Part:BBa_K4344040

pEGFP-C1

We used this part for evaluation of our liposomal formulation in an in vitro assay.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1359
  Illegal XbaI site found at 1402
  Illegal PstI site found at 1364
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1359
  Illegal NheI site found at 591
  Illegal PstI site found at 1364
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1359
  Illegal BglII site found at 1339
  Illegal BamHI site found at 1390
  Illegal XhoI site found at 1343
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1359
  Illegal XbaI site found at 1402
  Illegal PstI site found at 1364
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1359
  Illegal XbaI site found at 1402
  Illegal PstI site found at 1364
  Illegal NgoMIV site found at 1972
  Illegal NgoMIV site found at 3255
  Illegal NgoMIV site found at 3538
  Illegal AgeI site found at 600
  
• 1000
  COMPATIBLE WITH RFC[1000]

Analysis of GFP expression pattern

As described in our methods we monitored eGFP expression over the course of three days. Figure 1 visualizes the GFP expression. Logistic course of eGFP expression can be observed with already continuous eGFP expression before the cells were placed in the IncuCyte. Red bars indicate the value and time stamp of maximum eGFP expression. Blue line indicates the half maximum time stamp of eGFP expression.

<figure>

     <img class="img-fluid" alt="MD simulation of lipids as gif" style="filter: none;" src="">
     <figcaption>
       Figure 1: GFP Expression over time. HeLa cells were transfected with pEGFP and kept in transfection media as described in methods.</b> The course of eGFP expression can be observed with already continuous eGFP expression before the cells were placed in the IncuCyte. Red bars indicate the value and time stamp of maximum eGFP expression. Blue line indicates the half maximum time stamp of eGFP expression.
     </figcaption>
   </figure>

Visual progression of eGFP expression can be seen in Figure 2. During expression measurement pictures were taken at 10h, 24h, 34h, 48h and 58h post transfection. Increase in fluorescence can be observed.

<figure>

     <img class="img-fluid" alt="MD simulation of lipids as gif" style="filter: none;" src="">
     <figcaption>
       Figure 2: Fluorescent microscopic images for different tímestems of eGFP expression. Images were taken at different time points during the eGFP Quantification. An increase in eGFP expressing cells can be observed for the time frame.
     </figcaption>
   </figure>

Analysis of eGFP knockdown

In the same manner as the eGFP expression pattern we evaluated the knockdown capabilities of our liposomal formulation (vehicle). As comparison served delivery of the anti eGFP siRNA in a commercially available transfection reagent. Figure 3 visualizes the eGFP expression after treatment over time. Green line indicates the eGFP control, which received no treatment. The blue line indicates cells treated with the empty vehicle and the red line shows cells treated with the anti eGFP- siRNA encapsulated in our vehicle.The black line is the positive control, therefore anti eGFP siRNA in transfection reagent. Whilst green and blue increase over time, red reaches a high and maintains this level, black line shows a deep decrease of eGFP fluorescence.

   <figure>
     <img class="img-fluid" alt="MD simulation of lipids as gif" style="filter: none;" src="">
     <figcaption>
       Figure 3:  Visualization of GFP knockdown efficiency. Green line indicates the eGFP control, which received no treatment,blue line: cells treated with the empty vehicle, red line: cells treated with the anti eGFP- siRNA encapsulated in our vehicle, black line: positive control, therefore anti eGFP siRNA in transfection reagent. Whilst green and blue increase over time, red reaches a peak and maintains this level, black line shows a deep decrease of eGFP fluorescence.
     </figcaption>
   </figure>

We decided to select a time point before reaching 50% of the max value of eGFP expression as time to treat our cells with our formulations due to the fact that at this time, RNA is already prescribed and therefore present in the cell and can be knocked down, therefore hindering protein translation due to mRNA degradation. The reason why mRNA levels are that high after a short time frame after transfection is found in the continuous CMV promoter driven transcription of said mRNA, which is further enhanced by the CMV enhancer element present on the eGFP plasmid (Norman et al., 2010). These findings are transferable onto the pRK5 UL19 expressing plasmid since both plasmids carry identical promoter, enhancer, terminator and kozak sequences. A disadvantage of the chosen plasmid system is that without selection pressure cells will lose their plasmid upon division because of HeLa cells lacking large T-antigen of SV40 Virus for replication of the plasmid. The transfection is therefore only transient. This has to be considered in the evaluation of all time dependent results.