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Cre or Flp^e Electroporation in Targeted ES Cells

The methods for this procedure were first established in the laboratory of Dr. Magnuson, with the assistance of this resource and involve coupling site-specific recombination to homologous recombination to remove selection cassettes or swap or knock-in gene sequences. Replacement vectors are configured in a similar fashion as standard knockout vectors except that the selection cassettes and/or the heterologous sequences are flanked by loxP or FRT sites. In later microinjection or electroporation experiments utilizing a supercoiled Cre or Flp^e expression plasmid, these floxed or flrted segments of DNA can be selectively removed from the genome. This allows the investigator to study, in a time efficient manner, the effects of having the gene present or absent.

The same steps are followed to initiate a conditional knockout or knock-in experiment. The responsibilites are identical for the investigator and shared resource and due to the technical difficulty, the progress of each experiment is monitored closely by a member of our staff. The cost of the pronuclear microinjection experiments using a supercoiled Cre or Flp^e expression plasmid to remove the "floxed" or "flrted" segments of DNA from the genome is $4,800. Manipulations that require elecroporations utilizing this same supercoiled Cre expression plasmid to convert the gene-targeted allele directly within the ES cells costs $12,000 per electroporation. Ten successful conditional knockout experiments have been completed to date and nine other experiments, in various stages of progress, are currently being completed in the ES Cell laboratory.