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. 2012 Sep 28;7(9):e46093. doi: 10.1371/journal.pone.0046093

Figure 3. Generation of Pno1 KO mice.

Figure 3

A. Targeting strategy to generate Pno1 KO mice. The black square marked as 3′ probe represents the sequence used as probe in Southern blotting for genotyping. A 18.6-kb BamH1 fragment detected by this probe represents the WT allele, and a 7.5-kb BamH1 fragment, the KO allele. B. Genotyping of Pno1 mutant mice . Tail DNA was digested with BamHI, and analyzed by Southern blotting (left panel), with a 3′ probe whose sequence location is indicated in A. A 18.6-kb band representing the WT allele and a 7.5-kb band representing the recombinant allele are indicated by arrows. Ear lobe DNA without digestion was analyzed by PCR for routine genotyping (right panel). A 548-bp band representing the WT allele and a 224-bp band representing the recombinant allele are indicated by arrows. C. Reduced Pno1 mRNA expression in Pno1+/− tissues. mRNA from the Li, Th and Spl of WT and heterozygous Pno1+/− (HET) mice were analyzed by reverse transcription-real time PCR (RT-qPCR) for Pno1 mRNA levels. The results are expressed as ratios of Pno1 versus β-actin signals with means ± SD indicated. D. Reduced Pno1 mRNA up-regulation in Pno1+/− T cells upon activation. T cells from WT and HET Spl were stimulated with solid phase anti-CD3 mAb and anti-CD28 mAb (0.5 µg/ml and 4 µg/ml respectively for coating) for 1 to 24 h, and their Pno1 mRNA levels were quantified by RT-qPCR. The results are expressed as ratios of Pno1 versus β-actin signals with means ± SD indicated.