C57BL/6 mice deficient in Db or Kb and were generated by gene targeting in the laboratory of Dr. Francois Lemonnier of The Institut Pasteur, Paris France. 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 129/OLA 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/6Ji 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 129/OLA origin. After injection into B6 blastocysts, the resultant germline chimeras were backcrossed twice to C57BL/6Ji 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/6Ji mice another 10 times
Pascolo S, Bervas N, Ure JM, Smith AG, Lemmonier FA and Pérarnau B. HLA-A2.1-restricted education and cytolytic activity of CD8+ T lymphocytes from β2-microglobulin(β2m) HLA-2.1 monochain trangenic H2Dbβ2m double knockout mice. J. Exp. Med. 1997. 185: 2043-2051
Pérarnau B, Saron MF, San Martin BR, Bervas N, Ong H, Soloski MJ, Smith AG, Ure JM, Gairin JE and Lemonnier FA. Single H-2Kb, H-2Db and double H-2KbDb knockout mice: peripheral CD8+ T cell repertoire and antilymphocytic choriomeningitis virus cytolytic responses. Eur. J. Immunol. 1999. 29: 1243-1252
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