Human Molecular Genetics, 2001, Vol. 10, No. 15 1581-1589
© 2001 Oxford University Press
A specific promoter of the sensory cells of the inner ear defined by transgenesis
Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
Received April 14, 2001; Revised and Accepted May 30, 2001.
DDBJ/EMBL/GenBank accession no. AF384559.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To date, no promoter sequence specific to the inner ear sensory cells (hair cells) has been reported. In an effort to understand the molecular mechanisms that determine hair cell fate in the inner ear, and with the goal of developing a valuable tool for gene therapy and for the generation of conditional knockouts, we initiated a search for cis-acting DNA sequences that regulate the expression of the murine Myo7a and human MYO7A genes. These genes encode the unconventional myosin VIIA which is expressed in hair cells and in some other epithelial cells. We generated lines of transgenic mice expressing the green fluorescent protein (GFP ) reporter gene under the control of several 5'-truncated versions of the Myo7a/MYO7A promoter region and intron 1. We obtained transgenic mice with a GFP expression restricted to the hair cells of the inner ear, cochlea and vestibule. Successive deletions of the promoter allowed us to define a minimal sequence of 118 bp that is sufficient, in the presence of intron 1, to target the transgene expression to hair cells. In addition, the deletion of intron 1 from the transgenes abolished hair cell expression, thus indicating the presence of a strong enhancer in the intron. This is the first report of regulatory sequences sufficient to target the expression of a gene exclusively in sensory cells of the inner ear. It also opens up the possibility for the analysis of the hair cell transcriptome.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the past 10 years or so, there have been many studies aimed at characterizing the DNA sequences that control the specific expression of genes in the visual and olfactory sensory cells. In particular, the analysis of the genes encoding rhodopsin and olfactory marker protein (OMP) has led to the definition of short regulatory sequences that are sufficient to direct specific transgene expression in photoreceptors and olfactory receptor neurons, respectively (1–3). Not only have these studies provided promoters that permit the expression of any transgene in these sensory cells, but they have also led to the discovery of transcription factors that control cell type- and lineage-specific gene expression. For instance, based on promoter analysis, the transcription factor Crx has been identified as binding to a DNA site present in the promoters of numerous ‘retinal genes’ (4,5). Similarly, transcription factors of the Olf-1/EBF-like family have been recognized as being involved in gene expression restricted to the olfactory receptor neurons (6–8).
In contrast, our understanding of the functioning of the inner ear sensory cells in molecular terms is hampered by the lack of characterized promoter sequences driving specific expression in these cells. Such an analysis encounters two major difficulties. First, although some cell lines derived from the inner ear and expressing some hair cell markers have been generated (9), expression in these cell lines is generally low and heterogeneous. Secondly, only one gene with an expression restricted to hair cells has been reported so far, i.e. the gene encoding prestin (10). However, this gene is only transcribed in a subpopulation of the hair cells, namely the outer hair cells (OHCs) of the cochlea (the auditory organ). It is expressed neither in the inner hair cells (IHCs) of the cochlea, the genuine auditory sensory cells, nor in the hair cells of the vestibular end organs (balance organs). The deciphering of the molecular bases of hereditary hearing loss has identified a few genes which, in the inner ear, are exclusively expressed in hair cells, even though they are also expressed in various other organs (reviewed in refs 11 and 12). Among these is the myosin VIIA (Myo7A) gene, which is expressed in all the inner ear hair cells, both cochlear and vestibular (13,14). This gene is, in addition, transcribed in several epithelial cell types that possess microvilli, i.e. in the retinal pigment and olfactory epithelium, choroid plexus, liver, kidney, testis and small intestine (15).
