**4. Identification of the genetic modifiers in mice**

We have described in the previous section that several mouse inbred strains developed ARHL by genetic background effects. In this section, we introduce approaches to identify genetic modifiers and susceptibility loci of hearing loss underlying the genetic backgrounds.

#### **4.1. Classical forward genetics approach**

by interaction between *Cdh23c.753G>A* and *Csp.His55Asn* mutations. The CDH23 is a member of the calcium-dependent cell-cell adhesion and tip link component [21, 29, 30]. In contrast, CS is the first enzyme of the tricarboxylic acid cycle, generating citrate and free coenzyme A [31]. Therefore, both proteins seem to contribute different functions in the inner ear for hearing, suggesting that A/J mice develop early onset hearing loss from additive effects of different functional mutations, as described in the previous section (**Figure 1B**). In addition, A/J mice carried a single adenine insertion in the mitochondrial tRNA-Arg gene (*mt-TrArg*) [32]. CS is transported into the mitochondrial matrix and plays an important role in condensing mitochondrial acetyl-coenzyme A and oxaloacetate for transporting of acetyl-coenzyme A from the mitochondrial matrix to the cytosol [31], suggesting that mitochondrial dysfunction by epistasis (**Figure 1B**) between *Csp.His55Asn* and *mt-TrArg* mutations is accelerated in ARHL of A/J mice. The DBA/2J mice also have a strain-specific mutation (p.Arg109His) in the fascin 2 gene (*Fscn2*) [33]. DBA/2J mice exhibit progressive shortening of the stereocilia, and this phenotype only develops with homozygosity of both the *Cdh23c.753G>A* and *Fscn2p.Arg109His* mutations [34]. FSCN2 is an actin crosslinking protein and localizes along the length of stereocilia at especially high concentration around the stereocilia tips [33, 34]. Although the pathological mechanisms in the genetic interaction between the *Cdh23c.753G>A* and *Fscn2p.Arg109His* mutations are widely unknown, the degeneration of stereocilia in DBA/2J mice may be explained by epistasis. Moreover, the other QTLs related to ARHL were detected in DBA/2J mice. The QTLs, *Ahl9* [35] and *Chr5* QTL [25, 36], are likely to contribute to frequency-specific ARHL. Although the causative genes and mutations are still unknown, these QTLs lead to severe hearing loss by

**Figure 3.** Comparison of hearing levels among the mouse inbred strains. The means (circles, squares, diamonds, and upper and lower triangles) and standard deviations (error bars) of ABR thresholds for 4, 8, 16, and 32 kHz sound stimuli are shown for MSM/Ms, C3H/HeN, DBA/2J, C57BL/6J, and A/J mice at 4 months of age. The graph was created by using

data from our previous studies [23–25].

8 An Excursus into Hearing Loss

The forward genetics approach is phenotype-driven, with a foundation that associates the detection of chromosomal location with phenotype by linkage analysis. The start of the experiments included production of chromosomal recombinants, F<sup>2</sup> and N2 mice, by crossing between the susceptible and resistant strains in phenotype (**Figure 4A**). The linkage analysis is based on meiotic recombination events that occur in sperm and egg precursor cells of F<sup>1</sup> hybrid, which have heterozygous chromosomes derived from both the susceptible and resistant strains. Accordingly, the F<sup>2</sup> and N2 progenies were produced by intercrossing between F<sup>1</sup> mice and backcrossing of F1 mice to one parental strain, respectively, and inherited chromosomes that underwent recombination events. The recombinant region on the chromosomes was detected by using a genetic marker, such as microsatellites and SNPs, which recognized genetic polymorphisms compared to parental strains. Finally, the phenotypes of F<sup>2</sup> and N2 mice were investigated to determine whether there is linkage with the recombinant regions of F2 and N2 mice. This approach has been a powerful and productive method to identify QTL-associated ARHL. Johnson et al. [37] detected the first QTL *ahl* locus for ARHL that displayed a *Cdh23c.753G>A* mutation by using N2 backcross mice between ARHL-susceptible C57BL/6J and -resistant CAST/Ei.

