Animals were marked with ear tags and separated by sex into full (27 × 27 cm) cages. Animals of the two genotypes (Mycand Myc) were housed together. No animals were lost to fighting or accidental death. The first death occurred at 498 days (16.4 months) due to an apparent respiratory illness. Dermatitis did occur in very few of the animals but was successfully treated by spraying the affected area with 70% ethanol. No animals had to be euthanized or were deemed to have died due to dermatitis. A few animals (n = 12) were euthanized for humane reasons (these animals were deemed to have lived to the end of their natural life spans and were thus not censored in the demographic analysis). The guidelines for euthanasia developed by the NIA Interventions Testing Program (ITP) were used (), also see. Seven animals (4 Mycfemales and 3 Mycfemales) were sacrificed at approximately 30 months of age for the collection of tissue specimens, at which time they were in apparent good health. These animals were censored (at the age of sacrifice) in the demographic analysis ( Table S1 ). No other animals were censored. The only procedures that were performed were periodic weighings (weekly in the first 6 months of life, and monthly thereafter), rotarod tests on a few animals, and tail bleeds at a young age on a few animals. The lifespans of the Mycanimals in our colony are among the longest reported ().

Matings between either Mycmales and Mycfemales or Mycfemales and Mycmales produced normal numbers of pups of each genotype. Animals for the aging study were produced by mating Mycmales with C57BL/6NCrl females purchased from Charles River. Females were purchased at 12 weeks of age and bred immediately. Two females were housed continuously with one male in a full (27 × 27 cm) cage. Females were replaced after producing 3-4 litters. A total of 7 males and 23 females were used to produce the cohorts for the aging study. Four cohorts were entered into the study: 42 Mycmales, 41 Mycfemales, 42 Mycmales, and 42 Mycfemales. None of the animals entered into the aging study were allowed to breed. Most of these animals were produced over a period of 7 months (11/17/07 to 9/9/08) by 6 males and 22 females ( Table S2 ). A few animals (9 pups, 2 litters) were produced over the subsequent months by a new pair and used to top up the cohorts. Litters were weaned at 21 days of age, at which time tail snips were taken for PCR genotyping. Genomic DNA was prepared from the tails as follows. A piece of tail ∼5 mm in length was cut and lysed in 0.5 ml of buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 mM NaCl, 0.2% SDS) with 140 μg/ml proteinase K. Samples were shaken at 55°C until the tail was mostly dissolved, spun briefly to pellet bones and hair, and DNA was precipitated with an equal volume of isopropanol). Reactions were performed using the Promega GoTaq system (Fisher PRM7122) and primer pairs 1 and 2, using protocols provided by the manufacturer, run out on 1% agarose gels, and photographed after staining with ethidium bromide.

Animals are housed in plastic 27 cm x 27 cm cages which are held in HEPA-ventilated cage racks (maximum of 7 animals per cage). Animals are also housed in half-cages (maximum of 3 animals). Cages, bedding (Sani-chip hardwood bedding) and food (Purina Lab Chow # 5010) are sterilized by autoclaving. Food and water (also sterilized) are provided ad libitum. The same food is used for all animals, including pregnant and nursing mothers and weaned pups. Bedding is changed once per week. A light-dark cycle of 12 hr is used (7 AM On, 7 PM Off). Temperature is maintained at 70°F, and humidity at 50%.

Mice were produced and housed in a specific pathogen-free (SPF), AAALAC-certified barrier facility. The following pathogens are tested on a quarterly basis: Sendai virus (SEND), Pneumonia virus of mice (PVM), Mouse hepatitis virus (MHV), Minute virus of mice (MVM), Theiler’s murine encephalomyelitis virus (TMEV), Reovirus 3 (REO), Mouse parvovirus (MPV), Mouse parvovirus (NS1), Epizootic diarrhea of infant mice (EDIM), Lymphocytic choriomeningitis virus (LCMV), Hantavirus (HTN), and Mycoplasma pulmonis. In addition, the following pathogens are tested on a yearly basis: Mouse adenovirus 1 & 2 (MAV), Ectromelia virus (ECTRO), K virus (K), Polyoma virus (POLY), Mouse thymic virus (MTLV), Murine cytomegalovirus (MCMV), Mouse Noro virus (MNV), Encephalitozoon cuniculi (ECUN), Cilia-associated respiratory bacillus (CARB) and Helicobacter. The following strains of Helicobater are tested: Helicobacter bilis, Helicobacter ganmani, Helicobacter hepaticus, Helicobacter rodentium, Helicobacter sp., Helicobacter trogontum, Helicobacter typhlonius.

Mice with one floxed allele of Myc (Myc) on a mixed 129 and C57BL/6 background () were obtained from Dr. Ignacio de Alboran, Department of Immunology and Oncology, University of Madrid, Spain. Upon receipt the mice were rederived and subsequently housed in a Helicobacter-free barrier facility at Brown University. The floxed allele was converted to a germline deletion allele (Myc, referred to here as Myc) by breeding heterozygous floxed animals with the Cre deleter line TgN(CMV-Cre)1AN (), which was backcrossed by Dr. Tomas Prolla (Departments of Genetics and Medical Genetics, University of Wisconsin, Madison) to C57BL/6 for over 6 generations. After deleting the Mycallele Cre was segregated away, and the Myc heterozygous line was backcrossed more than 10 times to C57BL/6NCrl mice purchased from Charles River (strain code 027). The Myc deletion line has been propagated by breeding Myc heterozygous males (Myc) with C57BL/6NCrl females purchased from Charles River. These crosses produce the two genotypes (Mycand Myc) and the two sexes at the expected Mendelian ratio (1:1).