With the twin goal of understanding the molecular bases of hair cell-specific gene expression and developing an important tool for the field of hearing research, we generated several constructs with various truncated versions of the Myo7a/MYO7A promoter region fused to a GFP reporter gene, and studied their expression in transgenic mice. We report here the characterization of regulatory sequences which are sufficient to target gene expression specifically to sensory cells of the inner ear.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cloning the putative promoter sequences of the murine and human myosin VIIA genes
As a first step towards the isolation of the 5' end of myosin VIIA transcripts, RACE-PCR experiments were performed on a cDNA library generated from murine vestibular end organs (16), using primers located in the Myo7a coding region (Materials and Methods) (17). This resulted in a PCR product which extended 281 bp upstream of the previously described cDNA sequence (17). A yeast artificial chromosome (YAC) clone (M3H11), with
200 kb of sequence upstream of the start codon and containing the totality of the Myo7a sequence, was isolated from a murine YAC library (18) (Materials and Methods). Comparison of the RACE-PCR product and YAC clone sequences allowed us to define an additional 237 bp untranslated exon, exon 1, located 1.5 kb upstream of the first coding exon (Fig. 1A). The same analysis was performed to define the 5'-genomic region of the human MYO7A gene. This allowed us to confirm the size of the previously reported non-coding MYO7A exon 1 (19), which is separated from exon 2 by a 2 kb intron (Fig. 1A). DNA fragments covering 6 kb upstream of the first exon and first intron were generated, both in mouse and man. Sequence analysis revealed the presence of the Capn5 gene encoding a calcium-dependent intracellular non-lysosomal protease (GenBank accession no. U85020), 2 kb upstream of the Myo7a exon 1. The orthologous human gene, CAPN5 (GenBank accession no. XM 006193), was detected 4.4 kb upstream of the MYO7A transcription initiation site (20). However, we observed, by northern blot analysis, an alternative 3' end for this gene with a polyadenylation signal 2109 bp upsteam of the MYO7A exon 1 (data not shown). This suggested that the promoter regions of Myo7a and MYO7A might not extend beyond 2 and 2.1 kb, respectively. In these 2 kb murine and human sequences, the Neural Network promoter prediction program (Materials and Methods) did not predict any promoter element. In particular, no consensus signal for transcription initiation such as a CAAT or a TATA box was present.
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In an attempt to detect cis-acting elements that regulate Myo7a/MYO7A expression, the mouse and human myosin VIIA genomic sequences encompassing the putative promoter region and the first intron were compared. The pair-wise identity scores were 45.8% over the whole putative promoter region and 53% over the whole of intron 1. Ten highly conserved islands were detected (Fig. 1A) using the DNA motif elicitation program DBA (21). Three of them were located in the promoter region and extended from nucleotide –644 to nucleotide +1 in the murine sequence. The seven others were scattered throughout intron 1, suggesting the presence of regulatory elements in this region. In addition, the promoter region and intron 1 sequences shared some degree of homology within each species. A 100 bp sequence present in the promoter region and intron 1 of MYO7A showed 90% homology, and a 66 bp sequence in the promoter region and intron 1 of Myo7a showed 80% homology (Fig. 1A). However, no homology could be detected between the human 100 bp and murine 66 bp fragments.
YAC transgenesis
The green fluorescent protein (GFP) reporter gene was introduced into YAC M3H11 at the Myo7a ATG initiation site by homologous recombination in exon 2 (Materials and Methods). Because of the presence of Capn5, the sequence upstream of exon 1 was reduced to 5 kb by homologous recombination (Materials and Methods) (22). This recombinant YAC, M3H11-R (Fig. 1B), was injected into pronuclei of fertilized B6/SJ mouse eggs, and offspring were analysed by PCR and Southern blot hybridization. Two mice (M3H11-R1 and -R2) were found to have integrated the transgene. Long range PCR analysis (Materials and Methods) showed that intact 5'-upstream sequences of the M3H11-R DNA were integrated into the genome of these two transgenic mice. The two recombinant lines showed the same GFP expression pattern. In the adult inner ear, both in the vestibule and in the cochlea, only the sensory hair cells were labelled. A systematic analysis of GFP expression in tissues of embryonic day (E)9.5, E16, P0-P2 and adult transgenic mice failed to detect fluorescence in any of the other organs and tissues where Myo7a is expressed, including the eye, liver, kidney, olfactory epithelium, testis and choroid plexus (15). No labelling was observed in the rest of the mouse either (data not shown).
