PMC

Weight gain of +/+, tub/tub, and −/− mice: (A) Weight gain of male wild-type (+/+), tubby (tub/tub), and tub-deficient (−/−) mice. The weights of 10 mice of each genotype were measured at the indicated times. (B) Weight gain of female wild-type, tubby, and tub-deficient mice. The weights of 10 mice of each genotype were measured at the indicated times.

(A) Anti-Tub immunoblot of whole cell lysates from CHO cells transfected with the empty vector (Vec; lanes 1 and 2), HA-Tub (WT; lanes 3 and 4), or HA-mutant Tub (Mut; lanes 5 and 6). (B) Brain lysates were resolved by SDS-PAGE and probed with the anti-Tub antiserum as for Fig. . Each lane represents an individual mouse of the indicated genotype. KO, knockout; MC4R, melanocortin 4 receptor.

Histological section and TUNEL assay of wild-type, tub/tub, and tub^−/− mouse retinas. (A to C) Histological sections of retinas from wild-type (A), tub/tub (B), and tub^−/− (C) mice at 8 weeks of age. Photoreceptors (Ph) and outer nuclear layers (ONL) are reduced in tub/tub and tub^−/− mice (arrows) compared to wild-type mice. All other layers appear normal. Magnification, ×400. OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (D to F) In situ TUNEL assay of retinas from wild-type (D), tub/tub (E), and tub^−/− (F) mice at 8 weeks of age. Apoptotic figures (indicated by arrowheads) are present in retinas derived from tub/tub and tub^−/− but not wild-type mice. Magnification, ×40.

Expression of the Tub protein in wild-type (WT) and tub^−/− mice. (A) Anti-Tub immunoblot of HA-Tub expressed in CHO cells. Lanes 1, 3, and 5, transfection with the empty vector (V); lanes 2, 4, and 6, transfection with HA-Tub. Samples were resolved by SDS-PAGE in a 10% gel, transferred to nitrocellulose, and probed with the anti-Tub antibody. Lanes 1 and 2, blot of whole cell lysate (WCL); lanes 3 and 4, blot of anti-HA immunoprecipitates; lanes 5 and 6, blot of anti-Tub immunoprecipitates. Sizes are indicated in kilodaltons. (B) Brain and spleen lysates from wild-type and tub knockout mice were resolved by SDS-PAGE in an 8% gel and transferred to nitrocellulose. The membrane was probed with anti-Tub antibody. The Tub protein, indicated by the arrow, is detectable in brain lysates from wild-type (lane 1) but not tub knockout (KO; lane 2) mice. No Tub expression is detected in spleen lysates from either wild-type (lane 3) or tub knockout (lane 4) mice.

Disruption of the mouse tub locus. (A) Schematic representation and partial restriction maps of the tub wild-type locus, targeting vector, and targeted allele. Closed boxes represent tub exons 1 and 12, the hatched box indicates the 5′ flanking probe, the shaded box is the PGK-neo expression cassette, and the horizontal dotted line is plasmid DNA. The arrow below neo indicates the direction of transcription. (B) Southern blot analysis of EcoRI-digested tail DNA from F₂ progeny hybridized with the radiolabeled probe shown in panel A. The wild-type (+/+) hybridizing band is 6 kb; the band from homozygous mutant mice (−/−) is 4 kb; mice heterozygous for the mutation (+/−) show both 6- and 4-kb bands. (C) PCR analysis of tail DNA of F₂ +/+ and −/− mice. Each PCR was done in duplicate. The primers are from exons 2 and 3 (lanes 1 to 4) or 7 and 8 (lanes 5 to 8) and 12 (all lanes). Exons 2, 3, 7, and 8 are absent in −/− mice. Exon 12 is present in both +/+ and −/− mice.