Gene Trap - 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 α-myosin heavy chain (α-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 are submitted to puromycin selection to kill all cell types except cardiomyocytes. Reporter gene expression is then measured specifically on cardiomyocytes.
Ventura C., Zinellu E., Maninchedda E., and Maioli M. Dynorphin B is an agonist of nuclear opioid receptors coupling nuclear protein kinase C activation to the transcription of cardiogenic genes in GTR1 embryonic stem cells. Circ Res. 92(6):623-9, 2003. [PDF]
Ventura C., Zinellu E., Maninchedda E., Fadda M., and Maioli M. Protein kinase C signaling transduces endorphin-primed cardiogenesis in GTR1 embryonic stem cells. Circ Res. 92(6):617-22, 2003. [PDF]