Modeling insertional mutagenesis using gene length and expression in murine embryonic stem cells.
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.


RESULTS
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
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.
Nord
Alex S
AS
Department of Medicine, MacDonald Medical Research Laboratories, University of California at Los Angeles, California, USA. nordalex@u.washington.edu
Vranizan
Karen
K
Tingley
Whittemore
W
Zambon
Alexander C
AC
Hanspers
Kristina
K
Fong
Loren G
LG
Hu
Yan
Y
Bacchetti
Peter
P
Ferrin
Thomas E
TE
Babbitt
Patricia C
PC
Doniger
Scott W
SW
Skarnes
William C
WC
Young
Stephen G
SG
Conklin
Bruce R
BR
D000818:Animals; D002874:Chromosome Mapping / Q000379:methods; D053595:Embryonic Stem Cells / Q000502:physiology*; D005091:Exons / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D016678:Genome; D016013:Likelihood Functions; D051379:Mice; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D020411:Oligonucleotide Array Sequence Analysis; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction