Targeted, non-conditional (Promoter-driven)

Further details at the International Mouse Phenotyping Consortium: https://www.mousephenotype.org/data/alleles/MGI:2684972/tm1e%2528KOMP%2529Wtsi

KOMP Targeting Strategies

• Chromosome Spreads and Counting KOMP ES cell clones
• Long Range PCR protocol for KOMP CSD targeted alleles
• Genotyping of KOMP CSD targeted alleles
• Electroporation into JM8 cells with FlpE plasmid
• Electroporation into JM8 cells with Cre plasmid
• Germline Testing of KOMP JM8A (agouti) clones
• Microinjection of blastocysts for CSD JM8A clones
• Culturing and media conditions for KOMP CSD JM8A (Agouti) ES cell clones

• EPD0422_4_D05

Instructions for ordering multiple clones:

1. Before leaving this page, you may want to copy the list of clones to paste into the special requests field as described below in Step 4.*
2. At the bottom of this page, click "Request this Strain" button to add to your cart and proceed with the ordering process.
3. In Step 1 of the Checkout process, select the number of additional clones desired per cell line under "Additional Clones" (ex. if ordering 3 different clones, and 1 vial of each clone, the total quantity of Additional Clones would be 2). Select any desired additional products/services for the cell line(s).
4. In Step 4 of the Checkout process, in the "Shipping Instructions/Special Requests" box, make a note of which specific clones you are requesting.
   *Note: unless you select specific clones, the MMRRC will choose the clones.

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.

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 KOMP ES Cell clones includes development of genotyping protocols, genetic verification at the 3' and 5' ends, vector copy number = 1, presence or loss of Y chromosome, and mycoplasma and MPV testing. Type of genetic testing varies for CSD and Velocigene targets. Approximately 30% of KOMP clones tested fail genetic analysis and will require a new clone be selected for expansion and testing. Based on the average germline rate for KOMP targets, we recommend requesting and injecting 2-3 clones per targeted allele. All mutant ES cell lines are heterozygous for the mutation, meaning they carry one modified allele and one wild type allele. Please note, there is high incidence of mixed cell populations in the CSD targeted cell lines, and while our testing can detect this in most cases, it is not 100%, and there may be a very small percent of non or incorrectly targeted ES cells in the population; therefore we strongly recommend targeting verification of the derived mice with 5’ or 3’ LRPCR and/or Loss of Allele analysis. MMRRC also recommends performing chromosome counts or karyotyping prior to injection of clones.

Estimated timelines: Processing ES cells (including genetic verification) prior to injection or shipping requires 12-16 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.

Distribution of this strain requires submission of the KOMP MTA. The MTA should be signed by an authorized official from your institution, generally this is someone in your Technology Transfer or Intellectual Property offices. A link to this form will be provided via email when an order is submitted; the PDF form should be downloaded or the email forwarded to the appropriate institutional representative for completion following directions in the document.