Gene Targeting

Information

The facility produces mutated pluripotent embryonic stem cells (ESc) clones by electroporation with the targeting construct provided by the investigator.

The cells are stocked in liquid nitrogen and they are thawed and expanded in culture, prior to electroporation. The ES cell lines are growing on feeder cells which are mouse embryonic fibroblasts (MEFs), being derived, maintained and inactivated by the facility. In case of feeder independent cells, we are using feeder- free conditions for growth of these cells.

A fresh preparation of the linearized DNA construct should be submitted in two samples of 20-30μg each. The investigator should provide evidence that the screening strategy by Southern blot can detect the locus to be targeted. For this purpose, the facility can supply genomic DNA extracted from the cell line used.

Following 7-10 days under selection, a maximum of 500 clones are picked up and expanded. Duplicates of all colonies are provided to the investigator for DNA analysis. During the screening process, duplicates of all clones will be safely stored at -80°C in the Facility’s cryostorage.

Upon verification of successful recombination, the clones of interest are expanded and kept at low passage status in liquid nitrogen.