Publications

BACKGROUND

High-throughput mutagenesis of the mammalian genome is a powerful means to facilitate analysis of gene function. Gene trapping in embryonic stem cells (ESCs) is the most widely used form of insertional mutagenesis in mammals. However, the rules governing its efficiency are not fully understood, and the effects of vector design on the likelihood of gene-trapping events have not been tested on a genome-wide scale.

METHODOLOGY/PRINCIPAL FINDINGS

In this study, we used public gene-trap data to model gene-trap likelihood. Using the association of gene length and gene expression with gene-trap likelihood, we constructed spline-based regression models that characterize which genes are susceptible and which genes are resistant to gene-trapping techniques. We report results for three classes of gene-trap vectors, showing that both length and expression are significant determinants of trap likelihood for all vectors. Using our models, we also quantitatively identified hotspots of gene-trap activity, which represent loci where the high likelihood of vector insertion is controlled by factors other than length and expression. These formalized statistical models describe a high proportion of the variance in the likelihood of a gene being trapped by expression-dependent vectors and a lower, but still significant, proportion of the variance for vectors that are predicted to be independent of endogenous gene expression.

CONCLUSIONS/SIGNIFICANCE

The findings of significant expression and length effects reported here further the understanding of the determinants of vector insertion. Results from this analysis can be applied to help identify other important determinants of this important biological phenomenon and could assist planning of large-scale mutagenesis efforts.

Homocysteine may play a causal role in cognitive decline. The authors analyzed the 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T genotype, a correlate of plasma homocysteine levels, among 6,653 participants in the Study of Osteoporotic Fractures, a community-based, prospective cohort study of older women in four US states. During the years 1986-1998, the authors assessed whether the distribution of MTHFR C677T genotypes was independent of potential confounders and whether persons with the TT genotype had lower baseline performance or showed greater longitudinal declines on standard cognitive tests. Although ethnicity was associated with MTHFR genotype distribution within the entire cohort (p < 0.001), all measured confounders appeared independent of MTHFR genotype within the largest ethnically homogenous subgroup, persons of Northern and/or Central European ancestry (n = 5,668) (Kolmogorov-Smirnov p = 0.97). In this subgroup, the TT genotype was associated with lower scores on the Digit Symbol Substitution Test (p = 0.034) and the Trails B test (p = 0.020) and with a small excess annual decline on a modified version of the Mini-Mental State Examination (p = 0.035). Although the strength of the observed associations was modest, these results lend some support to the theory that an elevated homocysteine level contributes to cognitive decline.

T cell activation and differentiation is accompanied and mediated by transcriptional reprogramming. The NFATc1 transcription factor is strongly induced upon T cell activation and controls numerous genes involved in the T cell effector function. However, its regulation by physiological stimuli in primary T cells has not been well understood. We previously found that ICOS synergizes with TCR and CD28 to greatly enhance NFATc1 expression in primary T cells. In this study, we have examined the signaling mechanisms whereby costimulation regulates NFATc1 expression. We found that CD28 and ICOS regulate sustained PI3K activity in primary T cells, which is required for NFATc1 up-regulation. CD28 and ICOS costimulation, possibly through Itk, a Tec kinase downstream of the PI3K, enhanced phosphorylation of phospholipase C gamma1 and increased and sustained Ca(2+) flux in T cells. Costimulation of T cells potentiated transcription of the Nfatc1 gene P1 promoter in a PI3K-dependent manner. This work demonstrates an important role for costimulatory receptors in sustaining T cell activation programs leading to Nfatc1 gene transcription and has implications in our understanding of the immune response and tolerance.