As mentioned earlier, there are many modifiers in the genome of inbred strains. To evaluate the effect of a single QTL identified in the mapping by avoiding the effects from other modifiers, congenic mice have become a powerful tool. Congenic mice are defined as having part of the mutation or a chromosomal segment from one inbred genetic background (donor) to another (host) [38] (**Figure 4B**). The creation of congenic mice is based on backcrossing the system for at least seven times. Although this process is long, the resolution of the phenotype greatly improves when compared with F<sup>2</sup> and N2 mice. The most successful example of this strategy is the study of *moth1* locus [39, 40]. The *tubby* mice, which are a mutant of tubby bipartite transcription factor gene (*Tub*), exhibit severe hearing loss caused by cochlear degeneration in C57BL/6J background [39]. However, some F<sup>2</sup> mice produced by intercrossing with AKR/J and CAST/Ei showed normal hearing. Ikeda et al. [39] mapped the modifier, *moth1*, via linkage analysis using both F2 mice and confirmed the locus by creating congenic mice. This study led to the successful identification of the association of the modification of hearing loss with a strain-specific mutation in microtubule-associated protein 1 gene (*Map1a*), which was the first elucidated causative gene caused by the background effect in hearing [40].

introduce the public genetic reference populations of mice. These populations have several

Effects of Genetic Background on Susceptibility and the Acceleration of Hearing Loss in Mice

Recombinant inbred strain (RIS) panel is a genetic reference population of mice and can serve

ing progeny, at least 20 generations later, is fully inbred and displays a mosaic of parental genomes (**Figure 5A**) [41, 42]. RIS panel has several advantages for QTL mapping; if the genotyping is performed once, it does not require genotyping in each individual and is available in public databases; individual, environmental, and measurement variability can be reduced; it has greater mapping resolution because the breakpoints in the genome are denser than those

and N2

RIS panels have been successfully applied to several QTL mappings for ARHL. The strategy includes only evaluating the hearing abilities of each individual RIS panel performed by

**Figure 5.** Schematic representation of the virtual genomic structures of the recombinant inbred strains (RIS) (A), consomic strain (CSS) (B), and hybrid mouse diversity panel (HMDP) (C) for QTL analysis and genome-wide association study (GWAS). The rectangles and circles represent chromosomes and mitochondrial DNA, respectively. Each strain-derived

mice [41].

mice until the result-

11

http://dx.doi.org/10.5772/intechopen.72469

as a powerful tool for QTL mapping. It is produced by mating sibling F2

advantages owing to established QTL mapping.

*4.2.1. Recombinant inbred strain (RIS)*

that occur in any one meiosis, such as F<sup>2</sup>

chromosomal region is distinguished by a different color.

**Figure 4.** Schematic representation of the virtual genomic structures of experimental cross (F2 and N2 ) (A) for genetic mapping and congenic mouse (B). The rectangles and circles represent chromosome and mitochondrial DNA, respectively. The different strain-derived chromosomal regions are distinguished by light and dark gray colors. Bidirectional arrows indicate quantitative trait locus (QTL) regions.

#### **4.2. Forward genetics approaches using genetic reference populations of mice**

Currently, we believe that a classical forward genetics approach using F2 , N2 , and congenic mice is useful to identify the modifiers from genetic background in mice. However, productions of the F<sup>2</sup> , N2 , and congenic mice consume great amount of time and costs for breeding. Large numbers of mice are required to increase mapping resolution. Moreover, the F<sup>2</sup> and N2 mice must be used for genome-wide genotyping because their genome architectures are uniquely mixed between parental chromosomes. Therefore, the genotyping cost is enormous. Here, we introduce the public genetic reference populations of mice. These populations have several advantages owing to established QTL mapping.

