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Gene Trap Primer

Gene trapping in ES cells is a random insertional mutagenesis approach to functional genomics. The gene trap vector is designed to generate sequence, expression and functional information for each clone. The basic gene trap vector contains a splice acceptor site immediately upstream of a promoterless reporter gene and a selectable marker, shown below.

The gene trap vector is introduced into ES cells by electroporation or retroviral infection and clones containing gene trap insertions are selected by antibiotic resistance. Antibiotic resistant clones are then picked into 96-well master plates, which are later replicated to generate frozen stocks for long-term storage of clones, cell lysates for RNA and DNA isolation followed by PCR-based gene identification techniques. In some cases, replicates are tested for reporter expression in expression screens.

Gene trap ES cell lines are not knockouts. These random insertions are often mutagenic, even leading to null mutations. However, the insertion site in a clone is a critical determinant of the type of mutation generated. Insertions occurring in the 5’ coding region will likely generate a null mutation, while other types of insertions lead to hypomorphic or neomorphic mutations, even dominant negative mutations. This is dependent upon the gene structure and function as well as the type of gene trap vector used. The types of mutations generated by polyA trap vectors are further explained in the PolyA trapping & NMD and PolyA Trap Vectors pages.

 

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