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Availability & Fees Order this Strain
KOMP repository Mutant Mouse Resource and Research Centers (MMRRC) Thtm1a(KOMP)Wtsi ES cells were re-engineered using dual recombinase-mediated cassette exchange (dRMCE) with a targeting vector that included splice acceptor and exons 7 to 13 of Th locus, the autocatalytic peptide sequence 2A, WPRE sequence, and a polyadenylation signal followed by a rox-flanked puromycin selection cassette. Since the two coding sequences are separated by virtue of a ribosome-skipping event that occurs at the glycyl-prolyl peptide bond at the C terminus of the 2A peptide (effectively, an autocatalytic ‘cleavage’), by design, any cell expressing TH protein also synthesizes Flpo. The targeting vector was electroporated into KOMP ES cells as well as the plasmid pDIRE (Addgene #26745) to induce a high level of Cre and Flp expression. Selected for construct using puromycin. 62 clones were screened using PCR; 63% were positive for Flpo and WPRE and of those, 26% were lacking Neo and LacZ, suggesting RMCE occurred. The re-engineered clones were injected into blastocysts by Northwestern University's Transgenic and Targeted Mutagenesis Laboratory (TTML). Subsequent backcrossing to C57BL/6NCrl for 4 generations.
Removal of the puromycin cassette is suggested by crossing to a Dre-expressing line (e.g., Th-2A-Flpo mice to B6;129-Tg(CAG-dre)1Afst/Mmucd, MMRRC:32246).
Poulin JF, Caronia G, Hofer C, Cui Q, Helm B, Ramakrishnan C, Chan CS, DombeckDA, Deisseroth K, Awatramani R. Mapping projections of molecularly defineddopamine neuron subtypes using intersectional genetic approaches. Nat Neurosci.2018 Sep;21(9):1260-1271. doi: 10.1038/s41593-018-0203-4. Epub 2018 Aug 13.(Medline PMID: 30104732)
Colony Surveillance Program and Current Health Reports
Limited quantities of breeder mice (up to 2 males and 2 females or 4 mice) per investigator per month are available from a live colony, usually available to ship in under 12 weeks. Larger quantities may be available, please contact the distributing center directly at mmrrc@missouri.edu for more details.
Cryopreserved material may be available upon request, please inquire to mmrrc@missouri.edu for more information.
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.
The donor or their institution limits the distribution to non-profit institutions only.
Additional charges may apply for any special requests. Shipping costs are in addition to the basic distribution/resuscitation fees. Information on shipping costs and any additional charges will be provided by the supplying MMRRC facility.
1 The distribution fee covers the expense of rederiving mice from a live mouse; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.
2 An aliquot contains a sufficient number of embryos (in one or more vials or straws and based on the transfer success rate of the MMRRC facility) to transfer into one to three recipients. The MMRRC makes no guarantee concerning embryo transfer success experienced in the recipient investigator's laboratory. Neither gender nor genotype ratios are guaranteed.
3 An aliquot is one straw or vial with sufficient sperm to recover at least one litter of mice, as per provided protocols, when performed at the MMRRC facility. The MMRRC makes no guarantee concerning the success of these procedures when performed outside the MMRRC facilities.
4 The distribution fee covers the expense of resuscitating mice from the cryo-archive; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.