ES Cell-Mediated Gene Targeting Services

Introduction

Electroporation and clone-picking service includes:
Linearization and purification of the targeting construct DNA
Electroporation into ES cells
Growth of cells under selection
Picking up to 384 drug-resistant colonies into 96-well plates
Splitting the plates into 2 sets
Freezing one set and extracting DNA from the other set

Summary

Gene targeting is the process of disrupting or mutating a specific genetic locus in embryonic stem (ES) cells, usually with the intention of making knock-out or knock-in mice. Other methods of mutating the mouse genome include chemical mutagenesis, insertion of viral vectors or transposons, and random integration of gene-trap or promoter-trap vectors. Use of Cas9 nuclease is probably the most popular method to mediate genome engineering directly in mouse embryos, without the need to use ES cells. Note, CRISPR engineering can also be used in ES cells to engineer changes in the genome, including insertion or substitution of relatively large regions of DNA.

Turnaround Times

The minimum time needed to go from electroporation of the targeting construct to clone DNA ready for genotyping is about 4-5 weeks. Factors that affect this time include growth rates of different ES cell lines, selection times of different antibiotics, and the number of clones picked.
Southern blotting turnaround times can be quite variable, depending on the status of concurrent projects and the number of clones picked. The DNA of clones from two 96-well plates can be digested, electrophoresed, and blotted by one person in one week. Hybridization and autoradiography require several more days.
Expansion of positive clones from 96-well plates, freezing back multiple vials, and extracting more DNA for a second genotyping assay require about 2 weeks.

CD Genomics has already updated its technology platforms to mainstream NGS, long-read sequencing, and microarray instruments. We continue to strive to offer the same reliable services to pharmaceutical and biotechnology companies, as well as academia and government agencies, for all your sequencing or array needs. If you are interested, please contact us directly for assistance.