#### *4.2.1. Recombinant inbred strain (RIS)*

**4.2. Forward genetics approaches using genetic reference populations of mice**

**Figure 4.** Schematic representation of the virtual genomic structures of experimental cross (F2

numbers of mice are required to increase mapping resolution. Moreover, the F<sup>2</sup>

is useful to identify the modifiers from genetic background in mice. However, productions

mapping and congenic mouse (B). The rectangles and circles represent chromosome and mitochondrial DNA, respectively. The different strain-derived chromosomal regions are distinguished by light and dark gray colors.

must be used for genome-wide genotyping because their genome architectures are uniquely mixed between parental chromosomes. Therefore, the genotyping cost is enormous. Here, we

, and congenic mice consume great amount of time and costs for breeding. Large

, N2

and N2

, and congenic mice

) (A) for genetic

and N2

mice

Currently, we believe that a classical forward genetics approach using F2

Bidirectional arrows indicate quantitative trait locus (QTL) regions.

of the F<sup>2</sup>

, N2

10 An Excursus into Hearing Loss

Recombinant inbred strain (RIS) panel is a genetic reference population of mice and can serve as a powerful tool for QTL mapping. It is produced by mating sibling F2 mice until the resulting progeny, at least 20 generations later, is fully inbred and displays a mosaic of parental genomes (**Figure 5A**) [41, 42]. RIS panel has several advantages for QTL mapping; if the genotyping is performed once, it does not require genotyping in each individual and is available in public databases; individual, environmental, and measurement variability can be reduced; it has greater mapping resolution because the breakpoints in the genome are denser than those that occur in any one meiosis, such as F<sup>2</sup> and N2 mice [41].

RIS panels have been successfully applied to several QTL mappings for ARHL. The strategy includes only evaluating the hearing abilities of each individual RIS panel performed by

**Figure 5.** Schematic representation of the virtual genomic structures of the recombinant inbred strains (RIS) (A), consomic strain (CSS) (B), and hybrid mouse diversity panel (HMDP) (C) for QTL analysis and genome-wide association study (GWAS). The rectangles and circles represent chromosomes and mitochondrial DNA, respectively. Each strain-derived chromosomal region is distinguished by a different color.

measurements of ABR thresholds and then performing QTL linkage analysis using WebQTL [43], which collects genotypes of microsatellite and SNPs in each RIS panel. By this strategy, the QTLs *ahl4* (*Csp.His55Asn*) [44], *ahl8* (*Fscn2p.Arg109His*) [45], *Ahl9* [35], and *Snhl* [18] were mapped using the AXB/BXA [46], BXD [47], BXD and LXS [48], and RIS panels, respectively (**Table 1**). The other RIS panels, such as CXB [49], BXH [50], and SMXA [51], have been established by sibling mating between several inbred strains and will become useful resources to identify new loci associated with ARHL and NIHL.

been mapped to a single chromosome by ABR measurements in CSSs of partial and full sets (**Table 1**). Moreover, congenic mice for QTL mapping can be easily and quickly produced

(**Figure 5B**). The genotyping is only required in one chromosome for progeny. By this strategy, we have previously mapped the *Ahl3* [54] and a modifier [24] responsible for the accel-

In addition, CSS may be used to study epistasis in detail. An example is *Ahl3* [54], which was mapped to chromosome 17 using C57BL/6J-Chr17MSM/Ms CSS. The donor strain, MSM/Ms, is an inbred derived from the Japanese wild mouse, *Mus musculus molossinus*. By introgression of the genomic segment including *Ahl3*, the ARHL of C57BL/6J was dramatically suppressed despite having the homozygous *Cdh23c.753A* allele in the genome. Although we predicted that *Ahl3* is the resistance allele for ARHL in MSM/Ms mice, the resistant effects of *Ahl3* were not detected in the other mapping system of the C57BL/6J and MSM/Ms strain [24]. The genetic divergence between C57BL/6J and MSM/Ms is extremely high [55]; therefore, the resistant effect of *Ahl3* was caused by incompatibility by chromosomal substitution. We detected a similar situation in CSSs of another chromosome [54] (Yasuda et al. unpublished data). There is some possibility that the incompatibility is caused by a *cis*/*trans* change via chromosomal substitution; therefore, the analysis of CSS may allow for the study of cross talk between genes on different chromosomes, namely, *cis*- and *trans*-regulation of the variation of gene expression in hearing research.

