Cell line and culture conditions
DLD-1 cells were purchased from American Type Cell Culture (ATCC, Manassas, VA). The integrated DLD-1 clone one (DLD-1-1) was obtained by integrating the pEGFP-N3 vector containing a single point mutation (TAG) in the chromophore region of the eGFP gene, and "clone one," containing 2–3 copies of the mutated eGFP, was isolated through serial dilution [see [18]]. For these experiments, cells were grown in RPMI 1640 medium supplemented with 2 mM glutamine, 4.5 g/l glucose, 10 mM HEPES, 1 mM sodium pyruvate, and supplemented with 10% FBS. Cells were maintained at 37°C and 5% CO2 and kept under selection by the addition of 200 μg/ml G418 to the media (Gibco, Invitrogen Co., Carlsband, CA).
Oligonucleotide Designs, eGFP targeting, and flow cytometry analysis
EGFP3S/47NT (IDT, Coralville, IA) is single stranded DNA oligonucleotides 47 nucleotides long containing three phosphorothioate linkages at both the 5' and 3' ends. This oligonucleotide was designed to be complimentary to the non-transcribed strand and contains a mismatch at the site of the desired base change, which converts the mutant stop codon to the wild type TAC (encoding tyrosine), restoring the expression of eGFP in corrected cells. Previous publications have shown that neither a non-specific nor a perfectly matched oligonucleotide is able to foster this conversion [18].
For the eGFP targeting experiments, DLD-1 cells were subcultured at a density of 1.6 × 106 cells per 100 mm dish 24 hours prior to targeting. Following this incubation, cells were washed with PBS, trypsinized, centrifuged, and re-suspended in serum-free medium at a concentration of 2 × 106 cells/100 μl; 100 μl was transferred to a 4 mm gap cuvette (Fisher Scientific, Pittsburgh, PA). The EGFP3S/47NT oligonucleotide was added at a concentration of 1 μM and the cells were electroporated (LV, 250 V, 13 ms, 2 pulses, 1 s interval) using a BTX Electro Square Porator™ ECM830 apparatus (BTX, Holliston, MA). The cells were then transferred to a 60 mm dish (100 mm for incubations exceeding 48 hours) containing fresh medium supplemented with 10% FBS and incubated for the indicated time points at 37°C before harvesting for FACS or other analysis, as indicated.
eGFP fluorescence of corrected cells was measured by a Becton Dickinson FACS calibur flow cytometer (Becton Dickinson, Rutherford, NJ.). For eGFP fluorescence, cells were harvested after electroporation (between 8 and 144 hours, as indicated) and re-suspended in FACS buffer (.5% BSA, 2 mM EDTA, 2 μg/ml propidium iodide in PBS). 50,000 cells were analyzed for each sample and the cells that were eGFP positive (corrected) and PI negative (live) were scored as "corrected." Settings for the FACS calibur were determined as previously described [21].
Thymidine treatment
Thymidine was obtained from Sigma-Aldrich (St. Louis, MO.) with a stock solution prepared in distilled water to a final concentration of 500 mM and added to incubating cells at a final concentration of 1–12 mM, as indicated. The term "pre-treatment" indicates addition of thymidine at the start of the 24 hour incubation prior to electroporation; "post-treatment" refers to the addition of thymidine to the 60 mm or 100 mm dishes following electroporation, in the recovery phase. Washing out of thymidine refers to the removal of the agent from the cell culture by aspirating off medium, washing the cells twice with PBS, and adding fresh medium containing FBS. For incubations exceeding 72 hours in the presence of thymidine, cells were washed twice with PBS and fresh medium was added in addition to thymidine at the 72 hour mark, to maintain desired thymidine concentration.
BrdU cell replication assay, PKH26 cell division, and cell cycle analysis
BrdU, 5-Bromo-deoxyuridine, was obtained from Roche Applied Science (Indianapolis, IN). Cells were treated as for gene targeting and the time points indicated, 10 μM BrdU was added to the growth medium. Cells were incubated for 30 minutes at 37°C and then washed three times with PBS and DNA was denatured in 0.2 M HCl for 1 h at 37°C. After two washes in 0.1 M sodium borate and three washes in PBS, cells were blocked in PBS-0.5%BSA-0.1% Tween-20 for 20 min. Cells were then incubated for 1 hour at room temperature with Anti-BrdU-fluorescein antibody (Roche Diagnostics, Switzerland) diluted in PBS-0.1% BSA at a final concentration of 25 μg/ml.
PKH26 (MINI-26 Red Fluorescent Cell Linker Mini Kit) was obtained from Sigma-Aldrich (St. Louis, MO.) and the supplied protocol was optimized for the DLD-1 cells. About 5 × 106 cells were collected and washed once with PBS, cells were resuspended in 200 μl Diluent C, equivolume of PKH26 at 8 × 10-6 M in Diluent C was added, and incubated at room temperature for 5 minutes, with occasional agitation. 400 μl 1% BSA was added for 1 minute at room temperature to stop the reaction. 800 μl DLD-1 medium containing FBS was added, cells were spun down at 1500 rpm for 5 minutes, medium was aspirated off and cells were re-suspended in 800 μl PBS and transferred to a clean tube. Cells were washed twice more with PBS and re-suspended in DLD-1 medium prior to plating. An aliquot was removed to determine mean fluorescence intensity (m.f.i.) immediately following staining (T0), the remaining cells were plated in the conditions noted and their fluorescence was measured at 48 or 144 hours (T48 and T144, respectively), as indicated. Detection of PKH26 was done via flow cytometry, using a rhodamine filter.
For cell cycle analysis, cells were treated as indicated, harvested and washed in PBS. They were then spun at 1200 rpm for 5 minutes, re-suspended in 500 μl cold PBS and fixed by adding 5 ml of 70% cold ethanol while vortexing. Following a 4°C incubation, the cells were centrifuged for 5 minutes at 2000 rpm, washed once with PBS and then re-suspended in 300 μl FACS buffer (50 μg/ml RNase A, 2.5 μg/ml PI, 1% FBS in PBS). The cells were incubated at 37°C for 1 hour and at 4°C over night before analysis by flow cytometry. DNA content was analyzed and data were plotted as DNA content versus cell number; cell cycle modeling was performed and the percent of cells in G0–G1, S, and G2-M phase was calculated using Modfit (Verity Software House, Topsham, ME.).
Senescence associated β-Gal assay
At the indicated times, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. β-gal staining solution (40 mM citric acid/sodium phosphate, pH 6.0; 150 mM NaCl; 2 mM MgCl2; 5 mM potassium ferrocyonide; 5 mM potassium ferricyonide; 1 mg/ml X-gal) was added and plates were incubated over night at 37°C. Following this, cells were washed twice with PBS and re-suspended in PBS for microscopy studies. Cells were viewed under phase contrast and to enhance the blue staining, a bright field illumination was used. Images were obtained by a Nikon Coolpix 995 digital camera at 20× magnification.
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