Figure 2.

Gene knockout of COX4i2. A) Schematic representation of the gene-knockout strategy. PCR-based amplification of the 5′ and 3′ COX4i2 regions (o-PCR, outer PCR; arrows) was followed by an inner PCR (i-PCR) with primers containing the indicated restriction sites (sphere-tailed arrows). Two fragments are generated, the promoter region to the end of intron I (1572-bp fragment), and the 6459-bp region spanning from the beginning of intron III to the end of exon IV. These fragments were cloned into the respective sites of the pPNT vector (pPNT4-2), leading to a replacement of exons II and III by the neo cassette in the recombinant. B) Northern blot analysis shows absence of COX4i2 transcripts in the knockouts. Total RNA (40 μg) derived from mouse lung tissues from knockout (KO), heterozygous (HT), and wild-type (WT) mice were loaded per lane. The blots were probed with the COX4i1 (left panel) and COX4i2 (right panel) digoxigenin-labeled antisense RNA. The size of the resulting signal was calculated to be ∼950 bases for both isoforms with the ribosomal bands consistent with the cloned cDNAs, allowing a poly(A) tail of 200–300 bases. KO and HT mice show absence and reduced COX4i2 transcript levels, respectively. C) Western blot analysis using lung and liver tissue from WT and KO mice indicates absence of COX4i2 protein in lung tissue of the KO mice.