Although the classical genetic crosses, RIS and CSS, are powerful tools to identify modifiers in genetic backgrounds, the phenotypic and genetic variations are low because of the inclusion of only two parental strains. Moreover, the low genetic variation is not suitable for GWAS. The hybrid mouse diversity panel (HMDP) was developed to increase the statistical power and resolution of the classical QTL mapping [56]. The HMDP consists of 30 classical inbred strains and four set of RISs (AXB/BXA, BXD, BXH, and CXB) (**Figure 5C** and **Table 1**), which are genotyped with 140,000 SNPs [57]. By using the HMDP, a high statistical power and high resolution of QTL mapping were provided from the RISs and classical inbred strains, respectively.

HMDP contributed to the identification of the gene and locus associated with NIHL. Lavinsky et al. [17] investigated the noise susceptibility in five-week female mice in 64 strains selected from HMDP. The noise susceptibilities of strains varied widely, and GWAS analysis picked up five quantitative trait nucleotides (QTNs) with statistically significant p values (p < 4.1E−06) [17]. Consequently, candidate genes were screened by expression QTL (eQTL) analysis after which NADPH oxidase-3 gene (*Nox3*) was selected as a candidate (**Table 1**). Moreover, the study identified that the *Nox3* KO mice showed noise sensitivity [17]. This is the first study to have reported the NIHL-related gene. Till date, noise sensitivity of 100 stock strains from HMDP has been reported [58], and phenotype data have also been collected continuously [59]. Additionally, a

QTN at chromosome 6 associated with noise susceptibility was detected (**Table 1**) [60].

QTL analysis using inbred strains is limited to the phenotypes and alleles associated with ARHL and NIHL. Although the genotyping is required, outbred stock (OS) is a colony with

eration of ARHL of heterozygotes of Jackson shaker mice (*Ush1gjs*) (**Table 1**).

and backcrossing (CSS × host strain) F1

Effects of Genetic Background on Susceptibility and the Acceleration of Hearing Loss in Mice

× host strain

13

http://dx.doi.org/10.5772/intechopen.72469

by intercrossing (CSS × host strain) F1

*4.2.3. Hybrid mouse diversity panel (HMDP)*

*4.2.4. Outbred stock (OS)*

#### *4.2.2. Consomic strains (CSS)*

Consomic strains (CSS), also called chromosome substitution strains, are combined genomes of two founder inbred strains that have a substitution of one whole chromosome pair from the donor strain into the genetic background of the host strain (**Figure 5B**) [52, 53]. The productive strategy is the same with congenic mice. Usually, a full set of CSSs will consist of 22 strains, which includes 19 pairs of autosomal chromosomes, X and Y sex chromosomes, and a mitochondrial genome, although the introgression into the host background of a whole chromosome from the donor is difficult in some cases [53]. The main advantage of CSS is mapping of the phenotype to single chromosomes. The *Ahl3* [54] and *ahl4* [44] loci have


\* Inbred long sleep (ILS) and inbred short sleep (ISS) mouse strains, which were derived from a multi-generation cross of eight inbred strains.

\*\*Modifier of *js* (tentative symbol).

**Table 1.** Genetic reference population contributed for identification of the loci and SNPs associated with hearing loss underlying in genetic background of mice by QTL analysis and GWAS.

been mapped to a single chromosome by ABR measurements in CSSs of partial and full sets (**Table 1**). Moreover, congenic mice for QTL mapping can be easily and quickly produced by intercrossing (CSS × host strain) F1 and backcrossing (CSS × host strain) F1 × host strain (**Figure 5B**). The genotyping is only required in one chromosome for progeny. By this strategy, we have previously mapped the *Ahl3* [54] and a modifier [24] responsible for the acceleration of ARHL of heterozygotes of Jackson shaker mice (*Ush1gjs*) (**Table 1**).

