Reverse transfection

Transfection is the process in which foreign genetic material is introduced into a host organism using recombinant DNA technology. Reverse transfection refers to the procedure of adding cells to the transfection reaction second to the transfecting reagents, whereas regular transfection is performed by growing cells in plates until they reach the desired confluence prior to adding transfection reagents. Reverse transfection was used because it was consistent with a high-throughput assay, as the additional plating and growing of cells necessary in regular transfection was no longer required. An added benefit of reverse transfection was the increased cell surface available, which may result in higher transfection efficiency.

Lipofectamine 3000

Lipofectamine 3000 is a lipid based transfection agent comprised of two components;

Lipofectamine 3000 and the helper-lipid P3000. An illustration of lipid based transfection is displayed in Figure 15.

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Figure 15. A schematic illustration of cationic lipid mediated transfection. The positively charged head groups of the lipids interact with the negatively charged phosphate backbone of DNA. This interaction forms a DNA-cationic lipid complex that can enter the cell through endocytosis (Thermo Fischer Scientific).

Transfection procedure

The pcDNA3 GAL4 DBD:BRCA1 plasmids were co-transfected into HEK293T, MDA-MB-231 or MDA-MB-436 cells together with two reporter plasmids; phRG-TK (Renilla) and pGAL4-e1b-Luc (Firefly), to investigate the effects of BRCA1 variants on TA-activity.

Transfection was done using Lipofectamine 3000, according to the manufacturers procedure.

The amount of, and ratio between the plasmids, were taken from Jarhelle et al. (2016), and scaled down from a 6-well plate to a 96-well plate setup. The ratios between the Lipofectamine 3000 kit reagents and plasmid-amount previously found by the Iversen, N. research group for a similar experiment, were tested and deemed suitable. The NucleoCounter® NC-100™ was used for cell quantification in all transfection experiments.

96-well plate experiment

The transfection experiment described by Jarhelle et al. (2016) was scaled to a 96-well experiment, in order to increase efficiency and sample number per setup.

Wild type and variant plasmids were transfected in six parallels. For every experiment, transfection with reporter plasmids pGAL4-e1b-Luc (Firefly) and phRG-TK (Renilla) only, was included as a measure of background expression. Three parallels of cells transfected with reporter plasmids and pMaxGFP were included to verify successful transfection, as well as three parallels of non-transfected cells. A reaction mixture of plasmids and helper lipid P3000 was prepared (Table 8), and mixed with a Lipofectamine 3000 dilution (Table 9), before 15 minutes of room temperature incubation and subsequent addition of 30 µL of OptiMem, bringing the total transfection mixture volume to 50 µL pr. well (20 µL plasmid and Lipofectamine 3000 mixture + 30 µL OptiMem).

33 Table 8. Reaction mixes used in 96-well transfection of HEK293T, MDA-MB-231 and MDA-MB-436 cells. pcDNA3 GAL4 DBD:BRCA1 was the plasmid containing either wild type or variant BRCA1 BRCT domain. Samples refer to the mixture containing either wild type or variant plasmid. GFP was the mixture containing the Green Fluorescent Protein, and Reporters contained reporter plasmids Firefly and Renilla only. The volumes listed per column were for one well.

HEK293T MDA-MB-231/436

Table 9. Lipofectamine 3000 dilutions in OptiMem used in 96-well transfection of the cell lines HEK293T, MDA-MB-231 and MDA-MB-436. Volumes listed were for one well.

Cell line OptiMem (µL) Lipofectamine 3000 (µL)

HEK293T 10 0.35

MDA-MB231/436 10 0.50

A DMEM suspension of 4x10⁵ cells/mL of HEK293T and MDA-MB231 cells, and 4.5x10⁵ of MDA-MB436 cells were prepared using the NucleoCounter® NC-100™ for quantification of cell numbers. To each well 50 µL of transfection mixture was added, before 100 µL of cell suspension were transferred to the well. The plate was incubated at 37 °C with 5 % CO2 for 24 or 48 hours before harvesting. The experiment was repeated three-four times for each variant and incubation time.

12-well plate experiment

The transfection procedure described for a 96-well experiment was scaled to a 12-well plate setup in order to yield the amount of protein and RNA needed to perform protein- and RNA-based assays. The 12-well experiment was performed similar to the 96-well experiment, but in one parallel and without GFP.

A reaction mixture of plasmids and helper lipid P3000 was prepared (Table 10), and mixed with a Lipofectamine 3000 dilution (Table 11), before incubation for 15 minutes at room temperature.

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Table 10. Reaction mixes used in 12-well transfection of HEK293T, 231 and MDA-MB-436 cells. pcDNA3 GAL4 DBD:BRCA1 was the plasmid containing either wild type or variant BRCA1 BRCT domain. Samples refer to the mixture containing either wild type or variant plasmid. GFP was the mixture containing the Green Fluorescent Protein and Reporters contained reporter plasmids Firefly and Renilla only. The volumes listed per column were for one well.

HEK293T MDA-MB231/436

Table 11. Lipofectamine 3000 dilutions in OptiMem used in 96-well transfection of the cell lines HEK293T, MDA-MB-231 and MDA-MB-436. Volumes listed were for one well.

Cell line OptiMem (µL) Lipofectamine 3000 (µL)

HEK293T 50 1.75

MDA-MB-231/436 50 2.50

A DMEM suspension of 2x10⁵ cells/mL of HEK293T and MDA-MB-231 cells, and 2.5x10⁵ of MDA-MB436 cells were prepared using the NucleoCounter® NC-100™ for quantification of cell numbers. To each well, a 100 µL of transfection mixture was added, before 1 mL of cell suspension was transferred to the well. The plate was incubated at 37 °C with 5 % CO2 for 48 hours before harvesting.

3.3.4 Cell harvesting