Targeted gene trapping in mouse ES cells

Roland Friedel et al. report a method allowing efficient generation of targeted gene knockouts by homologous recombination in mouse ES cells. Current gene targeting techniques use vectors with a strong promoter to drive expression of a selectable antibiotic resistance marker, but such methods usually yield low homologous recombination frequencies because nonhomologous insertions (false positives) also result in antibiotic resistance. Friedel et al. improved upon the previously developed strategy of targeted trapping, in which promoterless gene trap vectors depend on the endogenous promoter of the targeted gene for expression of the antibiotic resistance marker. The authors achieved targeting frequencies as high as 90%, purportedly higher than any other approach reported to date. Although targeted genes must be expressed in ES cells at sufficient levels to confer antibiotic resistance, the authors found a threshold level of expression, and they estimate that about half of all genes are expressed above this level. — P.D.