1. 1 day prior to transfection, seed approximately 1.0 x 10^5-3.0 x 10^5 cells in each well of a 6-well plate containing 2 mL of complete growth medium.
2. Grow cells overnight to approximately 90%-95% confluence.
1. Equilibrate DNA, Lipofectamine 2000 reagent, and Opti-MEM® I Reduced Serum Medium (Life Technologies. Catalog number: 31985-088) to room temperature.
2. Dilute 2 μg plasmid DNA (total of all plasmids being co-transfected) with 125 μL Opti-MEM® I.
3. Incubate the mixture for 5 minutes at room temperature. Once the transfection reagent is diluted, combine it with the DNA within 30 min.
4. Combine the diluted DNA with the diluted transfection reagent. Incubate at room temperature for 5 to 20 min. to allow DNA-transfection reagent complexes to form.
5. Add the DNA-transfection reagent complexes directly to the well and mix gently by rocking the plate back and forth.
6. Incubate the cells at 37°C in a CO2 incubator for a total of 4 h.
We recommends isolating single cells. The reason is that if you want to obtain a cell line with your gene product completely modified, single clone isolation will get rid of the cells in which the gene is either incompletely modified or that went untransfected, and that carry unwanted background mutations. Clonal isolation is followed by an expansion period to establish a new clonal cell line. Note that cell types can vary substantially in their responses to single-cell isolation, so literature specific to the cell type of interest should be consulted.
1. 24-48 hours after transfection, trypsinize the cells and transfer to a sterile 15 mL centrifuge tube.
2. Centrifuge the cells at 1,000 x g for 3 minutes.
3. Resuspend cell pellet in 5 mL complete growth medium.
4. Count the number of live cells in a hemocytometer to determine the cell density.Use trypan blue to exclude dead cells, which take up the dye and turn blue.
5. Dilute cells in 40 mL of complete growth medium containing antibiotic that selects for the donor plasmid in a sterile 50 mL centrifuge tube.
6. Make 4 10-fold serial dilutions from the starting.
7. Dispense 10 mL of each diluted cell suspension into each of 4 10 cm tissue culture dishes. If your transfection efficiency is low and you did not enrich for transfected cells, you will need to scale this up accordingly in order to obtain and screen more clones.
8. Incubate the plates at 37°C until cells have grown into colonies that are large enough to remove using sterile cloning cylinders, from plates carrying 10-20 colonies each. This can take several (3-6) weeks, depending on the growth rate of the parent cells.
9. Remove 20 single clones from the plates using sterile cloning cylinders. Transfer each clone to 1 well of a 24-well plate containing 0.5 mL of complete growth medium.
1. Fill each well of a 4 sterile 96-well plates with 100 μL of medium, except for well A1, which should remain empty.
2. Add 200 μL cell suspension to well A1. Mix 100 μL from A1 with the medium in well B1. Avoid bubbles. Continue this 1:2 dilution through column 1. Add 100 μL of medium back to column 1 so that wells A1 through H1 contain 200 μL.
3. Mix cells and transfer 100 μL of cells from column 1 into column 2. Mix by gently pipetting. Avoid bubbles. Repeat these 1:2 dilutions through the entire plate. Bring the final volume to 200 μL by adding 100 μL of medium to all but the last column of wells.
4. Incubate plates undisturbed at 37°C.
5. Cells will be observable via microscopy over 3 days and be ready to score in 5-8 days, depending on the growth rate of cells. Mark each well on the cover of the plate indicating which well contains a single colony. These colonies can later be subcultured from the well into larger vessels.
1. Design PCR primers that flank both donor/chromosome junctions. At the upstream junction, the forward primer must lie in the sequence outside of the region of upstream homology arm. The reverse primer sequence will only be found in the plasmid (for example, from GFP gene). Conversely, at downstream junction, the forward primer sequence will only be found in the plasmid (for example, from GFP gene), while the reverse primer must lie in the sequence outside of the region of downstream homology arm.
2. Candidate clones grow to approximately 95% confluency (for adherent cells) or to a density of ~1 x 10^7 cells /mL (for suspension cells).
3. Split cells to 2 new plates or flasks for each candidate clone.
4. Harvest cells of each clone and extract genome. Successful PCR amplification using this strategy will occur only if the donor plasmid integrated at the correct site.
5. PCR product DNAs are ligated to clonal vectors.
6. Transform vectors into E. coli and sequence the insert DNA. Sequence 3-4 E. coli clones for each junction.
7. Analyze the insret sequence of each E. coli clone to ensure that the junction sequences are correct.
8. Do another PCR experiment to confirm that the sequence knocked in is present by using appropriate PCR primers.
9. Rule out random integration of the donor plasmid. To detect random integrations, design one or more pairs of primers that recognize the plasmid backbone only. Positive results indicate that the cell line clone sustained one or more random integrations of the donor plasmid.