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Recommendations for Users of EUCOMM Alleles
- Southern blot: The EUCOMM resource is a high-throughput gene targeting effort and for practical reasons, all clones have been characterised by sequence-verified PCR only (3’ screen and/or 5’ screen)
In a random sampling of the resource, Southern blot analysis with internal probes revealed complex events in approximately 15% of the ES cell clones. As such we recommend that customers order at least 3 different ES cell clones per gene, and use Southern blot analysis to verify correct targeting for all clones. - Removal of floxed region (when used as complete knockout (targeted trap)): EUCOMM strongly recommends the removal of the floxed region prior to phenotypic analysis (especially for promoter-containing targeting cassettes as the promoter-driven neo is also removed) as this will ensure a true null allele.
- Germline transmission: The clonal germline transmission rate of EUCOMM targeted ES cells obtained by EUCOMM mouse production centers is approximately 50%. To maximize the chance of achieving germline transmission, EUCOMM strongly recommends microinjections of three independent ES cell clones. Optimal results are obtained from injections of C57BL/6 host blastocysts.