In addition, CSS may be used to study epistasis in detail. An example is *Ahl3* [54], which was mapped to chromosome 17 using C57BL/6J-Chr17MSM/Ms CSS. The donor strain, MSM/Ms, is an inbred derived from the Japanese wild mouse, *Mus musculus molossinus*. By introgression of the genomic segment including *Ahl3*, the ARHL of C57BL/6J was dramatically suppressed despite having the homozygous *Cdh23c.753A* allele in the genome. Although we predicted that *Ahl3* is the resistance allele for ARHL in MSM/Ms mice, the resistant effects of *Ahl3* were not detected in the other mapping system of the C57BL/6J and MSM/Ms strain [24]. The genetic divergence between C57BL/6J and MSM/Ms is extremely high [55]; therefore, the resistant effect of *Ahl3* was caused by incompatibility by chromosomal substitution. We detected a similar situation in CSSs of another chromosome [54] (Yasuda et al. unpublished data). There is some possibility that the incompatibility is caused by a *cis*/*trans* change via chromosomal substitution; therefore, the analysis of CSS may allow for the study of cross talk between genes on different chromosomes, namely, *cis*- and *trans*-regulation of the variation of gene expression in hearing research.

#### *4.2.3. Hybrid mouse diversity panel (HMDP)*

measurements of ABR thresholds and then performing QTL linkage analysis using WebQTL [43], which collects genotypes of microsatellite and SNPs in each RIS panel. By this strategy, the QTLs *ahl4* (*Csp.His55Asn*) [44], *ahl8* (*Fscn2p.Arg109His*) [45], *Ahl9* [35], and *Snhl* [18] were mapped using the AXB/BXA [46], BXD [47], BXD and LXS [48], and RIS panels, respectively (**Table 1**). The other RIS panels, such as CXB [49], BXH [50], and SMXA [51], have been established by sibling mating between several inbred strains and will become useful resources to identify

Consomic strains (CSS), also called chromosome substitution strains, are combined genomes of two founder inbred strains that have a substitution of one whole chromosome pair from the donor strain into the genetic background of the host strain (**Figure 5B**) [52, 53]. The productive strategy is the same with congenic mice. Usually, a full set of CSSs will consist of 22 strains, which includes 19 pairs of autosomal chromosomes, X and Y sex chromosomes, and a mitochondrial genome, although the introgression into the host background of a whole chromosome from the donor is difficult in some cases [53]. The main advantage of CSS is mapping of the phenotype to single chromosomes. The *Ahl3* [54] and *ahl4* [44] loci have

**Population Panel Origin QTL or QTN Gene**

Consomic strain (CSS) C57BL/6J-Chr#A/J [52] Host: C57BL/6J, donor: A/J *ahl4* [44] *Cs* [28]

MSM/Ms

strains

Outbred stock (OS) Black Swiss [62] NIH Swiss, C57BL/6J *Ahl5* [63] *Gipc3* [14]

Inbred long sleep (ILS) and inbred short sleep (ISS) mouse strains, which were derived from a multi-generation cross

**Table 1.** Genetic reference population contributed for identification of the loci and SNPs associated with hearing loss

30 classic inbred strains and 70 recombinant inbred

NIH Swiss [64] NIH GP colony *Hfhl1* [15] Unknown

C57BL/6J-Chr#MSM/Ms [53] Host: C57BL/6J, donor:

BXA [46] (C57BL/6J × A/J) F1

LXS [48] \*

Hybrid mouse diversity panel (HMDP) [56]

underlying in genetic background of mice by QTL analysis and GWAS.

