DNA Delivery Systems and Data Collection

Transfection Using Lipofectamine 2000 (Invitrogen)

To introduce our engineered genetic parts into mammalian cells, we employed the Lipofectamine 2000 reagent from Invitrogen [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Transfection-Selection/lipofectamine-2000.html], and obtained at best an 80% transfection efficiency for Hek293 cells and 10% transfection efficiency for CHO cells.

Figure 1: Hek293 Lipofectamine Transfection Results

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(1A) This scatter plot shows the distribution of the Hek293 population after it was transfected with the following DNA parts: Hef1a-LacO:eYFP and Hef1a:mKate, both of which were constitutively active, expressing yellow and red fluorescent proteins respectively. We observe a distinct shift of approximately 83% of the population in their eYFP fluorescence (FITC channel), while we observe a 69% shift in mKate fluorescence (PE-TexasRed channel). (1B) Histogram of signal intensities of the above data. (1C) The negative control sample was transfected with plasmids containing no functional promotor-gene pairs.

We were then interested in the efficiency of multiple plasmid transfections. Previous work suggests that the probabilities of uptaking different plasmids is not independent. Cells that have been succesfully transfected with one plasmid are more likely to also have been transfected with another. To show this correlation, we decided to see what percentage of cells that took up the Hef1a:mKate plasmid also took up the Hef1a-LacO:eYFP plasmid. In order to evaluate the correlation, we took all events above the observed basal PE-TexasRed fluorescence level and examined their FITC profile. We observe in the below graph that more than 99% of the red population display yellow fluorescence above basal levels (2A). We make use of this high level of correlation later.

Figure 2: Hek293 Lipofectamine Multiplasmid Transfection Results

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Figure 2: Hek293 cells were transfected with equal amounts by weight of Hef1a:mKate and Hef1a-LacO:eYFP plasmids. (2A) Scatterplot displaying fluorescence levels of events as measured by flow cytometry. Events falling below the basal threshold for red fluorescence were omitted to observe "red" population. We observe that along the FITC axis, more than 99% of the red population fall above the threshold. (2B) Histogram of the same data

Although each of our constructed DNA parts contains Gibson sequences flanking the promoter and gene of interest, we have not yet used the Gibson reaction to piece together two or more promoter-gene pairs. The construction involved in doing so would involve the generation of a large library of promoter-gene pairs with various Gibson sequences that would be compatible with those of other parts that we intend to experiment with. In short, we have not yet engaged in Gibson-reacting parts together, and instead preserve our experimental flexibility through co-transfection of multiple plasmids.

CHO Lipofectamine Transfection Results

Control CHO cells:

In the flow cytometry scatter plot and histogram above we see the fluorescence distribution of a population of CHO cells transfected with Lipofectamine 2000 and Hef1a_eBFP2, a constitutive blue color. We note that CHOs naturally emit autofluorescence, as seen in the small tail pointing upwards in the DAPI (blue) channel. This tail accounts for 8% of the population, and after transfection, only 14.5% exhibit blue fluorescence beyond the majority of the CHO population, giving us thus approximately 6.5% transfection efficiency.

Transfection By Nucleofection

Although Hek293 cells are very easy to transfect and are therefore a very suitable target for demonstration, we found during the summer that cadherins are endogenously expressed, and this limits their experimental flexibility when it comes to cell-cell adhesion. CHO cells, however, do not have the same problem. Seeing also that the Notch-Delta system was previously characterized by the Elowitz group using CHO cells, we decided to open up a parallel research channel with CHO cell transfections. As documented above, however, lipofectamine proved to be an exceedingly difficult and somehow unsuccessful method of transfection into CHO cells, so we thus moved to nucleofection.

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