Microarray Analysis of Apoptosis-associated Pathways in Connexin30-deficient Cochlea Wenxue Tang 1,...
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Microarray Analysis of Apoptosis-associated Pathways in Connexin30-deficient Cochlea Wenxue Tang 1 , Yu-Hua Li 2 , Yan Qu 1 , Nicholas Wagar 2 and Xi Lin 1 Departments of Otolaryngology, School of Medicine 1 , Yerkes Microarray Core, Yerkes National Primate Research Center 2 , Emory University, Atlanta Molecula r Diagnost ics #14 Apoptosis Cells in Cochlear Epithelium of Cx30 -/ Mice Conclusions Introduction Connexins (Cxs) are a family of membrane proteins that form intercellular channels known as gap junctions (GJs). At least four different Cxs have been reported to be present in the mammalian inner ear, including Cx26 and Cx30. Mutation in Cx genes is one of the most common forms of human genetic defects resulting in hearing losses in millions of patients with either autosomal dominant or recessive deafness. The Cx30 deficient (Cx30 -/- ) or knockout mice have been created by deletion of the Cx30 coding region, to be used as a model to study the molecular mechanism of the hearing loss (Teunner et al., 2003). These Cx30 -/- mice have hearing loss accompanying hair cell degeneration and apoptosis in cochlear epithelium. Recently, we genetically modified a mouse strain in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome (BAC) incorporated in the Cx30 -/- mouse genome (named BACCx26Cx30 -/- mice). The over-expression from extra alleles of Cx26 gene completely prevented the hair cell degeneration and apoptosis of supporting cells and the hearing sensitivity of BACCx26Cx30 -/- mice was completely restored to normal. To understand and identify molecular events responsible to the cochlear death and hearing loss, changes in gene expression in the cochlea of Cx30 Knockout (KO) mice versus wild-type (WT) mice was analyzed using ColdLink mouse whole genome bioarrays compared to those in Cx26 rescue (RES) mice versus wild-type mice. Genes that showed greater or less than 1.5-fold changes were selected for Ingenuity Pathway Analysis. Preliminary analyses showed that the most affected genes included genes in apoptosis, cell death receptor and calcium signaling pathways, etc. TUNEL staining also demonstrated the apoptosis and gradual loss of hair cells in the cochlea of Cx30 -/- mice. This microarray-based analysis gives candidate genes, some of them novel, which may be responsible for cochlear cell deaths in Cx30 -/ mice. The apoptosis-associated key components will be validated by using quantitative real-time PCR and Western blot. Materials and Methods DNA microarray and Ingenuity pathway analysis Total RNA was isolated from cochleae of adult wild-type, Cx30 knockout and BAC Cx26 rescue mice (n=3) using the PicoPureTM RNA Isolation Kit (Arcturus Bioscience, Mt View, CA). The integrity and concentration of total RNA were examined using Agilent Bioanalyzer 2100 with RNA 6000 Naco LabChips Kit (Agilent, Palo Alto, CA). The 0.2 µg of total RNA was analyzed on ColdLink mouse whole genome bioarrays which represents 36227 transcripts (GE Healthcare, St. Giles, UK) following Codelink system manual . Briefly, biotin-labeled cRNA was synthesized by in vitro transcription in the presence of biotinylated nucleotides (Biotin-11-UTP, PerkinElmer, Boston, MA). 10 g of the cRNA was used for the hybridization on the boarray. Microarray slides were scanned using a GenePix 4000B scanner (Molecular Devices, Palo Alto, CA) with these parameters: wavelength=635 nm , PMT=600V, 100% of laser power, pixel size=5 m. The gene features were extracted from the images using CodeLink Expression Analysis Software (version 4.1, GE Healthcare), and then the signal intensities were imported into GeneSpring 7.3 (Silicon Genetics, Redwood City, CA) for analyses. The data were normalized to the 50th percentile of the measurements taken from the chip to reduce chip-wide variations in intensity. Each gene was normalized to the average measurement of the gene throughout the experiment to enable comparison of relative changes in gene expression levels between different conditions. Data filtration was performed based on flags present or marginal in at least three of the nine samples, and a corresponding gene list based on those flags was generated. The gene expressions of connexin family members and glucose transporter family members in WT mice were analyzed separately. Genes that showed greater or less than 1.5-fold changes in KO or Res versus