Novel PMCA2 Mutation and Hearing Loss in Mouse Figure 8. Immunolocalisation of PMCA2 in cochlear hair cells. Confocal imaging immunoassays of PMCA2 (green) in cochlear whole mounts from wild type (+/+), heterozygous (Obl/+), and homozygous (Obl/Obl) mice (red, actin). For details, see Materials and Methods. doi:10.1371/journal.pgen.1000238.g008 anaesthetised (urethane 2 mg/g) and subcutaneous needle elec- trodes inserted on the vertex (active), and over the left (reference) and right (ground) bullae. A calibrated sound system was used to deliver free-field click (0.01 ms duration) and tone pip (various frequencies from 3–42 kHz of 5 ms duration, 1 ms rise/fall time) stimuli at a range of intensity levels in 3 dB (or multiple) steps. Averaged responses to 512 stimuli, presented at 21.1 s21, were analysed and thresholds established as the lowest sound intensity giving a visually-detectable ABR response. ABR recordings were obtained from a total of 61 mice, 28 at P20 (+/+, n = 9; Obl/+, n = 19), 14 at P59–62 (+/+, n = 5; Obl/+, n = 9) and 19 at P89–91 (+/+, n = 5; Obl/+, n = 14). Mapping the Obl Mutation Obl/+ mutants on a C3HeB/FeJ background were outcrossed to C57BL/6J wild type females. Obl/+ F1 progeny were then backcrossed to +/+ animals from the original C3HeB/FeJ strain. Offspring from these backcross matings were examined at 2 months of age or older (as this is the age at which Obl/+ mice on the original genetic background show profound hearing loss) using the Preyer reflex. Tail and pinna tissue were collected for DNA preparation. A total of 255 backcross mice were analysed (129 Obl/+, 126 +/+). A genome-wide scan was conducted with 60 microsatellite markers approximately 25 cM apart, that had been shown to be polymorphic between C3HeB/FeJ and C57BL/6J inbred strains (Table S2). Additional microsatellite markers used for fine mapping of the Obl mutation were: D6Mit36; D6Mit104; D6Mit150; D6Mit115; D6Mit218; D6Mit254. PCR was performed using standard techniques. Mutation Screening and Confirmation Sequence analysis was performed on genomic DNA in Obl/+ mutants and littermate controls using primers designed to amplify the coding exons and splice sites of the Atp2b2 gene. The primer sequences are listed in Table S3. PCR was performed using standard techniques and products were cleaned using magnetic bead separation (Ampure) and sequenced using BigDye Termi- nator Cycle sequencing kit (Applied Biosystems). Sequence traces were analysed using Gap4 software [38]. To confirm the mutation identified in exon 15 and for genotyping of the colony, a PCR- based genotype test was designed. The 2630CRT missense mutation did not change a restriction enzyme recognition site, so primers were designed flanking the mutation site that would incorporate a StyI recognition site in the wildtype (C) allele, but not in the mutant (T) allele. Primers OblRTF (59-CTT CTT CTC CCT GCC ACT GTC GTA G) and OblRTR (59-CCA CCG AGA CAC CGG TCC CGG TTC) were used for PCR. The 111 bp PCR product was digested with StyI (New England Biolabs) which cuts the wildtype allele giving an 89 bp fragment while the mutant allele remains uncut. This genotyping tool was used to establish whether the sequence change in Obl DNA was a polymorphism in a total of 17 inbred strains: BALB/C, CBA/Ca, C3HeB/FeJ, DDY/Jc1, 129X1/SvJ, A/J, Bxd-1/Ty, C58/J, CE/ J, DA/HuSn, DBA/2J, FL/1Re, LP/J, NON/LtJ, RBG/Dn, St/ bJ and SWR/J. Cloning and Mutagenesis of pmRFP-PMCA2 w/a mRFP was amplified from pCDNA3.1/zeo-mRFP (kindly donated by Dr. M. Zaccolo, Padua, Italy) using the following primers, forward: 59-GCGCTAGCATGGCCTCCTCCGAG- GACGTCA-39 and reverse: 59-GCAGATCTGAGGCGCC- GGTGGAGTGGCGG-39, bearing restriction sites for NheI and BglII, respectively (in bold). The PCR product was then digested with NheI and BglII and inserted in pEGFP-c1 (Clontech, Palo Alto, CA) digested with NheI and BglII to create pmRFP-c1. PMCA2 w/a in pMM2 (kindly provided by Dr. Strehler, Rochester, MN) was excised PLoS Genetics | www.plosgenetics.org 9 October 2008 | Volume 4 | Issue 10 | e1000238