>0.8 ul</td>
<td>4ul</td>
<td>-</td>
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<tr>
<td>Buffer</td>
<td>49.2ul</td>
<td>46ul</td>
<td>50ul</td>
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<tr>
<td>Reagent</td>
<td>1ul</td>
<td>1ul</td>
<td>1ul</td>
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</li>
<li>3CB is the batch number of the EGFP mRNA given to us from the lab that is known to be functional. This is our positive control.</li>
<li>25 ul of transfection mix was added to one well containing HEK cells with +- 50% confluency, 2 wells per sample.</li>
<li>Cells were incubated with the transfection mix for +- 19h</li>
<p>The 3CB control shows visible expression indicating transfection was successful. However, the expression of the control was expected to be higher. Low expression can be explained by the fact that the cells were not very healthy when the transfection was performed.
The expression of our synthesized mRNA is a lot lower, but some cells expressing GFP can be found - like the one indicated by the red arrow. The negative control is empty.
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<p><b>Conclusion:</b>We produced functional mRNA. However, the expression is not optimal. From this we can conclude that our method for synthesizing mRNA works, but could be improved. Due to the low expression of our mRNA, it might be beneficial to use the 3CB GFP mRNA to test the efficiency of the LNPs.</p>
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<h2>RAD302: Transfection of mRNA in HEK cells, attempt 2</h2>
<p>
<b>Background:</b> It will be attempted to insert mRNA from experiment RAD133 and LNPs from experiment RAD206. The following scheme of transfection will be implemented:
<p>mRNAs used: GFP 1, GFP2, F8 1, 5x FV F8 2, control GFP; LNPs used: LNPs with no mRNA, GFP1 LNPs, GFP2 LNPs, F8 1 LNPs, 5xFV F8 2 LNPs, GFP cont LNPs; one slot with just cells, one slot with method for mRNA transfection.</p>
<p>
LNP transfection was done by using the following procedure:
<p>Figure 4. Cells without fluorescence - survived</p>
<p>Only the sample with control GFP was expressed. This implies that the mRNA samples are nonfunctional (GFP 1, which was slightly expressed in RAD301, was not visible here, potentially due to denaturation). Since control was expressed by regular transfection, and not with LNPs, the LNP synthesis procedure in the moment is not efficient.</p>
<p>SDS-PAGE gel was considered to be unnecessary for analysis of F8 protein presence, since if there was no expression of control GFP, than yes, but since there is no expression of the experiment with GFP whose RNA also looked better from the start, I think it is a waste of time and resources. In the final effort, I would put my money on getting the transfection both with JetMessenger ánd LNPs to work before analyzing protein extracts of cells.</p>
<p>
<b>Conclusion:</b>Combining the results from the RAD206, RAD133, and RAD203, it can be said that the reason for the following transcription result is a combination of the results from all 3 factors: HEK cells are used instead of the cells used in the reference [1] - HeLa or LSEC cells, different lipid composition was used: instead of DSPE-PEG 2000, C16-PEG ceramide (PEG-lipid) and DSPE-PEG-mannose lipids were used, and the mRNA experiments provided very low yield of functionable mRNA.
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<p>This resulted in no transfection fluorescence of just GFP mRNA samples (except for the control one), since the mRNA from RAD133 also was not visible on the gel, and in no transfection fluorescence of GFP in LNPs. However, the LNP procedure was also not efficient - very little amount of LNPs produced in combination with different compounds used for production.</p>
<p>It is advised to make more mRNA samples after consultation on the current results, procedure and storage should be revised. Different cell cultures should be used: either HeLa or LSEC cell lines. Also, for LNPs, it is recommended to use higher concentration of lipids, and implement a pulsification method or microfluidics device to prevent low yields of LNPs in the future. It is also recommended to purchase more expensive compounds for LNP formulation, as in the referenced article [1]: instead of DSPE-PEG 2000 - C16-PEG ceramide (PEG-lipid) and DSPE-PEG-mannose lipid which might provide for the better targeting during transfection.</p>
