Gene Trap Paradigm
Gene trapping in ES cells is a random insertional mutagenesis approach to functional genomics developed here at The Samuel Lunenfeld Research Institute. 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 and clones containing gene trap insertions are selected by G418 (neomycin) resistance. NeoR 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 isolation and RACE, and to assay reporter expression in the expression screens developed around the in vitro differentiation assays described below.
ES Cell Lines
Our gene trap initiative is being performed using the R1 ES cell line and a derivative of this cell line, GTR1. The R1 cell line was derived by Andras Nagy here at the SLRI (http://www.mshri.on.ca/nagy). R1 cells and their derivatives can be introduced into the murine germline by aggregation of 6-10 ES cells and 8-cell stage embryo into a microdepression made in the bottom of the tissue culture dish. This technique is simple and inexpensive compared to conventional microinjection of blastocysts. R1 cell lines also can be aggregated with tetraploid embryos. The tetraploid embryo develops into the placenta and some extraembryonic membranes while the embryo proper develops entirely from the ES cells.
Most of our gene trap screens exploit the morphological differences between the various lineages that develop in the differentiation cultures. Although cardiomyocytes readily differentiate in EB cultures, they are not distinguishable by morphology alone and are normally assayed by immunohistochemistry. Because immunohistochemistry is not easily amenable to high throughput analysis, we have developed a reporter ES cell line for cardiomyocyte gene trapping. A transgene encoding the cardiomyocyte-specific a-myosin heavy chain (a-MHC) promoter driving the puromycin-resistance gene was introduced into the R1 cells to generate the GTR1 ES cell line. To screen for gene trap insertions into cardiomyocyte-specific genes, replicate attached EB cultures (see below) are submitted to puromycin selection to kill all cell types except cardiomyocytes. Reporter gene expression is then measured specifically on cardiomyocytes.