AXB [46] (A/J × C57BL/6J) F1 *ahl4* [44] *Cs* [28]

BXD [47] (C57BL/6J × DBA/2J) F1 *ahl8* [45] *Fscn2* [34]

*Ahl9* [35] Unknown

*Ahl3* [54] Unknown

*mjs*\*\* [24] *Cdh23* [24]

rs33652818 [17] *Nox3* [17]

rs37517079 [60] Unknown

*Ahl6* [63] Unknown

*Hfhl2* [15] Unknown *Hfhl3* [16] Unknown

(ILS × ISS) F1 *Snhl* [18] Unknown

new loci associated with ARHL and NIHL.

*4.2.2. Consomic strains (CSS)*

12 An Excursus into Hearing Loss

Recombinant inbred strain (RIS)

Inbred strain population

\*

of eight inbred strains.

\*\*Modifier of *js* (tentative symbol).

Although the classical genetic crosses, RIS and CSS, are powerful tools to identify modifiers in genetic backgrounds, the phenotypic and genetic variations are low because of the inclusion of only two parental strains. Moreover, the low genetic variation is not suitable for GWAS. The hybrid mouse diversity panel (HMDP) was developed to increase the statistical power and resolution of the classical QTL mapping [56]. The HMDP consists of 30 classical inbred strains and four set of RISs (AXB/BXA, BXD, BXH, and CXB) (**Figure 5C** and **Table 1**), which are genotyped with 140,000 SNPs [57]. By using the HMDP, a high statistical power and high resolution of QTL mapping were provided from the RISs and classical inbred strains, respectively.

HMDP contributed to the identification of the gene and locus associated with NIHL. Lavinsky et al. [17] investigated the noise susceptibility in five-week female mice in 64 strains selected from HMDP. The noise susceptibilities of strains varied widely, and GWAS analysis picked up five quantitative trait nucleotides (QTNs) with statistically significant p values (p < 4.1E−06) [17]. Consequently, candidate genes were screened by expression QTL (eQTL) analysis after which NADPH oxidase-3 gene (*Nox3*) was selected as a candidate (**Table 1**). Moreover, the study identified that the *Nox3* KO mice showed noise sensitivity [17]. This is the first study to have reported the NIHL-related gene. Till date, noise sensitivity of 100 stock strains from HMDP has been reported [58], and phenotype data have also been collected continuously [59]. Additionally, a QTN at chromosome 6 associated with noise susceptibility was detected (**Table 1**) [60].

#### *4.2.4. Outbred stock (OS)*

QTL analysis using inbred strains is limited to the phenotypes and alleles associated with ARHL and NIHL. Although the genotyping is required, outbred stock (OS) is a colony with maintained phenotypic and genetic diversity kept in laboratory settings and thus exhibits a high degree of both genetic and phenotypic diversity, allowing high-resolution genetic mapping for a wide variety of traits by crossing with another population [42, 61].

In a study of ARHL, Black Swiss [62], which is derived from NIH Swiss outbred stock and C57BL/6J, was first used in OS for QTL linkage mapping. Drayton and Noben-Trauth [62] performed QTL linkage mapping using [(Black Swiss × CAST/Ei) F1 × Black Swiss] N2 backcross mice and mapped two QTLs: *Ahl5* and *Ahl6*. Subsequently, the responsible mutation for *Ahl5* was detected in the GIPC PDZ domain containing family member 3 gene (*Gipc3*), which is associated with audiogenic seizures and sensorineural hearing loss in mice and humans [14]. The *Gipc3p.Gly115Arg* mutation was absent in inbred strain, indicating that OS is a powerful tool to identify new ARHL-related genes. Moreover, NIH Swiss mice [63] also contributed to the detection of three loci (*Hfhl1*–*3*) associated with high-frequency hearing loss (HFHL) (**Table 1**) [15, 16].

Heterogeneous stock (HS) and diversity outcross stock (DO) could be considered variants of OS and display a similar advantage with OS for QTL mapping [42, 61]. These populations are available and probably will contribute to the identification of QTL and genes responsible for ARHL and NIHL.