Procedures Performed on Live Animals

Danilovich et al., 1999 Danilovich N.

Wernsing D.

Coschigano K.T.

Kopchick J.J.

Bartke A. Deficits in female reproductive function in GH-R-KO mice; role of IGF-I. To score vaginal patency, female mice were examined daily from weaning (21 days) until vaginal opening was observed (). Mice were held with the back feet against the cage wall and tail stretched up, and a sterile pipet tip was used to gently probe for vaginal opening.

Reproductive longevity and output were assessed for both males and females. For males, young (12 weeks old) males of either genotype were housed with Myc+/+ females from our colony or purchased from Charles River (C57BL/6NCrl). Males and females (1 pair per cage) were co-housed continuously for the entire experiment. Litters were removed when ready for weaning and either used in other experiments or euthanized. Females were replaced approximately every 6 months with younger animals. If a male did not produce any litters for 3 months, he was given a proven breeder female. Litters were counted by date of birth of the pups and the number of pups was tracked for each litter. In cases where a litter was born but did not survive, we counted the dead pups if possible; if we were not able to count the pups, the number of pups was entered as “0.” Males were left in with females until they ceased reproducing (did not produce a litter with a proven breeder for 8 weeks) or until their death. For females, young (12 weeks old) females of either genotype were continuously mated with one Myc+/+ male from our colony or purchased from Charles River (C57BL/6NCrl). If a female did not produce a litter within 3 months of being mated, a proven breeder male (taken from our colony) was put in to replace the original male. Litters were tracked the same way as with the males. A female was considered no longer fertile if she had not produced a litter for 3 months.

For rotarod tests, the apparatus used was from MedAssociates (St. Alban, VT). A mouse was placed on the cylinder (rod) with high walls on both sides that forced the mouse to ambulate on the rod. The rod was at a height of 35 cm above the base, and was accelerated continuously from 4 to 40 revolutions per minute in 5 min. The trial was terminated at 6 min or when the animal fell from the rod. Falls were detected by a photobeam detector. Each mouse received three trials per day for three consecutive days.

To assess metabolic rates, O 2 consumption and CO 2 production were measured using a fully automated comprehensive lab animal monitoring system (CLAMS, Oxymax Open Circuit Calorimeter, Columbus Instruments) equipped with 4 mouse chambers. Animals were transferred to the chambers (one per chamber) and allowed to acclimate for one day. Measurements were taken every 10 min continuously for a period of 3 days (72 hr). Food and water were provided ad libitum, and the animals were maintained on a normal light-dark cycle.

Jhuang et al., 2010 Jhuang H.

Garrote E.

Mutch J.

Yu X.

Khilnani V.

Poggio T.

Steele A.D.

Serre T. Automated home-cage behavioural phenotyping of mice. Spontaneous home cage activity was monitored using fully automated computer vision analysis of continuous video recordings (). Mice were housed in individual cages under standard conditions, and recorded continuously for a period of 5 days. The analysis software uses a database of annotated behaviors and machine learning to distinguish and score 9 distinct behaviors, which can be grouped into 4 categories: sleeping, micromovements (sniffing, looking from side to side, moving bedding around, etc.), consumption (drinking, eating, eating from hands), and active behaviors (grooming, hanging, rearing, walking). Micromovements would not be scored as activity in a CLAMS apparatus since the movements are too small to break any of the monitoring beams, however, the animals are not motionless and hence are not sleeping or resting. The output is the number of video frames an animal is engaged in any one of the behaviors at any point in time. Statistical significance was computed using a general linear model (GLM) repeated-measures ANOVA to assess main effects. Post hoc analysis was performed using independent sample t tests.

For body temperature measurements an implantable temperature transponder system (IPTT-300, Bio Medic Data Systems) was used. The transponders were implanted under the skin on the back of the animals between the shoulder blades using the provided instrument. The animals were allowed to heal and recover for a minimum of one week after the implantation before measurements were commenced. The transponders were read by a hand-held IPTT 7009 reader without disturbing the animals (taking them out of their cages; when needed animals were individually housed). The transponders were kept in the animals until they died or were euthanized for other reasons (such as collection of tissue specimens).

Wound healing experiments were performed by introducing 6 mm diameter full thickness skin punches (using a disposable skin biopsy punch, Jorgensen Labs). One wound was made on each animal, located on the back between the shoulder blades. To perform the procedure, animals were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg). The general area to be punched (1-2 cm2) was first depilated using depilation wax (GiGi Sugarbare). This was done so that the hair would not interfere with making a clean punch, and also to allow accurate visual monitoring of the healing process. The depilation was done under isofluorane anesthesia one day before the punch. The wax was warmed up, spread on the area to be depilated, allowed to cool for 10 min, and then peeled off. After the punch the animals were allowed to regain consciousness and were then housed under standard housing conditions. To monitor the healing, mice were photographed every second day at various magnifications using a dissecting microscope. An internal calibration ruler was included in each image. Animals were lightly anesthetized with ketamine/xylazine for each imaging session. This was necessary to minimize their movement so that high quality close-up images could be acquired. Images were processed using ImageJ software.