Generation of Murine (M)CCR2 Knock-Out/Human (H)CCR2 Knock-In Mice

supplemental data

Generation of murine (m)CCR2 knock-out/human (h)CCR2 knock-in mice

Transgenic mCCR2 knock-out/hCCR2 knock-in mice were generated in collaboration with Lexicon Pharmaceuticals Incorporated (USA). The strategy applied yielded replacement of the coding sequence of murine CCR2, encoded by a single exon, by the coding sequence of human CCR2B (Supplemental Figure A). Using a PCR probe genomic clones were isolated by screening the 129SvEvBrd derived lambda pKOS genomic library (Wattler et al., 1999). A 10.7 kb genomic clone spanning the single coding exon (ENSEMBLE gene ID: ENSMUSG00000049103) was used to generate the targeting vector via yeast-mediated homologous recombination. In this vector a 1178 bp genomic fragment, with the ATG start codon at its 5’ prime end and spanning the entire 1122 coding sequence of murine CCR2, was replaced by the coding sequence of human CCR2B and a floxed version of the PGK-neo selection cassette. Targeted 129Sv/Evbrd (LEX1) embryonic stem (ES) cell clones were injected into C57Bl/6 (albino) blastocysts, and the resulting chimeras were mated to C57Bl/6 females to generate N1/F0 animals heterozygous for the CCR2B knock-in allele. These were subsequently crossed to Protamine-Cre mice, which are on a C57Bl/6 background (O'Gorman et al., 1997), and N2/F0 male descendants heterozygous for both the CCR2B knock-in allele, including the floxed PGK-neo selection cassette and the Protamine Cre transgene were crossed to C57Bl/6 females to obtain N3/F0 heterozygous CCR2B knock-in animals devoid of the selection cassette. These were subsequently interbred to generate both N3/F1 genotypes. Backcrossing into C57Bl/6 genetic background continued to the 8th generation to provide a stock for the generation of the study population used in these experiments. Expression of the hCCR2B transcript was confirmed by quantitative RT-PCR performed on spleen, kidney, thymus and lung total RNA from wild type (WT/WT), heterozygous (WT/KI) and homozygous hCCR2B knock-in mice (KI/KI) (n=3) (Supplemental Figure B). The hCCR2B primer-probe pair (primer 5’-CCT GAA CAC CTT CCA GGA ATT C-3’ primer 5’-CGT GGC TTG GTC CAG TTG A-3’, probe 5’-CGG CCT GAG TAA CTG TGA AAG CAC C-3' [5']FAM [3']TAMRA) relative to actin (primer 5’-CAT CTT GGC CTC ACT GTC CAC-3’, primer 5’-GGG CCG GAC TCA TCG TAC T-3’, probe 5’-TGC TTG CTG ATC CAC ATC TGC TGG A-3’ [5']FAM [3']TAMRA) was used to asses expression levels.

Supplemental Figure A Human CCR2B knock-in generation. Structure of the targeting vector, wild type locus, targeted locus and cre-excised locus. Boxes represent the exons, in white and colour (red and blue) indicated are the non-coding and coding regions. A lambda pKOS based targeting construct was generated by replacing the coding region of mCCR2, located in a single exon by the coding sequence of hCCR2B and a floxed version of the PGK-neo selection cassette. Cre-mediated recombination results in deletion of the PGK-neo selection cassette.

Supplemental Figure B Expression of the hCCR2B transcript in lung, spleen, thymus and kidney was detected in heterozygous (WT/KI) and homozygous (KI/KI) knock-in CCR2B mice as determined by quantitative RT-PCR. No hCCR2B transcripts were detected in wild type C57Bl/6 mice. Values expressed are average ± stdev relative expression levels after normalization to β-actin(n=3).

References

Wattler S, Kelly M, and Nehls M (1999) Construction of gene targeting vectors from lambda KOS genomic libraries. Biotechniques26:1150-6, 1158, 1160.

O'Gorman S, Dagenais NA, Qian M, and Marchuk Y (1997) Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells. Proc Natl Acad Sci U.S.A94:14602-14607.