Male and female LmnaHG/+
mice were bred by mating female C57BL/6 mice with male chimeric mice (generated with LmnaHG/+
mouse embryonic stem cells). Because all of the mice in this study were bred from chimeras, the LmnaHG/+
mice were genetically identical (except for the targeted mutation). Genotyping was performed by PCR with genomic DNA prepared from biopsies of the tail [6
]. The mice were fed a chow diet and housed in a virus-free barrier facility with a 12-h light-dark cycle. UCLA's Animal Research Committee approved all procedures.
We administered an FTI, ABT-100 [13
], to groups of 8−13 male and female LmnaHG/+
mice and 7−16 male and female Lmna+/+
mice. ABT-100 was mixed in drinking water containing 0.4% hydroxy methyl propyl cellulose and 1.0% ethanol at a concentration of 0.3 mg/ml, so as to deliver an approximate dose of 39 mg/kg/day. The vehicle consisted of drinking water with 0.4% hydroxy methyl propyl cellulose and 1.0% ethanol. The FTI was initiated at four weeks of age and was continued for up to 45 weeks of age (when the last of the LmnaHG/+
mice died). Previous studies have revealed that this dosage of ABT-100 (~39 mg/kg/day) yields mean plasma ABT-100 concentrations of approximately 0.7 μg/ml [12
]. This FTI dosage did not elicit liver pathology, as judged by routine histological studies. In one series of experiments, we administered a higher concentration of ABT-100 to Lmna+/+
= 14) and LmnaHG/+
= 10), so as to deliver 117 mg/kg/d.
The accumulation of prelamin A in liver was assessed by western blots. Liver samples were collected and frozen in liquid nitrogen, and urea-soluble extracts were prepared and analyzed by SDS-polyacrylamide gel electrophoresis and western blotting. The antibody dilutions were 1:6000 for a rabbit anti-prelamin A antiserum [6
], 1:400 for a goat anti-lamin A/C antibody (Santa Cruz Biotechnology), 1:500 for a mouse anti-HDJ-2 antibody (NeoMarkers), 1:1000 for a goat anti-actin IgG (Santa Cruz Biotechnology), 1:6000 for anti-goat IgG-HRP antibody (Santa Cruz Biotechnology), 1:6000 for anti-rabbit IgG-HRP antibody (Santa Cruz Biotechnology), and 1:6000 for HRP-labeled anti-mouse IgG (Amersham Biosciences). Antibody binding was detected with the ECL Plus chemiluminescence system (Amersham) and exposure to X-ray film.
Body weights of Lmna+/+ and LmnaHG/+ mice were assessed weekly. Body weight curves were compared with repeated-measures ANOVA and the log rank test. The number of surviving male and female mice was recorded weekly and expressed as a percentage of the total number of mice. The significance of differences was determined by the Kaplan-Meier method.
At the time of death, the number of spontaneous rib fractures in LmnaHG/+ mice was documented. After opening the thoracic cavity and removing the heart and lungs, the interior of the thorax was photographed with a digital camera, and rib fractures were counted. The number of rib fractures in FTI- and vehicle-treated LmnaHG/+ mice was compared with a two-tailed Student's t test.