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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: J Immunol. 2013 Mar 4;190(7):3600–3612. doi: 10.4049/jimmunol.1201933

Figure 1. Generation of mice homozygous for the lox P allele and the macrophage specific KO mice.

Figure 1

a, Design of the targeting construct and identification of the recombinant mouse embryonic stem (ES) cells harboring the conditional null allele. The positions of the lox P sites, Neo Cassette, long and short homology arms and the primers used in genotyping are shown (a, left panel). The recombinant ES cells were genotyped by PCR using Lox1/SDL2 (see above) primers and identified on the basis of the appearance of a doublet band of 325 bp/263 bp (a, right panel). b, Confirmation of the presence of conditional null allele in the recombinant ES cells. The recombinant ES cells were infected by Adenovirus expressing Cre recombinase (Ad-CMV-Cre, Vector Biolabs, USA). The Cre-dependent depletion of L13a was confirmed by immunoblot analysis of the infected cells using anti-L13a antibody. c, Identification of the F4 mice homozygous for the lox P allele (lox P+/+) and Neo deletion allele. Tail DNA samples of the pups were screened by PCR with the Lox1/SDL2 primer pair for the lox P allele (upper panel) and NDEL3/Anti AT2 primer pair for the Neo deletion allele (lower panel). The appearance of the 280 bp band shows the presence of Neo deletion allele. d, Identification of the F4 mice harboring the conditional null allele. Fibroblasts were isolated from the lung and infected with Adenovirus expressing Cre recombinase. The Cre dependent depletion of L13a was confirmed by immunoblot analysis with anti-L13a antibody. e, The macrophage-specific KO mice were generated by crossing the lox P+/+ with the lox P+/−LysM Cre+/− mouse and genotyping by PCR with the Lox1/SDL2 primer pair for the lox P allele and Cre specific primer pair (Jackson Laboratory) for Cre allele. f, Confirmation of macrophage specific depletion of L13a. Lysates were made from the peritoneal macrophages, liver and kidney harvested from L13aflox/floxLysMCre+, L13aflox/flox and wild type (WT) mice. Lysates were subjected to immunoblot analysis using anti-L13a antibody (10). The blot from the macrophage lysates was re-probed with anti-Cre recombinase antibody and anti-actin antibody.