Part:BBa_K1442023:Experience

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Experimentation

We compared efficiency of EMCV and NKRF in both HeLa and Huh 7.5 cells. EMCV and HeLa have been previously investigated in HeLa cells however have not been directly compared in Huh 7.5 cells. We compared each of these at the peak of fluorescence for most of the cells, 1800 seconds after loading. Due to the nature of the measurement equipment we were unable to supply CO2 to the cells during measurement and the cells gradually died during the 25 hour measurement period explaining the exponential decline in fluorescence.

This shows an exponential decline as the cells die due to lack of CO2 supply and shows the same overall trend in all the cells with maximal fluorescence occurring at approximately 1800 seconds

Method

We transfected the cells with 0.1μg of the IRES or control RNA, transcribed in vitro using Invitrogen RNA transcription kit, into each well of cells in a 96-well plate. The cells were plated at a concentration of 0.15x10⁵ determined using a haemocytometer. The cells were transfected using lipofectamine delivery in which lipid nanoparticles details of which can be found at Biontex K2 transfection system. Flourescence of the media and cells themselves were also taken into account. Each IRES had 10 biological repeats in each cell while the control had 5. Technical repeats were very difficult to achieve as it required splitting of the cells in each well as both HeLa and Huh 7.5 are attached cells, therefore the likelihood of spillage and contamination between the wells was too high to warrant the added accuracy of technical repeats.

Results

The fluorescence of each type of transfected cell was taken at 1800 seconds and averaged across the groups while fluorescence was adjusted for media and cell alone fluorescence and consolidated in this table.

We measured fluorescence using a tecan Magellan plate reader at wavelength 465nm - 530nm (that corresponding to GFP). Each IRES had ten replicates in each cell and hence error bars had an accuracy of 0.1. The fluorescence of media and cells themselves were accounted for in each case and the negative control used was the human optimised forwards GFP part:BBa_K1442106 without IRES included.

HeLa cell experimentation

In HeLa cells the NKRF IRES have previously been directly compared with firefly luciferase association in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC85491/#__ffn_sectitle Oumard et al 2000]

This clearly indicates that the NKRF IRES is significantly more efficient at initiating translation of GFP following this IRES conferring 2.4 times greater intensity than EMCV. Strangely the negative control showed higher fluorescence than the EMCV IRES also. This may have been due to increased growth rate of the cells or slower depletion of the growth media hence increasing overall fluorescence. Due to time restraints and limited number of wells in the plate we did not have as many replicates with this control.It may also be due to inefficient transfection of the cells with EMCV DNA strand.

Huh 7.5 experimentation

This is, to our knowledge, the first investigation of NKRF relative efficiency in Huh 7.5 cells. The results indicated that the NKRF IRES would be more efficient for use in our replicon but is conferred a less significantly increased relative fluorescence intensity than in HeLa cells as seen in the bar chart below.

This bar chart indicates that the results of the IRES efficiency in Huh cells is not significantly different as the standard error bars overlap somewhat. The negative control, as in the HeLa cells, indicates that NKRF does work efficiently and significantly in HeLa cells.

These results indicate that overall the NKRF transfection was more efficient and would be more appropriate and reliable for use in our system.<p>

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