Estimation of mutation rates in cultured mammalian cells

Abstract

The factors that affect reliable estimations of mutation rates (mu) in cultured mammalian somatic cell populations by fluctuation analysis are studied experimentally and statistically. We analyze the differential effect of the final cell population size in each culture (Nt) and the number of parallel cultures (C) on the variation in the rate estimates (mu) inferred from the P0 method. The analysis can be made after the derivation of the variance of mu, which is a measure of variation of mu for a given combination of Nt and C in a number of repeat experiments. The variance of mu is inversely proportional to C and to the square of Nt . Nt determines the probability of occurrence of mutations in a cell culture. By influencing the size of P0, Nt also determines whether a rate estimate is obtainable from the experiment. Since Po is estimated from the fraction of cultures containing no mutation in a set of C cultures, C becomes a determining factor for the accuracy of mu. The rate estimated from P0 is biased, but the bias is in general 2 orders of magnitude smaller than mu. By the selection of an appropriate combination of Nt and C for the experiment, this bias can be reduced even further. Based on the notion of comparing two proportions, we propose a test statistic and have applied it to experimental results for a test of equality of mutation rates in different cell lines. This development places the comparison of mutation rates on a statistical basis.

Mutation of cancer driverMLL2results in transcription stress and genome instability

“…6B). In agreement with an estimated mutation rate of ∼6 × 10 −8 mutations per nucleotide site per generation for mouse cells in culture (Li et al 1983), only one mutation was detected at the end of this growth period in the F/F control cells (P = 0.13, binomial test). In the MLL2 FC/FC cells, however, we found seven mutations (P = 1.4 × 10 −10 ), again indicating that MLL2 mutation indeed results in a hypermutation phenotype.…”

Loss of Werner syndrome protein function promotes aberrant mitotic recombination

“…The statistical significance of recombination rate differences was determined by applying Welch's t-test for equality of means between groups with unequal variances. Confidence intervals were calculated as described by Li et al (1983). The statistical significance of differences in the distribution of numbers of Lac+ cells in Lac+ WRN and control colonies was determined by a 2 test for equality of the distributions of Lac+ cells/colony, using quartiles of the combined data to determine the category cutpoints.…”

Functional immunoglobulin light chain genes are replaced by ongoing rearrangements of germline V kappa genes to downstream J kappa segment in a murine B cell line.

“…In addition, since only the presence or absence of variants in a clone is scored, it is irrelevant whether they grow faster or slower than the parental cells . The variance (var) of RIV and the coefficient of variance (CV), a measure of the precision in the estimate of RIV were calculated as described by Li et al (11):…”