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Analysis of frame-shift mutations in [AAAG]17- and [CA]13-repeats in MSH3-silenced primary colon epithelial cells.

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posted on 27.11.2012 by Christoph Campregher, Gerald Schmid, Franziska Ferk, Siegfried Knasmüller, Vineeta Khare, Benedikt Kortüm, Kyle Dammann, Michaela Lang, Theresa Scharl, Andreas Spittler, Andres I. Roig, Jerry W. Shay, Christopher Gerner, Christoph Gasche

2×104 EGFP-negative/RFP-positive cells were sorted into 24-well plates and mutant fractions were analyzed after 3, 6 and 9 days. At day 4 cells were expanded to 6-well plates to sustain exponential growth (A) Cell count of HCEC-1CT-[AAAG]17-control, HCEC-1CT-[AAAG]17-scrambled and HCEC-1CT-[AAAG]17-shMSH3 cells at day 3, 6 and 9 revealed exponential growth. (B) Analysis of EGFP-positive mutant fractions at day 3, 6 and 9 showed a 6- to 10-fold elevated mutant fraction in HCEC-1CT-[AAAG]17-shMSH3 cells compared to HCEC-1CT-[AAAG]17-scrambled. No difference was observed between HCEC-1CT-[AAAG]17-scrambled and HCEC-1CT-[AAAG]17-control cells. (C) Cell count of HCEC-1CT-[CA]13-control, HCEC-1CT-[CA]13-scrambled and HCEC-1CT-[CA]13-shMSH3 cells at day 6. (D) Analysis of EGFP-positive mutant fractions at day 6 showed a 3-fold increase in HCEC-1CT-[CA]13-shMSH3 cells compared to HCEC-1CT-[CA]13-scrambled. No difference was observed between HCEC-1CT-[CA]13-scrambled and HCEC-1CT-[CA]13-control cells. Mean of triplicate cultures for each clone, bars, SE. Asterisks indicate statistical significance of p<0.05. n.a. = not analyzed. White bars = control, grey bars = scrambled, black bars = shMSH3.

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