David LePage's Gene Targeting Protocols

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Electroporation Of Targeting/Transfection Vector

We generally follow the Wurst and Joyner transfection protocol (chapter 2 from Gene Targeting: a practical approach) with slight modification.

-On Wednesday I thaw one vial of R1 p11s onto 1 x 60 mm dish as usual. This is best done late before you leave to allow more leeway in preparing the cells.

-On Thursday, feed.

-On Friday split 1:6, usually onto 2 x 100 mm dishes. (The ratio can be varied. For instance, if you won't be able to get to them till late on Saturday do 1:8 to allow more leeway).

-On Saturday, feed.

-On Sunday split cells 1:2 for transfection by the afternoon on Monday. Split them to a larger extent if you're planning to get to them later (if, for instance, you need more time to get the DNA resuspended).

-Early on Monday feed the cells 4 hours before electroporation. The procedure will take an hour to half a day to complete. (A dense plate of R1s will contain 15-20 million cells, enough for 2-3 electroporations per plate)

-Cells are trypsinized with 3-4 ml of fresh trypsin and spun down as usual. Gently resuspend cells in ~1.5 ml ice cold PBS per dish and pool them into one tube. Remove 0.1 ml for counting and store the rest on ice.

-The 0.1 ml of cells is put into 0.9 ml of erythrosin B (Sigma E-7379; 0.1 g dissolved in 100 ml PBS). Mix and apply a small volume to hemocytometer.

-Count the four 4x4 squares, usually the areas numbered 1-4 on the diagram:

-Take the average of the four counts. Multiply this by 105 to yield the number of cells/ml. The final desired concentration is 7x106 cells/ml. It's OK to re-spin if too dilute, but not preferred.

-Usually one does 6-12 electroporations at once. It's usually easier to make cocktails of cells + DNA and aliquot 0.8 ml into cuvettes. Each cuvette should contain:

0.8 ml of cells at 7 x 106 cells/ml

40 µg linearized DNA of usually negligible volume

-When adding DNA and cells avoid bubbles (air is a resistor). Gently mix each cuvette. Allow to rest 5 min room temperature to allow cells and DNA to mix completely. Mix again half way through the 5 minutes.

-Remove the cuvette holder from the Tissue Culture Freezer and put a cuvette into the holder. Slide into the electroporator and pulse at 0.240 kV and 500 µF. Repeat until all the cuvettes are electroporated. (Some familiarity with electroporation, and/or reading the manual is warranted for basic safety).

-Note the tau value of each electroporation--this will vary by tissue culturist and DNA. I normally average 6.8 when I do everything. (Each individual will establish their average as they go along. It's mostly irrelevant unless there are problems, in which case you'll want to be more meticulous and discard outliers)

-Each cuvette then goes on ice for 20 min. During this time you can warm up your media.

-Using a 1 ml pipette gently mix each cuvette, draw up and put into 1.5 ml of medium per cuvette. Wash each cuvette with 1.0 ml of medium and pool all the electroporations and washes.

-Remove several 0.050 ml aliquots and plate out on 6-well plates for quantification.

-Plate out 3.0 ml of the pooled electroporations into 7.0 ml of medium on 1 x 100 mm dish. Repeat for all the electroporations (the last plate is usually a little short)

-The next day observe plates to see if they have been evenly distributed. The expectation is that 50% of the cells died under the conditions of electroporation. (This was explicitly determined once when the electroporator was brand new with mock electroporated cells. If severe systemic problems are occurring with routine electroporations it may be worth re-verifying the conditions)

-Approximately twenty-four hours after the electroporations were plated out feed the cells with medium + G418 + Gancyclovir (or whatever drugs are appropriate: see notes on drugs above). Also feed the quantification plates.

-Feed daily with media supplemented with fresh drugs. In G418 cells will take 3-4 days to start dying appreciably. After about 7-10 days of selection colonies will usually be visible.

-When the quantification plates have grown up they can be stained with methylene blue solution (0.33 g methylene blue, 0.11 g basic fuchsin in 100 ml of methanol). Aspirate medium from plates and wash once with PBS. Apply methylene blue to cover completely and incubate 5 min room temperature. Wash liberally with distilled water, dry the plates, and count colonies.

-Rates of transfection usually vary, especially between individuals. I would expect 500-1000 stable tranfectants per electroporation. Negative enrichment varies considerably as well. Using gancyclovir and single tk negative selection an average of 5-10 fold enrichment is expected. Enrichments that are much higher or much lower (that is 100 fold or 2 fold) are suggestive of problems and usually warrant repeating immediately, if for nothing else than to establish expectations for scaling up.

-Regardless of the numbers generated, at least 300 to 500 colonies need to be screened in total before concluding that a construct does not work. Often additional constructs are made in parallel as a back up against this possibility.


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