The GFP expression pattern within the inner ear was investigated in the offspring, from E9.5 to adult stage. At E9.5, the expression of the transgene mimicked that of Myo7a in the epithelium of the otic vesicle and stato-acoustic ganglion (Fig. 2A and B) (15). At E16, myosin VIIA expression is restricted to the hair cells in the cochlea and the five vestibular end organs (utricle, saccule and the three ampullae) (14,23). On the same day, in the two transgenic lines, GFP expression was restricted to the sensory patches of the inner ear. A strong labelling of the vestibular hair cells was observed (Fig. 2C and D); the GFP staining was marginal in the cochlear hair cells. At P2, hair cells in the utricle (Fig. 3A), saccule and three ampullae (data not shown) expressed both Myo7a and GFP. In the cochlea, all IHCs expressed Myo7a and were GFP-positive (Fig. 4A). However, GFP was detected only in the OHCs of the apical half-turn of the cochlea, whereas all OHCs were labelled by anti-myosin VIIA antibodies. From P0 onward, the GFP expression pattern remained unchanged (Fig. 4B–D). From these results, we conclude that the 6.5 kb sequence upstream of Myo7a exon 2 is sufficient to direct specific expression of a transgene in inner ear sensory cells.
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Deletion mapping of the MYO7A/Myo7a promoter region
To define more precisely the DNA elements involved in the regulation of Myo7a transcription, transgenic mice expressing GFP under the control of several 5'-truncated versions of the promoter region were generated. A plasmid transgenesis approach based on sequence comparison between the murine and human MYO7A/Myo7a 5'-flanking regions was used. The following founder transgenic mice were obtained (Fig. 1B): four M(–2063 to +1776), one H(–2109 to +2370), two M(–1011 to +1776), one M(–507 to +1776) and four M(–118 to +1776). In this nomenclature, M and H refer to mouse and human sequences, respectively; the positions +1, +1777 and +2370 refer to the transcription initiation site and the translation start codons of Myo7a and MYO7A, respectively (Fig. 1B).
The construct M(–2063 to +1776) contained the sequence extending from the poly(A) signal of Capn5 to the Myo7a ATG initiation codon (GenBank accession no. AF384559). In three of the four M(–2063 to +1776) transgenic mice, GFP expression was restricted to the sensory cells of the inner ear in the postnatal stage. However, from one transgenic mouse to another, some differences were noted. Line 1 presented the more extensive staining of hair cells, with a spatiotemporal expression pattern of GFP during development similar to that of the M3H11-R lines. At P2, GFP was detected in the majority of the vestibular hair cells (Fig. 3B). It was also detected in IHCs but not in apical OHCs (data not shown). Line 2 had the same expression pattern as line 1 in the vestibule, but no staining was observed in the cochlea (data not shown). Line 3 expressed GFP only in a few hair cells of the vestibule. The last line (line 4) exhibited the same expression pattern as line 1 in the sensory cells, but GFP expression persisted in the spiral ganglion. This shows that the 2 kb promoter sequences (plus intron 1) are able to drive specific expression of a reporter gene in hair cells. The differences in the GFP expression pattern between lines are likely to result from differences in the site of insertion of the transgene.
In order to test whether the 5'-flanking region of human MYO7A is functional in the mouse, a human construct extending from the CAPN5 poly(A) region to the MYO7A start codon (Fig. 1B) was injected in fertilized mouse eggs. The single transgenic line H(–2109 to +2370) obtained showed the same GFP expression pattern as the M3H11-R lines (see above and Fig. 3C), i.e. GFP was expressed (i) in all the vestibular hair cells, (ii) in all IHCs but only in OHCs of the apical half-turn of the cochlea, and (iii) in no other organ. Therefore, the human MYO7A orthologous promoter was actually functional in the mouse. This result suggested that the regulatory sequences responsible for hair cell-specific expression belonged to the aforementioned islands conserved between mouse and human (Fig. 1A).
