Figure 1.

Generation of MEFs lacking MCLK1. (A) Schematic illustration of the conditional Mclk1 allele. A loxP/FRT-flanked Neo cassette (NEO, yellow) is inserted upstream of exon 2. A third loxP site is inserted downstream of exon 3. Exons are shown as blue boxes, and loxP sites are shown as orange triangles. Primers #1 and 2 were used to distinguish the conditional (Mclk1^loxP) allele and wild-type allele, whereas PCR using primers #2 and 3 reveal the presence or absence of the recombined allele (Mclk1^Δ). (B) Deletion efficiency in Mclk1^loxP/loxP MEFs infected with pBabe-puro-Cre retrovirus. The recombined allele (Δ) but not the floxed allele (loxP) was detected by PCR in Mclk1^loxP/loxP MEFs infected with Cre-expressing retrovirus (+). The results of the RT-PCR show loss of Mclk1 mRNA expression in Cre-expressing MEFs. β-Actin was used as RT-PCR control. Western blot (WB) indicates that MCLK1 protein is absent in Cre-expressing MEFs. Mitochondrial outer membrane protein Porin served as loading control. (C) HPLC traces of quinone extracts recorded at 275 nm. DMQ₉ is the only detectable quinone in Mclk1 knockout MEFs (Mclk1^Δ/Δ-Cre). The UQ₉ concentration in control cells (Mclk1^loxP/loxP MEF lines infected with empty retroviral vector; Mclk1^loxP/loxP -EV) is shown (normalized to protein content). (D) Growth curves of the indicated MEFs. Cells were seeded in triplicates in 48-well plates and viability was determined at the indicated time points with alamarBlue Assay. Cell viability is reported as the difference in absorbance at the two wavelengths (A_{570 nm}–A_{600 nm}) which changes with the number of living cells. Each growth curve represents three Mclk1^loxP/loxP MEF lines infected with either Cre-expressing retrovirus or retrovirus alone. No significant difference in proliferation rate is observed between Mclk1 knockout and control MEFs. (E) MitoTracker Green staining shows a typical reticular network of mitochondria in knockout MEFs. Scale bars = 20 µm.