Figure 1
Design and generation of the p15Ink4b conditional null allele. (A) Schematic of the mouse p15Ink4b gene (Wild type), targeted (Floxed), and Cre-driven deleted (Deleted) p15Ink4b alleles. Genomic sequences encoding 2 p15Ink4b exons (ex1 and ex2), a neomycin resistance cassette (Neo), and 3 loxP sites (▶). The size of DNA fragments resulting from BamHI (B1) digestion and the probe used for Southern blot analyses (Pr B) are shown. Positions of primers pg1 and pg2 used for genotyping of experimental mice and primers p1, p2, and p3 used for evaluation of Cre-driven efficiency of recombination by PCR are also illustrated. (B) Southern blot analysis of genomic DNA isolated from the tails of the p15Ink4b chimeras used for generation of the p15Ink4bfl/fl-LysMcre and p15ink4bfl/wt-LysMcre mice. (C) PCR genotyping of mice carrying floxed p15Ink4b and LysMcre alleles using pg1 and pg2 primers and the Cre-specific primer set. The picture shows PCR products from mice carrying the p15Ink4b alleles indicated on top of the lanes. (D) Southern blot analyses of LysMcre-driven recombination of floxed p15Ink4b. Genomic DNAs were isolated from BM, spleen (SP), liver (LV), and kidney (KD) of p15Ink4bfl/wt-LysMcre mouse, digested with BamH1 and hybridized with genomic probe B (Pr B). (E) Proliferation of splenocytes isolated from p15Ink4bfl/fl-LysMcre and p15Ink4bwt/wt-LysMcre mice. Untreated (0) and concanavalin A (conA, 10 μg/mL; 2 days)-treated splenocytes were analyzed by flow cytometry for BrdU incorporation. Data are mean ± SD (error bars) for BrdU incorporations. (F) Triple-primer PCR assay with primers p1, p2, and p3 (positions in the p15Ink4b gene are shown in panel A). Targeted p15Ink4b alleles produce a 700-bp band as a PCR product of primers p1 and p2. Extension cycles of the PCR program were chosen such that a large PCR product of primers p1 and p3 with an expected length of 5.5 kb was not amplified. After the deletion of exon 2, the priming site for primer p2 is lost and primer p3 is close to p1, which together amplify a 500-bp PCR product. The ratio of the band intensities was used to determine the approximate recombination efficiency in DNAs isolated from BM and purified BM monocytes (MO). DNA isolated from the BM of the p15Ink4bfl/fl mouse without (−) the LysMcre transgene was used as a negative control for Cre-driven recombination. (G) Reverse-transcribed PCR analyses for p15Ink4b, p16Ink4a, p19Arf, and gapd expression. Total RNAs were isolated from BM-derived monocytes purified from p15Ink4bwt/wt and p15Ink4bfl/fl mice with (+) or without (−) the LysMcre transgene.

Design and generation of the p15Ink4b conditional null allele. (A) Schematic of the mouse p15Ink4b gene (Wild type), targeted (Floxed), and Cre-driven deleted (Deleted) p15Ink4b alleles. Genomic sequences encoding 2 p15Ink4b exons (ex1 and ex2), a neomycin resistance cassette (Neo), and 3 loxP sites (▶). The size of DNA fragments resulting from BamHI (B1) digestion and the probe used for Southern blot analyses (Pr B) are shown. Positions of primers pg1 and pg2 used for genotyping of experimental mice and primers p1, p2, and p3 used for evaluation of Cre-driven efficiency of recombination by PCR are also illustrated. (B) Southern blot analysis of genomic DNA isolated from the tails of the p15Ink4b chimeras used for generation of the p15Ink4bfl/fl-LysMcre and p15ink4bfl/wt-LysMcre mice. (C) PCR genotyping of mice carrying floxed p15Ink4b and LysMcre alleles using pg1 and pg2 primers and the Cre-specific primer set. The picture shows PCR products from mice carrying the p15Ink4b alleles indicated on top of the lanes. (D) Southern blot analyses of LysMcre-driven recombination of floxed p15Ink4b. Genomic DNAs were isolated from BM, spleen (SP), liver (LV), and kidney (KD) of p15Ink4bfl/wt-LysMcre mouse, digested with BamH1 and hybridized with genomic probe B (Pr B). (E) Proliferation of splenocytes isolated from p15Ink4bfl/fl-LysMcre and p15Ink4bwt/wt-LysMcre mice. Untreated (0) and concanavalin A (conA, 10 μg/mL; 2 days)-treated splenocytes were analyzed by flow cytometry for BrdU incorporation. Data are mean ± SD (error bars) for BrdU incorporations. (F) Triple-primer PCR assay with primers p1, p2, and p3 (positions in the p15Ink4b gene are shown in panel A). Targeted p15Ink4b alleles produce a 700-bp band as a PCR product of primers p1 and p2. Extension cycles of the PCR program were chosen such that a large PCR product of primers p1 and p3 with an expected length of 5.5 kb was not amplified. After the deletion of exon 2, the priming site for primer p2 is lost and primer p3 is close to p1, which together amplify a 500-bp PCR product. The ratio of the band intensities was used to determine the approximate recombination efficiency in DNAs isolated from BM and purified BM monocytes (MO). DNA isolated from the BM of the p15Ink4bfl/fl mouse without (−) the LysMcre transgene was used as a negative control for Cre-driven recombination. (G) Reverse-transcribed PCR analyses for p15Ink4b, p16Ink4a, p19Arf, and gapd expression. Total RNAs were isolated from BM-derived monocytes purified from p15Ink4bwt/wt and p15Ink4bfl/fl mice with (+) or without (−) the LysMcre transgene.

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