• Donor's Genotyping Strategies Overview
• Donor's Protocol for Genotyping by Dot Blot Analysis
• 5'RACE Sequence Tag Generation Protocol
• 5'RACE Sequencing Outline
• 5'RACE Sequencing Protocol

• MMRRC's Cell Culture Protocol
• Donor's Cell Culture Protocol
• Electroporation and Isolation of ES Cell Lines
• Blastocyst Injections and Mouse Breeding
• PLAP Staining
• Taqman 7700 QPCR Assay to Assess Neo Dosage in Knockout Mice
• Paraffin Embedding and Sectioning of Mouse Embryos
• In Situ Hybridization with DIG-probes
• ALL experimental protocols from BayGenomics

When choosing a clone, we recommend blasting the sequence tag to estimate the location of a gene trap. Generally you will want to select a clone that traps the furthest upstream. The vast majority of gene trap cell lines have been correctly identified, as judged by DNA sequencing (i.e., repeat determination of the DNA sequence tag when the ES cell clone is requested). However, incorrectly identified ES cell clones are occasionally encountered, either because of human error or because a thawed ES cell line gets overgrown by a contaminating ES cell line. Therefore, we strongly recommend that you request verification of the identity of your ES cell line by DNA sequencing (there is an additional fee for DNA sequence verification of your clone). We also recommend that recipients of ES cells re-verify the identity of the ES cell line by RT-PCR and/or Southern blot analysis. If more than one ES cell line is available for your "gene-of-interest", we recommend that you order several of the ES cell lines. Ordering more than one ES cell line for a specific 'gene-of-interest' will increase the chances of success.

ES Cell Sex and Genotype: These ES Cells are expected to be male (XY) and heterozygous/hemizygous for the mutation.

Requested cell lines are tested by PCR analysis for mycoplasma spp. and Murine Parvoviruses (MPV) upon completion of expansion and prior to shipment. The parental cell line E14Tg2a.4, from which these cells were derived, was tested for multiple mouse pathogens; please see the Health Status Report.

Mice derived from requested cell lines via Microinjection services will be pathogen tested prior to delivery of mice and health reports will be provided. If you require additional health status information prior to requesting microinjection please email mmrrc@ucdavis.edu.

MMRRC Testing of the BayGenomics, SIGTR, and Soriano gene trap cell lines includes RACE sequencing and mycoplasma and MPV testing. All mutant ES cell lines are heterozygous for the mutation, meaning they carry one modified allele and one wild type allele. MMRRC also recommends performing chromosome counts or karyotyping prior to injection of clones.

Estimated timelines: The repository was sent only one or two vials per clone/cell line from the production center. Processing ES cells (including genetic verification) prior to shipping or injection requires 6-8 weeks. Chromosome counting (to determine % euploid cells) requires an additional 4-8 weeks. Microinjection requires an additional 11-15 weeks. Germline transmission testing (breeding of chimera) requires an additional 8-16 weeks. Germline transmission is not guaranteed.

The donor or their institution limits the distribution to non-profit institutions only.

Distribution of this strain requires submission of the MMRRC Conditions of Use (COU). A link to the COU web form will be provided via email after an order has been placed; the form should be completed then or the email forwarded to your institutional official for completion.