WT were selected for Ingenuity Pathway Analysis. Detection of apoptosis cells by TUNEL staining Adult Cx30 KO and WT mice were deep anesthetized by 4% chloral hydrate. After transcardiary perfusion with 4% paraformaldyhyde in phosphate-buffered saline solution (pH 7.4), animals were decapitated and inner ears were dissected out. Cleavage of genomic DNA in apoptotic cells were detected on 5 mm section by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), using an in situ cell death detection kit (Roche, Germany) according to the manufacturer's instruction. Briefly, Tissue is rinsed in 0.1 M PBS, pH 7.4, for twice and then incubated in TUNEL reaction mixture for 1 hr at 37oC. The tissue was then rinsed in 0.1 M PBS three times for 5 min and samples can be directly be analyzed under a fluorescence microscope. Positive control are treated with DNAse 1 (3u/ml) for 10 min at 25oC and negative controls for the TUNEL procedure are treated in the same manner as the test samples except that the TdT enzyme is omitted from the reaction mixtures in this kits and is replaced with label solution. Immunofluorescent staining of Cxs in the cochlea Cochlea collected from WT mice were used in this study. Details of immunolabeling protocol is given previously (Zhang et al., 2005; Sun et al., 2005). Cochlear tissues were further fixed in the 4% paraformaldehyde on ice for 1 hour. Decalcification was carried out in 10% EDTA solution for 72 hours. The dissected inner ear tissues were immersed in 20% sucrose phosphate-buffered saline solution (pH 7.4) overnight followed by being embedded in OCT overnight. The tissues were snap frozen in liquid nitrogen and 7μm sections were cut by Cryostat (2800 Frigocut E, Cambridge Instruments GmbH, West Germany). Cochlear sections were rinsed by 0.1% Triton in phosphate-buffered saline solution (pH 7.4,PBST) for 30 minutes. Then sections were blocked with 5% goat serum in PBST at room temperature for 1 hour. The antibodies against Glucose transporters 1and 10 (rabbit IgG, 1:200, Zymed Laboratories, Southern San Francisco ,CA) were used to label the cochlear sections at 4℃ overnight. After washed, the sections were incubated with secondary antibodies (1:400-1:800) at room temperature for 1 hour. Cy2- or cy3-conjugated secondary antibodies (Jackson ImmunoResearch Lab,West Grove,PA) were used to visualize the binding of the primary antibodies. The slides were mounted with fluoromount G (Electron Microscopy Sciences, Hatsfield PA ) and examined under a fluorescence microscope. Fig. 1. TUNEL staining shows a significant increased apoptosis cells in the Cx30 -/- mouse cochlea (B) and comparing to the WT mouse cochlea (A). DNA Microarray and Ingenuity Pathway Analysis A B Fig. 2. High-density oligonucleotide microarray and ingenuity pathway analysis shows the upregulated genes, which were selected with 1.5-fold changes from Cx30 KO vs WT and Cx26 RES vs WT, were identified that are involved in several signaling pathways (A) and functional networks (B). The genes that are involved in cell death and apoptosis signaling pathway (A and B) are also associated with the pathways of B cell receptor signaling, calcium signal, integrin signaling, JAK/STAT signaling, ERK/MAPK signaling SAPK/JNK signaling and Wnt/-catenin signaling (C and D). The further analysis indicated the key genes that are expressed with greater or less than 1.5-fold changes in the cochlear epithelium of Cx30 KO mice (C ) compared to those of Cx 26 RES mice (D). The Expression of Glucose Transporters in Mouse Cochlea A B Fig. 4. Immunofluorescence staining shows the expressions of glucose transporters 1 (Glu T1, A) and 10 (Glu T10, B) in wild-type mouse inner cells. Acknowledgement We thank GE Healthcare and Yerkes Microarray Core ( www.microarray.emory.edu ) for sponsoring a CodeLink Microarray competition program. 