Facility description

Creating genetically engineered ("knock-out") mice via homologous recombination in ES cells involves the following important steps:

• Engineering a gene targeting construct
• Transfection of ES cells
• Assesing ES cells for homologous recombination
• Generating chimeric mice by blastocyst injection
• Assesing germline transmission
• Breeding chimeric mice to homozygosity

We can help you design the targeting construct (if necessary) and tell you more about our expectations for you role in the gene targeting procedure.

Following targeting construct synthesis and validation, we will prepare ES cells and feeder cell dishes, electroporate the cells with your targeting construct. Following successful electroporation, selection and clonal expansion can be done. We will provide with approximately 200 clones picked on 24-well plates. Master plate will be hand out to you from which you will extract DNA and screen for homologous recombination.  In parallel, we will freeze 24-well plates at -70°C. So screening  must be performed promptly. Individual clones will be thawed at your request and you will receive a second hit of cells to confirm initial screening. ES cells will be tested for mycoplasma.