Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells.

Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous gene's polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5' intron. We also show that this relative positional independence is linked to the human beta-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting.

Differentiation of embryonic stem cells into anterior definitive endoderm.

Anterior definitive endoderm (ADE) is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other visceral organs. Here we describe a method for the differentiation of mouse embryonic stem (ES) cells to endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (aggregation or embryoid body) culture and in a serum-free adherent monolayer culture. Purified ES cell-derived ADE cells appear committed to endodermal fates and can undergo further differentiation in vitro towards liver and pancreas with enhanced efficiency.