0 5 10 15 20 25 GluT1 GluT2 GluT3 GluT4 GluT5 GluT6 GluT8 GluT9 GluT10 G luTs expression in the M ouse C ochlea SEM-W T Mean-W T b Fig. 3. Microarray analysis of gene expression of the Connexin family members (a) and Glucose Transporter members (b) in the cochlea of adult wide-type mice. 0 5 10 15 20 25 30 C x43 C x46 C x37 C x45 cx50 C x36 C x47 C x32 C x26 C x31 C x30.3 C x30 C x30.1 pannexin 1 pannexin 3 C onnexins Expression in the M ouse C ochlea (M icroarray) SEM -W T M ean-W T a The Gene Expression of Connexins in Mouse Cochlea The Gene Expression of Glucose Transporters in Mouse Cochlea C D Introduction Connexins (Cxs) are a family of membrane proteins that form intercellular channels known as gap junctions (GJs). At least four different Cxs have been reported to be present in the mammalian inner ear, including Cx26 and Cx30. Mutation in Cx genes is one of the most common forms of human genetic defects resulting in hearing losses in millions of patients with either autosomal dominant or recessive deafness. The Cx30 deficient (Cx30 -/- ) or knockout mice have been created by deletion of the Cx30 coding region, to be used as a model to study the molecular mechanism of the hearing loss (Teunner et al., 2003). These Cx30 -/- mice have hearing loss accompanying hair cell degeneration and apoptosis in cochlear epithelium. Recently, we genetically modified a mouse strain in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome (BAC) incorporated in the Cx30 -/- mouse genome (named BACCx26Cx30 -/- mice). The over-expression from extra alleles of Cx26 gene completely prevented the hair cell degeneration and apoptosis of supporting cells and the hearing sensitivity of BACCx26Cx30 -/- mice was completely restored to normal. To understand and identify molecular events responsible to the cochlear death and hearing loss, changes in gene expression in the cochlea of Cx30 Knockout (KO) mice versus wild-type (WT) mice was analyzed using ColdLink mouse whole genome bioarrays compared to those in Cx26 rescue (RES) mice versus wild-type mice. Genes that showed greater or less than 1.5-fold changes were selected for Ingenuity Pathway Analysis. Preliminary analyses showed that the most affected genes included genes in apoptosis, cell death receptor and calcium signaling pathways, etc. TUNEL staining also demonstrated the apoptosis and gradual loss of hair cells in the cochlea of Cx30 -/- mice. This microarray-based analysis gives candidate genes, some of them novel, which may be responsible for cochlear cell deaths in Cx30 -/ mice. The apoptosis-associated key components will be validated by using quantitative real-time PCR and Western blot. Introduction Connexins (Cxs) are a family of membrane proteins that form intercellular channels known as gap junctions (GJs). At least four different Cxs have been reported to be present in the mammalian inner ear, including Cx26 and Cx30. Mutation in Cx genes is one of the most common forms of human genetic defects resulting in hearing losses in millions of patients with either autosomal dominant or recessive deafness. The Cx30 deficient (Cx30 -/- ) or knockout mice have been created by deletion of the Cx30 coding region, to be used as a model to study the molecular mechanism of the hearing loss (Teunner et al., 2003). These Cx30 -/- mice have hearing loss accompanying hair cell degeneration and apoptosis in cochlear epithelium. Recently, we genetically modified a mouse strain in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome (BAC) incorporated in the Cx30 -/- mouse genome (named BACCx26Cx30 -/- mice). The over-expression from extra alleles of Cx26 gene completely prevented the hair cell degeneration and apoptosis of supporting cells and the hearing sensitivity of BACCx26Cx30 -/- mice was completely restored to normal. To understand and identify molecular events responsible to the cochlear death and hearing loss, changes in gene expression in the cochlea of Cx30 Knockout (KO) mice versus wild-type (WT) mice was analyzed using ColdLink mouse whole genome bioarrays compared to those in Cx26 rescue (RES) mice versus wild-type mice. Genes that showed greater or less than 1.5-fold changes were selected for Ingenuity Pathway Analysis. Preliminary analyses showed that the most affected genes included genes in apoptosis, cell death receptor and calcium signaling pathways, etc. TUNEL staining also demonstrated the apoptosis and gradual loss of hair cells in the cochlea of Cx30 -/- mice. This microarray-based analysis gives candidate genes, some of them novel, which may be responsible for cochlear cell deaths in Cx30 -/ mice. The apoptosis-associated key components will be validated by using quantitative real-time PCR and Western blot. 1. DNA microarray and Ingenuity pathway analysis are powerful tools for global analysis of molecular events and identification of new genes. 2. In this study, we have identified the pathways and their key components that are associated with the molecular events of cell death and apoptosis that may be responsible for the hearing loss of Cx30 knockout mice. 3. We also found two novel genes, GluT 10 and Pannexin 1, that are expressed in the mouse cochlea.