#### **4.3. Elucidation of genetic background effects by using reverse genetics approaches**

When the candidate gene and mutation are detected by forward genetics approach, the next step is elucidation of the real causative gene and mutation. The reverse genetics technique is immensely helpful with this elucidation. An approach is production and phenotypic analysis of KO mice of candidate gene *Nox3* as mentioned earlier [17]. However, rescue of phenotype is required for full proof.

Transgenic expression of bacterial artificial chromosome (BAC) clones in mice is commonly used for *in vivo* complementation tests. The test BAC contains wild-type allele and is simple since BAC contains the wild-type allele to be injected into the susceptible strain with the candidate mutation. The *Fscn2p.Arg109His* mutation of DBA/2J is confirmed by this complementation test [33]. The disadvantage of BAC transgenesis is that genes of large sizes exceeding the BAC clone (average size between 120 and 250 kb) cannot be rescued. Moreover, the rescue of the phenotype caused by dominant-negative and gain-of-function mutations is difficult. In addition, the strains of available BAC libraries are limited [64, 65].

An advanced approach is rescue by the knock-in (KI) method mediated by the CRISPR/Cas9 genome editing system. This system can efficiently and quickly repair the candidate mutation of the KI donor oligonucleotide containing the wild-type allele via a homology-directed repair (HDR) [66]. We previously reported utility of this method [24]. C57BL/6J-*Ush1gjs*/+ heterozygous mice exhibit severe early onset ARHL caused by progressive degeneration in the stereocilia of outer hair cells. We mapped a locus associated with early onset ARHL of *Ush1gjs*/+ mice in an interval of chromosome 10 that harbors the *Cdh23c.753G>A* mutation, which is also responsible for ARHL of C57BL/6J mice as mentioned above. We injected Cas9 mRNA, single guide RNA (sgRNA) and a donor oligonucleotide that contained *Cdh23c.753G* (**Figure 6A**) to produce KI mice. In KI mice, early onset ARHL and stereocilia degeneration were completely rescued (**Figure 6B**). This is the first report that confirms the phenotypic effect of modifiers at the mutation level in hearing research.

**Figure 6.** Phenotypic rescue of hearing loss in mice caused by genetic background effect using the CRISPR/Cas9 mediated KI method. (A) Schematic representation of the *Cdh23c.753A>G* KI using double-strand break within the vicinity of the *Cdh23c.753A* site of C57BL/6J mice by CRISPR/Cas9 and homology-directed repair of single-stranded donor oligonucleotide. The donor oligonucleotide was designed to include c.753A>G with a synonymous blocking of the c.714C>T mutation to avoid cleavage by Cas9 [24]. (B) Comparison of the hearing phenotypes among the C57BL/6J (top), C57BL/6J-*Ush1gjs*/+ (middle) and -*Ush1gjs*/+ *Cdh23c.753A/G* (bottom) mice. Illustrations of left panels represent combination of the *Cdh23c.753A>G* (★>☆) on chromosome 10 and *Ush1gjs* (●) on chromosome 11. Middle and right panels show ABR

Effects of Genetic Background on Susceptibility and the Acceleration of Hearing Loss in Mice

http://dx.doi.org/10.5772/intechopen.72469

15

waveforms and stereocilia phenotypes, respectively, in each mouse.

Effects of Genetic Background on Susceptibility and the Acceleration of Hearing Loss in Mice http://dx.doi.org/10.5772/intechopen.72469 15

maintained phenotypic and genetic diversity kept in laboratory settings and thus exhibits a high degree of both genetic and phenotypic diversity, allowing high-resolution genetic map-

In a study of ARHL, Black Swiss [62], which is derived from NIH Swiss outbred stock and C57BL/6J, was first used in OS for QTL linkage mapping. Drayton and Noben-Trauth [62] per-

mice and mapped two QTLs: *Ahl5* and *Ahl6*. Subsequently, the responsible mutation for *Ahl5* was detected in the GIPC PDZ domain containing family member 3 gene (*Gipc3*), which is associated with audiogenic seizures and sensorineural hearing loss in mice and humans [14]. The *Gipc3p.Gly115Arg* mutation was absent in inbred strain, indicating that OS is a powerful tool to identify new ARHL-related genes. Moreover, NIH Swiss mice [63] also contributed to the detection of three loci (*Hfhl1*–*3*) associated with high-frequency hearing loss (HFHL) (**Table 1**) [15, 16].

