The Mueller Laboratory

Mouse Mutants with Auditory Impairment: Protocols

All procedures have been published and are cited in the bibliography.

ENU Mutagenesis
Male C57Bl/6J mice are injected intraperitoneally in three consecutive weeks with ENU at 85 mg/kg. Following a period of sterility (70-100 days) that is caused by ENU cytotoxicity, the mice are bred with wild-type C57Bl/6J females to produce G1 mice. To obtain the widest variety of mutations, no more than five G1 males are produces from each founder. Male G1 mice are bred with C57Bl/6J wild-type mice to obtain the G2 generation. G2 females will be backcrossed with the parental G1 males to obtain a minimum of 20 offspring in the G3 generation for phenotypic characterization.

Phenotypic Screening
Initiall, mice were screened at 2 months of age for defects in the auditory startle reflex. More recently, we screen mice directly by measurements of the auditory brainstem response (ABR). Mice at 4 months of age are anethesized and placed on a heating pad in a sound-attenuating chamber. The stimulus presentation, ABR acquisition, equipment controls and data management are coordinated using a Tucker Davis Technologies, Inc. workstation (System III) and BioSig software. Filters are set to exclude signals above 3000 Hz and below 300 Hz. Analysis time is set at 10 msec. Needle electrodes are inserted subcutaneously at the vertex and pinna, with a
ground near the tail. A loudspeaker is inserted into the ear. Stimulus presentation consists of click stimuli (2-8 kHz, 12.1 clicks per second, duration 0.1 ms, alternating polarity) at 90 dB followed by decreasing the volume initially in 10 dB and subsequently in 5 dB steps. The number of acquisition trials is set at 512 averages with 256 rarefaction and 256 condensation stimuli. For each mouse, auditory thresholds are determined for both ears and compared to exclude errors from aberrantly placed electrodes or loudspeaker. The mean ± standard deviation are
determined and a Student's t-test is performed. Mice that show no response to up to 90 dB are considered deaf. Those with elevated thresholds are considered hearing impaired. Mice with elevated thresholds are re-tested at 6 and 10 months of age to evaluate progressive hearing loss.

Mice also evaluate vestibular function by analyzing circling behavior in the open field test. We use open field chambers (17" x 17" x 12"H; Med Associates, Inc.) that are equipped with infrared beams with 1" spacing, dividing the chamber into 256 quadrants. Open field behavior is monitored using video equipment and computer aided data analysis (Coulborne Instruments). Mice are placed in the same starting quadrant and allowed to roam the enclosure for 10 minutes. We record the total distance traveled to identify hyperactive mutants. To measure circling, we quantify
the number of small clockwise/counterclockwise rotations (MED; Rotational Analysis software). The baseline values have been determined (C57Bl/6J mice activity: 3855 ± 117 cm / 10 minutes; rotational movement of a radius of 2.75 cm: 10 ± 5 / minute [mean ± standard deviation]; 129S1SVIm/J, BALB/cByJ mice and intercrosses with C57Bl/6J in F1, F2, and F3 deviate less then 2 standard deviations from the mean in C57Bl/6J). Mice that score positive are retested. Mice are considered affected when they score 3 or more standard deviations above the mean.
Mice are also evaluated in the force swim test. Mice are placed in a glass cylinder (18 cm diameter) filled with water (25∞C). Over a period of 2 minutes, movements are registered and recorded with video equipment. Mice that have difficulty to swim are removed. The time to rescue is determined and defects in swimming behavior such as the ability to maintain upright posture are recorded.

Affected mice are subsequently also evaluated in a number of other tests (anxiety, learning and memory, fear conditioning) to identify those mice with general nervous system defects.

Positional Cloning
Affected G3 mice are crossed with 129S1/SvImJ and BALB/cByJ mice to obtain F1 offsping. For dominant traints, F1 offspring are crossed with 129S1/SvImJ and BALB/cByJ mice to obtain informative meiotic recombination events in F2. For recessive traits, F1 offspring are backcrossed to G3 parents. 10 affected and 10 unaffected siblings are identified by phenotyping. Our published data show that this number of mice is sufficient to carry out a genome scan. SNP assays are run for ~400 markers, which detects linkage anywhere in the genome. The assays are performed
following our published procedures using the Sequenome MassARRAY system. When
necessary, high resolution mapping is used to reduce the genomic interval, if necessary with additional affected/unaffected animals.

Subsequently, exons and exon-intron boundaries are sequenced. Primers for PCR amplification of genomic DNA and sequencing are designed using the Primer 3 software (MIT). Sequencing reactions are carried out with DNA samples from at least 2 affected and 2 unaffected mice. Subsequently, the presence of the mutation is confirmed in other available DNA samples from the same family (in different generations) and in cDNA generated by RT-PCR from RNA isolated from inner ears.

Only mutations that strictly segregate with the phenotype will be considered candidate mutations for causing the phenotype.