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Transcript of Microarray Analysis of Apoptosis-associated Pathways in Connexin30-deficient Cochlea Wenxue Tang 1,...
Microarray Analysis of Apoptosis-associated Pathways in Connexin30-deficient CochleaWenxue Tang1, Yu-Hua Li2, Yan Qu1, Nicholas Wagar2 and Xi Lin1
Departments of Otolaryngology, School of Medicine1, Yerkes Microarray Core, Yerkes National Primate Research Center2, Emory University, Atlanta
Molecular Diagnostics #14
Apoptosis Cells in Cochlear Epithelium of Cx30-/ Mice Conclusions
Introduction Connexins (Cxs) are a family of membrane proteins that form intercellular channels known as gap
junctions (GJs). At least four different Cxs have been reported to be present in the mammalian inner ear, including Cx26 and Cx30. Mutation in Cx genes is one of the most common forms of human genetic defects resulting in hearing losses in millions of patients with either autosomal dominant or recessive deafness. The Cx30 deficient (Cx30-/- ) or knockout mice have been created by deletion of the Cx30 coding region, to be used as a model to study the molecular mechanism of the hearing loss (Teunner et al., 2003). These Cx30-/- mice have hearing loss accompanying hair cell degeneration and apoptosis in cochlear epithelium. Recently, we genetically modified a mouse strain in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome (BAC) incorporated in the Cx30-/- mouse genome (named BACCx26Cx30-/- mice). The over-expression from extra alleles of Cx26 gene completely prevented the hair cell degeneration and apoptosis of supporting cells and the hearing sensitivity of BACCx26Cx30-/- mice was completely restored to normal. To understand and identify molecular events responsible to the cochlear death and hearing loss, changes in gene expression in the cochlea of Cx30 Knockout (KO) mice versus wild-type (WT) mice was analyzed using ColdLink mouse whole genome bioarrays compared to those in Cx26 rescue (RES) mice versus wild-type mice. Genes that showed greater or less than 1.5-fold changes were selected for Ingenuity Pathway Analysis. Preliminary analyses showed that the most affected genes included genes in apoptosis, cell death receptor and calcium signaling pathways, etc. TUNEL staining also demonstrated the apoptosis and gradual loss of hair cells in the cochlea of Cx30-/- mice. This microarray-based analysis gives candidate genes, some of them novel, which may be responsible for cochlear cell deaths in Cx30-/ mice. The apoptosis-associated key components will be validated by using quantitative real-time PCR and Western blot.
Materials and MethodsDNA microarray and Ingenuity pathway analysis
Total RNA was isolated from cochleae of adult wild-type, Cx30 knockout and BACCx26 rescue mice (n=3) using the PicoPureTM RNA Isolation Kit (Arcturus Bioscience, Mt View, CA). The integrity and concentration of total RNA were examined using Agilent Bioanalyzer 2100 with RNA 6000 Naco LabChips Kit (Agilent, Palo Alto, CA). The 0.2 µg of total RNA was analyzed on ColdLink mouse whole genome bioarrays which represents 36227 transcripts (GE Healthcare, St. Giles, UK) following Codelink system manual . Briefly, biotin-labeled cRNA was synthesized by in vitro transcription in the presence of biotinylated nucleotides (Biotin-11-UTP, PerkinElmer, Boston, MA). 10 g of the cRNA was used for the hybridization on the boarray. Microarray slides were scanned using a GenePix 4000B scanner (Molecular Devices, Palo Alto, CA) with these parameters: wavelength=635 nm , PMT=600V, 100% of laser power, pixel size=5 m. The gene features were extracted from the images using CodeLink Expression Analysis Software (version 4.1, GE Healthcare), and then the signal intensities were imported into GeneSpring 7.3 (Silicon Genetics, Redwood City, CA) for analyses. The data were normalized to the 50th percentile of the measurements taken from the chip to reduce chip-wide variations in intensity. Each gene was normalized to the average measurement of the gene throughout the experiment to enable comparison of relative changes in gene expression levels between different conditions. Data filtration was performed based on flags present or marginal in at least three of the nine samples, and a corresponding gene list based on those flags was generated. The gene expressions of connexin family members and glucose transporter family members in WT mice were analyzed separately. Genes that showed greater or less than 1.5-fold changes in KO or Res versus WT were selected for Ingenuity Pathway Analysis. Detection of apoptosis cells by TUNEL staining
