Promoterless gene trap vectors have beenudwidely used for high-efficiency gene targeting andudrandom mutagenesis in embryonic stem (ES) cells.udUnfortunately, such vectors are only effectiveudfor genes expressed in ES cells and this has promptedudthe development of expression-independentudvectors. These polyadenylation (poly A) trap vectorsudemploy a splice donor to capture an endogenousudgene’s polyadenylation sequence and provideudtranscript stability. However, the spectrum ofudmutations generated by these vectors appearsudlargely restricted to the last intron of target lociuddue to nonsense-mediated mRNA decay (NMD)udmaking them unsuitable for gene targeting applications.udHere, we present novel poly A trap vectorsudthat overcome the effect of NMD and also employudRNA instability sequences to improve splicingudefficiency. The set of random insertions generatedudwith these vectors show a significantly reducedudinsertional bias and the vectors can be targeteduddirectly to a 5’ intron. We also show that thisudrelative positional independence is linked to theudhuman b-actin promoter and is most likely a resultudof its transcriptional activity in ES cells. Takenudtogether our data indicate that these vectors areudan effective tool for insertional mutagenesis thatudcan be used for either gene trapping or geneudtargeting.
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