Measurements
Overview
- In our experiments we used the FACS machine that provided us with accurate measurements of the expression of the reporter fluorophores.
- For measuring gene expression we used either DNA or RNA transfection.
FACS machine
In order to test our two-module circuit under the same conditions used by Nissim et al. (Cell 171: 1138-1150, 2017), we employed DNA transfection to insert the expression plasmids carrying our new genes and control plasmids to HEK293T cells. All plasmids we tested encode fluorophores: mKate2, ECFP or EGFP. To monitor expression of these fluorophores we used the FACS machine, which gave us either qualitative results, determining whether a gene is expressed above background (usually irrelevant plasmid DNA) or quantitative measurements, that is, what is the actual level of fluorescence compared to background or reference plasmid.
The FACS machine is used to analyze diverse cell populations for data such as: size, granularity and fluorescence emitted by a wide spectrum of fluorophores, either expressed by the analyzed cells or conjugated to antibodies which bind distinct components on the cell surface or within the cell (‘intracellular staining’).
In our main experiments we used two fluorophores: mKate2 that was used to assess the expression driven from the combined action of the two modules of the circuit and emits in the red range and ECEP that was used as an internal control for the system and emits at the green-blue range.
The FACSCalibur instrument (BD) helped us with the qualitative tests. However, as in FACSCalibur there is overlap between the two colors of our fluorophores (ECFP and mkate2), we needed a device with better compensation capacity.
Fig 1 – shows the overlap between CFP and mkate2 (Spectrum viewer)
In order to solve this problem, we used the FACS machine in Lior Nissim’s laboratory (Attune NxT, Thermo Fisher Scientific), which possesses better compensation capacity in these ranges.
Figure 2 – shows the radiance intensity of mkate2 and the negative control (ECFP) (link to result page).
RNA transfection
In order to evaluate the amount and rate of the gene expression, we inserted it into a DNA plasmid (PGEM4Z). Then, we synthesized mRNA from the gene using a commercial kit (CELLSCRIPT) and transfected K562 cells (CML cell line) with this mRNA using electroporation (using Gene Pulser Xcell apparatus, Bio-Rad).
| Cell type | Amount of cells | Amount of mRNA | Pulse type | Volts | Ω | µF | Pulse duration | Cuvette width |
|---|---|---|---|---|---|---|---|---|
| K562 | 3x106 | 10µg | Exponential | 350V | - | 150µF | - | 4mm |
RNA transfection by electroporation is an effective method for expressing genes of interest in most cell types, usually yielding higher and more uniform expression compared to DNA transfection. In addition, since mRNA should only be introduced into the cell cytosol, only mild electroporation conditions are required, with only minimal effects on cell viability. In contrast, many DNA transfection protocols, which require entry of the DNA to the cell nucleus, fail to achieve sufficiently high yield and often result in high toxicity. Yet, the greater in-cell stability of DNA compared to RNA results allows longer period of effective expression of the gene of interest.
In the example shown here, 24 hours post-mRNA transfection we incubated the cells with antibodies: Anti-Human c-Myc- APC (R&D SYSTEMS, Cat: IC3696A) and anti Ha- Alexa Fluor 647 (R&D SYSTEMS, Cat: IC6875R) and subjected the cells to FACS analysis.
Here is our FACS measurement of the expression of our improved part, CAR-KIR3DL1 (BBa_K2946003), following mRNA electroporation.
Black- Irrelevant RNA
Blue- Positive control for C-myc
Red- CAR-KIR3DL1
Figure 3 – RNA electroporation of CAR-KIR3DL1, compared to negative (Irrelevant RNA) and positive (C-myc) controls.
Reference
1. Nissim, L. et al. Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy. Cell 171, 1138-1150.e15 (2017).