Adult Cx30 KO and WT mice were deep anesthetized by 4% chloral hydrate. After transcardiary perfusion with 4% paraformaldyhyde in phosphate-buffered saline solution (pH 7.4), animals were decapitated and inner ears were dissected out. Cleavage of genomic DNA in apoptotic cells were detected on 5 mm section by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), using an in situ cell death detection kit (Roche, Germany) according to the manufacturer's instruction. Briefly, Tissue is rinsed in 0.1 M PBS, pH 7.4, for twice and then incubated in TUNEL reaction mixture for 1 hr at 37oC. The tissue was then rinsed in 0.1 M PBS three times for 5 min and samples can be directly be analyzed under a fluorescence microscope. Positive control are treated with DNAse 1 (3u/ml) for 10 min at 25oC and negative controls for the TUNEL procedure are treated in the same manner as the test samples except that the TdT enzyme is omitted from the reaction mixtures in this kits and is replaced with label solution.Immunofluorescent staining of Cxs in the cochlea
Cochlea collected from WT mice were used in this study. Details of immunolabeling protocol is given previously (Zhang et al., 2005; Sun et al., 2005). Cochlear tissues were further fixed in the 4% paraformaldehyde on ice for 1 hour. Decalcification was carried out in 10% EDTA solution for 72 hours. The dissected inner ear tissues were immersed in 20% sucrose phosphate-buffered saline solution (pH 7.4) overnight followed by being embedded in OCT overnight. The tissues were snap frozen in liquid nitrogen and 7μm sections were cut by Cryostat (2800 Frigocut E, Cambridge Instruments GmbH, West Germany). Cochlear sections were rinsed by 0.1% Triton in phosphate-buffered saline solution (pH 7.4,PBST) for 30 minutes. Then sections were blocked with 5% goat serum in PBST at room temperature for 1 hour. The antibodies against Glucose transporters 1and 10 (rabbit IgG, 1:200, Zymed Laboratories, Southern San Francisco ,CA) were used to label the cochlear sections at 4 overnight. After washed, the sections were incubated with secondary antibodies (1:400-1:800) at room ℃temperature for 1 hour. Cy2- or cy3-conjugated secondary antibodies (Jackson ImmunoResearch Lab,West Grove,PA) were used to visualize the binding of the primary antibodies. The slides were mounted with fluoromount G (Electron Microscopy Sciences, Hatsfield PA ) and examined under a fluorescence microscope.
Fig. 1. TUNEL staining shows a significant increased apoptosis cells in the Cx30-/- mouse cochlea (B) and comparing to the WT mouse cochlea (A).
DNA Microarray and Ingenuity Pathway Analysis
A
B
Fig. 2. High-density oligonucleotide microarray and ingenuity pathway analysis shows the upregulated genes, which were selected with 1.5-fold changes from Cx30 KO vs WT and Cx26 RES vs WT, were identified that are involved in several signaling pathways (A) and functional networks (B). The genes that are involved in cell death and apoptosis signaling pathway (A and B) are also associated with the pathways of B cell receptor signaling, calcium signal, integrin signaling, JAK/STAT signaling, ERK/MAPK signaling SAPK/JNK signaling and Wnt/-catenin signaling (C and D). The further analysis indicated the key
genes that are expressed with greater or less than 1.5-fold changes in the cochlear epithelium of Cx30 KO mice (C ) compared to those of Cx 26 RES mice (D).
The Expression of Glucose Transporters in Mouse Cochlea
A
B
Fig. 4. Immunofluorescence staining shows the expressions of glucose transporters 1 (Glu T1, A) and 10 (Glu T10, B) in wild-type mouse inner cells.
AcknowledgementWe thank GE Healthcare and Yerkes Microarray Core (www.microarray.emory.edu) for sponsoring a CodeLink Microarray competition program.
Connexins Expression in the Mouse Cochlea
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GluT1 GluT2 GluT3 GluT4 GluT5 GluT6 GluT8 GluT9 GluT10
GluTs expression in the Mouse Cochlea
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Mean-WT
b
Fig. 3. Microarray analysis of gene expression of the Connexin family members (a) and Glucose Transporter members (b) in the cochlea of adult wide-type mice.
