Stable plasmid transfection

Background: A stable cell lines is one that has incorporated a plasmid into its genome, resulting in a stable over-expression of a protein of interest. This plasmid will also include a gene for antibiotic resistance, which is used to select the cells that have successfully incorporated the plasmid. Culturing transfected cells in medium containing the antibiotic will result in the death of all cells not expressing the plasmid. The surviving cells can then be cultured as normal but in medium containing this antibiotic. This means that all surviving cells should be expressing your gene of interest, removing the variability between cells which is seen with a transient transfection. If for any reason the plasmid stops being expressed the antibiotic will ensure these cells die, so there is no chance of you continuing with cells that no longer express the plasmid. As the cells are all expressing the gene they can be treated in the same way as wild-type, allowing treatment to be done immediately.

Reagents:

 DMEM-F12, supplemented with 10% FBS and 2 mM L-glutamine but no antibiotics.

 50mg/mL G418 in ddH2O, filtered with 0.22µm PES filter.

 FuGene HD (Promega, Wisconsin, United States)

 Serum-containing medium: DMEM-F12, 10 % (w/v) FBS, 2mM L-glutamine, 200IU/ml penicillin, 200µg/ml streptomycin.

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 Serum-free medium: DMEM-F12, 2mM L-glutamine, 200IU/ml penicillin, 200µg/ml streptomycin.

 Selective medium: DMEM-F12, 10 % (w/v) FBS, 2mM L-glutamine, 400 µg/mL G418.

 PCR clean up kit

 Restriction enzymes and relevant buffers.

Method: I used two different plasmids throughout this thesis, the first was

mEmerald-ALK1-N-13 and was a gift from Michael Davidson (Addgene plasmid

# 53981). The second was created in-house using the pcDNA3.1 plasmid (Thermo Fisher, Loughborough, UK). Both plasmids were selected for with the antibiotic G418. Prior to transfection the plasmid should be linearised. For this a 100µL reaction was set up contain 10µg plasmid and 50 units of either MfeI or Pvu1 (depending on the plasmid) along with the relevant buffer, these were then incubated for 4 hours at 37°C. An ethidium bromide gel was used to confirm the digestion had been successful. The remaining product was then purified using a PCR clean up kit, as per the instructions. A 6 well plate was seeded with 200,000 SW1353 cells then incubated overnight in Serum-containing medium. Plasmid DNA (2.75µg) was combined at a 1:3 ratio with 8.25µL FuGene HD and 137.5µL of Serum-free medium. A control was also created that had water instead of the plasmid. These mixtures were vortexed at full speed for 5 seconds then left for 10 minutes at room temperature, before being added to the cells along with 4ml of Serum-containing medium. After 24 hours the treatment medium was removed and replaced with Serum-containing medium overnight. The following day the medium was replaced with selective medium, this was then replaced every 2-3 days until the control cells were dead and the treated cells were fully confluent. Each well of the plate was then split and transferred to a T25 flask until confluent. They were then split and

transferred to a T75 flask. After which, they could be treated the same as wild-type SW1353 cells but incubated in selection medium instead of

Serum-containing medium. It is important to note that typically you need to test various concentrations of G418 for your specific cell type. However, this had already been done by our group for SW1353 cells.

50 2.1.18 Mini/maxi prep

Background: QIAprep Spin Miniprep Kits are designed to isolate high-purity plasmid DNA. Allowing the extraction of high quality DNA from bacterial cultures. The mini-prep results in lower quantities of DNA but is cheaper, allowing you to reduce risk when testing if a plasmid is as desired. The Maxi-prep then allows much higher quantities of DNA to be produced, allowing you to make sufficient plasmid DNA for regular transfection.

Reagents

 QIAprep Spin Miniprep Kit

 QIAprep Spin Maxiprep Kit

 QIAvac 24 Plus

 Antibiotic containing Lysogeny broth (LB) medium, autoclaved to prevent any unintended growth, also containing Kanamycin at 50mg/ml.

 DNase-free water.

Method: Three 50ml mixtures where made containing: the LB medium with no bacteria, bacteria containing the plasmid of interest or bacteria resistant to an alternative antibiotic. Bacteria were transferred to the tubes by pressing the colony with a 20µL pipette tip and then moving the tip to the medium, all under sterile conditions. The tubes were then incubated at 37ºC overnight with

agitation at 225-250 rpm in an orbital incubator. The following day only the bacteria containing the plasmid of interest should have grown.

Mini-prep: This tube is spun at 5400xg for 10 minutes at 4ºC and the

supernatant discarded. The pellet was then resuspended in 200µL P1 buffer and transferred to a 1.5ml microcentrifuge tube. 200µL P2 buffer was added and mixed by inverting gently. This was followed by adding 350µL P3 buffer and mixed by inverting gently. The tube was then spun at 17,900g for 10 minutes at 4ºC. The QIAvac 24 Plus filtering system was then used on the lysates. Lysates were passed through the column and then the eluant was discarded. Endotoxin removal wash was then eluted through the column. This was then followed by buffer PE to wash. The DNA was then eluted into a clean 1.5ml microcentrifuge tube using DNase-free distilled water, extra time was taken to ensure the

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column was completely dry. The eluted DNA could then be analysed on a Nanodrop ND 1000 spectrophotometer (Thermo Fisher, Loughborough, UK) to determine purity and concentration.

Maxi-prep: All the bacteria containing medium was then added to 250ml of fresh LB medium and incubated at 37ºC overnight with agitation at 225-250 rpm in an orbital incubator. This tube was spun at 5400xg for 10 minutes at 4ºC and the supernatant discarded. The pellet was then resuspended in 12ml resuspension buffer and vortexed until there were no clumps remaining. 12ml of lysis buffer was then added and inverted 3-5 times. After which, it was left for 3 minutes. I then added 12ml of neutralising solution and spun at 17,900g for 20 minutes at 4ºC. The QIAvac 24 Plus filtering system was then used on the lysate, with larger columns than used from the Mini-prep. Lysates were passed through the column and then the eluant was discarded. Endotoxin removal wash was then eluted through the column. This was then followed by buffer PE to wash. The DNA was then eluted into a clean 1.5ml microcentrifuge tube using DNase-free distilled water, extra time was taken to make sure the column was completely dry. The eluted DNA was then run on a Nanodrop ND 1000 spectrophotometer to detect purity and concentration. Plasmids were then stored at -20ºC until use.