Heterogeneous stock (HS) and diversity outcross stock (DO) could be considered variants of OS and display a similar advantage with OS for QTL mapping [42, 61]. These populations are available and probably will contribute to the identification of QTL and genes responsible for

When the candidate gene and mutation are detected by forward genetics approach, the next step is elucidation of the real causative gene and mutation. The reverse genetics technique is immensely helpful with this elucidation. An approach is production and phenotypic analysis of KO mice of candidate gene *Nox3* as mentioned earlier [17]. However, rescue of phenotype

Transgenic expression of bacterial artificial chromosome (BAC) clones in mice is commonly used for *in vivo* complementation tests. The test BAC contains wild-type allele and is simple since BAC contains the wild-type allele to be injected into the susceptible strain with the candidate mutation. The *Fscn2p.Arg109His* mutation of DBA/2J is confirmed by this complementation test [33]. The disadvantage of BAC transgenesis is that genes of large sizes exceeding the BAC clone (average size between 120 and 250 kb) cannot be rescued. Moreover, the rescue of the phenotype caused by dominant-negative and gain-of-function mutations is difficult. In addi-

An advanced approach is rescue by the knock-in (KI) method mediated by the CRISPR/Cas9 genome editing system. This system can efficiently and quickly repair the candidate mutation of the KI donor oligonucleotide containing the wild-type allele via a homology-directed repair (HDR) [66]. We previously reported utility of this method [24]. C57BL/6J-*Ush1gjs*/+ heterozygous mice exhibit severe early onset ARHL caused by progressive degeneration in the stereocilia of outer hair cells. We mapped a locus associated with early onset ARHL of *Ush1gjs*/+ mice in an interval of chromosome 10 that harbors the *Cdh23c.753G>A* mutation, which is also responsible for ARHL of C57BL/6J mice as mentioned above. We injected Cas9 mRNA, single guide RNA (sgRNA) and a donor oligonucleotide that contained *Cdh23c.753G* (**Figure 6A**) to produce KI mice. In KI mice, early onset ARHL and stereocilia degeneration were completely rescued (**Figure 6B**). This is the first report that confirms the phenotypic effect of modifiers at the mutation level in

**4.3. Elucidation of genetic background effects by using reverse genetics approaches**

× Black Swiss] N2

backcross

ping for a wide variety of traits by crossing with another population [42, 61].

formed QTL linkage mapping using [(Black Swiss × CAST/Ei) F1

tion, the strains of available BAC libraries are limited [64, 65].

ARHL and NIHL.

14 An Excursus into Hearing Loss

is required for full proof.

hearing research.

**Figure 6.** Phenotypic rescue of hearing loss in mice caused by genetic background effect using the CRISPR/Cas9 mediated KI method. (A) Schematic representation of the *Cdh23c.753A>G* KI using double-strand break within the vicinity of the *Cdh23c.753A* site of C57BL/6J mice by CRISPR/Cas9 and homology-directed repair of single-stranded donor oligonucleotide. The donor oligonucleotide was designed to include c.753A>G with a synonymous blocking of the c.714C>T mutation to avoid cleavage by Cas9 [24]. (B) Comparison of the hearing phenotypes among the C57BL/6J (top), C57BL/6J-*Ush1gjs*/+ (middle) and -*Ush1gjs*/+ *Cdh23c.753A/G* (bottom) mice. Illustrations of left panels represent combination of the *Cdh23c.753A>G* (★>☆) on chromosome 10 and *Ush1gjs* (●) on chromosome 11. Middle and right panels show ABR waveforms and stereocilia phenotypes, respectively, in each mouse.