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Cx43 Cx46 Cx37 Cx45 cx50 Cx36 Cx47 Cx32 Cx26 Cx31 Cx30.3 Cx30 Cx30.1 pannexin1
pannexin3
Connexins Expression in the Mouse Cochlea (Microarray)
SEM-WT
Mean-WT
a The Gene Expression of Connexins in Mouse Cochlea
The Gene Expression of Glucose Transporters in Mouse Cochlea
C D
Introduction Connexins (Cxs) are a family of membrane proteins that form intercellular channels known as gap
junctions (GJs). At least four different Cxs have been reported to be present in the mammalian inner ear, including Cx26 and Cx30. Mutation in Cx genes is one of the most common forms of human genetic defects resulting in hearing losses in millions of patients with either autosomal dominant or recessive deafness. The Cx30 deficient (Cx30-/- ) or knockout mice have been created by deletion of the Cx30 coding region, to be used as a model to study the molecular mechanism of the hearing loss (Teunner et al., 2003). These Cx30-/- mice have hearing loss accompanying hair cell degeneration and apoptosis in cochlear epithelium. Recently, we genetically modified a mouse strain in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome (BAC) incorporated in the Cx30-/- mouse genome (named BACCx26Cx30-/- mice). The over-expression from extra alleles of Cx26 gene completely prevented the hair cell degeneration and apoptosis of supporting cells and the hearing sensitivity of BACCx26Cx30-/- mice was completely restored to normal. To understand and identify molecular events responsible to the cochlear death and hearing loss, changes in gene expression in the cochlea of Cx30 Knockout (KO) mice versus wild-type (WT) mice was analyzed using ColdLink mouse whole genome bioarrays compared to those in Cx26 rescue (RES) mice versus wild-type mice. Genes that showed greater or less than 1.5-fold changes were selected for Ingenuity Pathway Analysis. Preliminary analyses showed that the most affected genes included genes in apoptosis, cell death receptor and calcium signaling pathways, etc. TUNEL staining also demonstrated the apoptosis and gradual loss of hair cells in the cochlea of Cx30-/- mice. This microarray-based analysis gives candidate genes, some of them novel, which may be responsible for cochlear cell deaths in Cx30-/ mice. The apoptosis-associated key components will be validated by using quantitative real-time PCR and Western blot.
Introduction Connexins (Cxs) are a family of membrane proteins that form intercellular channels known as gap
junctions (GJs). At least four different Cxs have been reported to be present in the mammalian inner ear, including Cx26 and Cx30. Mutation in Cx genes is one of the most common forms of human genetic defects resulting in hearing losses in millions of patients with either autosomal dominant or recessive deafness. The Cx30 deficient (Cx30-/- ) or knockout mice have been created by deletion of the Cx30 coding region, to be used as a model to study the molecular mechanism of the hearing loss (Teunner et al., 2003). These Cx30-/- mice have hearing loss accompanying hair cell degeneration and apoptosis in cochlear epithelium. Recently, we genetically modified a mouse strain in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome (BAC) incorporated in the Cx30-/- mouse genome (named BACCx26Cx30-/- mice). The over-expression from extra alleles of Cx26 gene completely prevented the hair cell degeneration and apoptosis of supporting cells and the hearing sensitivity of BACCx26Cx30-/- mice was completely restored to normal. To understand and identify molecular events responsible to the cochlear death and hearing loss, changes in gene expression in the cochlea of Cx30 Knockout (KO) mice versus wild-type (WT) mice was analyzed using ColdLink mouse whole genome bioarrays compared to those in Cx26 rescue (RES) mice versus wild-type mice. Genes that showed greater or less than 1.5-fold changes were selected for Ingenuity Pathway Analysis. Preliminary analyses showed that the most affected genes included genes in apoptosis, cell death receptor and calcium signaling pathways, etc. TUNEL staining also demonstrated the apoptosis and gradual loss of hair cells in the cochlea of Cx30-/- mice. This microarray-based analysis gives candidate genes, some of them novel, which may be responsible for cochlear cell deaths in Cx30-/ mice. The apoptosis-associated key components will be validated by using quantitative real-time PCR and Western blot.
1. DNA microarray and Ingenuity pathway analysis are powerful tools for global analysis of molecular events and identification of new genes.
2. In this study, we have identified the pathways and their key components that are associated with the molecular events of cell death and apoptosis that may be responsible for the hearing loss of Cx30 knockout mice.
3. We also found two novel genes, GluT 10 and Pannexin 1, that are expressed in the
mouse cochlea.
