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Genotyping Protocol
Generation of H2-Db knockout mice: The H2-Db gene was disrupted using an insert containing lacZ and a neo cassette flanked by a 4-kb HindIII-XbaI 5' and 1-kb KpnI-SpeI 3' fragment of the D gene resulting in the loss of the first three exons. The targeting construct was introduced into a CGR-8 embryonic stem cell of 129P2 origin. Clones that were confirmed for homologous recombination were injected into B6 blastocysts and germline chimeras were identified. The founders were crossed twice with C57BL/6J females and heterozygous N2 offspring were intercrossed to generate the H2-Db homozygous strain. Inactivation of the H2-Db gene was confirmed by Southern blot analysis on tail DNA and immunofluorescence assay.
Generation of H2-Db knockout mice: the H2-Db gene was disrupted by replacing the 2nd and 3rd exons with an HPRT minigene inserted in the opposite transcriptional orientation. The disrupted gene was introduced into an E14TGa(H-2b and b2m) HPRT-deficient embryonic stem cell of 129P2 origin. After injection into B6 blastocysts, the resultant germline chimeras were backcrossed twice to C57BL/6J mice and offspring heterozygous for the H2-Kb knockout allele were crossed to generate H2-Kb- deficient mice (Kb -/-). Inactivation of the H2-Kb gene was confirmed by Southern blot analysis on tail DNA and by FACS analysis.
Generation of H2-Kb/H2-Db double knockout mice: N2 Kb -/- and Db -/ - mice were intercrossed and the H2-Kb and H2-Db targeted mutations were genetically linked by crossing-over between the two mutated loci. The double targeted mutation mice were subsequently backcrossed to C57BL/6J mice another 10 times
Pascolo S, Bervas N, Ure JM, Smith AG, Lemonnier FA, Pérarnau B.HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes frombeta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m doubleknockout mice. J Exp Med. 1997 Jun 16;185(12):2043-51. (Medline PMID: 9182675)
Pérarnau B, Saron MF, Reina San Martin B, Bervas N, Ong H, Soloski MJ, SmithAG, Ure JM, Gairin JE, Lemonnier FA. Single H2Kb, H2Db and double H2KbDb knockoutmice: peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitisvirus cytolytic responses. Eur J Immunol. 1999 Apr;29(4):1243-52. (Medline PMID: 10229092)
Colony Surveillance Program and Current Health Reports
Limited quantities of breeder mice (recovered litter) are available from a cryoarchive; recovered litter usually available to ship in 3 to 4 months.
Cryopreserved material may be available upon request, please inquire to mmrrc@ucdavis.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.
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