Further 5' deletions were generated. Two independent M(–1011 to +1776) lines were obtained. The first line presented a labelling of sensory and non-sensory cells in the cochlea and vestibule (data not shown). The second line presented a GFP staining exclusively in some vestibular hair cells (data not shown).
A single M(–507 to +1776) recombinant transgenic mouse line was obtained. In this mouse, GFP expression was still observed in the sensory hair cells. However, only a small number of vestibular hair cells expressed GFP (Fig. 3D) which appeared randomly distributed in the neuro-epithelium. This led us to suspect that genomic DNA surrounding the transgene may have partly silenced its expression.
In order to minimize the influence of the position effect in further constructs, the insulator sequence of the chicken ß-globin gene, ins (Materials and Methods), was integrated upstream of the regulatory sequence of the M(ins –118 to +1776) transgene. Insulators have the capacity to prevent DNA methylation and histone deacetylation of the transgene (24). The four M(ins –118 to +1776) mouse lines obtained expressed GFP in vestibular hair cells. In one of these lines (Fig. 3E), the expression observed in the vestibular apparatus showed close similarity to that observed with M3H11-R and M(–2063 to +1777) lines. In only two of these lines, fluorescent IHCs were detected. They were restricted to the cochlear apex (Fig. 3F). In addition, in both lines some cochlear fibrocytes were also fluorescent (Fig. 3F). In all lines, a few scattered positive cells could be detected. Taken together, these results demonstrate that the murine construct which contains the 118 bp promoter sequences immediately upstream from the transcription initiation site and intron 1 still has the ability to target the expression of a reporter gene to the sensory cells of the inner ear.
Testing the putative promoter activity of the first intron
Evidence of a promoter activity of the first intron of MYO7A was reported (25) using luciferase reporter assay in ARPE-19 cells, a retinal pigment epithelial cell line. A promoter region has been described 160 bp upstream of exon 2 (Fig 1A). Moreover, transient transfection experiments, performed using the M(ins +238 to +1776) and M(ins +1530 to +1776) constructs in UB/UE cells which exhibit hair cell-specific markers (9), indicated that both the entire intron 1 and the last 204 bp of intron 1 were able to initiate the transcription of GFP (data not shown). We thus decided to test by transgenesis whether these constructs could activate in vivo the transcription of GFP in the inner ear sensory cells. Insulator sequences were added to the intronic sequences. A single line was obtained with the construct containing the entire intron 1, M(ins +238 to +1776) (Fig. 1B); no GFP labelling was observed in the inner ear. Six independent transgenic lines were obtained with the truncated intron 1 construct, M(ins +1530 to +1776). None of these mice expressed GFP in hair cells. These results show that the murine intron 1 is not sufficient to trigger the expression of a transgene in the inner ear sensory cells in vivo.
Evidence for the presence of a hair cell-specific enhancer in the first intron
All the previously described transgenes which are expressed in the sensory cells contained intron 1; this led us to investigate its putative role in Myo7A regulation. For this purpose, the entire intron 1 was deleted in the transgene construct M(ins –2063 to +75) (Fig. 1B). Four founder transgenic mice were obtained. None of these lines expressed GFP in hair cells of the vestibule or cochlea. Only one line presented a GFP labelling which was located in numerous organs, including some that did not express endogenous myosin VIIA (data not shown). These experiments demonstrate that intron 1 is essential for Myo7a expression in the inner ear hair cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In an effort to understand the molecular mechanisms that determine sensory cell fate in the inner ear and with the goal of developing a valuable tool in the field of hearing research, we have begun to define the cis-acting DNA sequences that regulate Myo7a expression in the ear. The Myo7a and MYO7A promoters were analysed using a transgenic mouse model system; the GFP gene was used as reporter gene.
Myo7a/MYO7A sequences regulating sensory hair cell expression
In transgenic lines carrying the YAC transgene construct, GFP was exclusively detected at the postnatal stage in inner ear sensory cells, i.e. in all cochlear IHCs, in the OHCs located at the apex of the cochlea and in all vestibular hair cells. The absence of GFP in other organs that express Myo7a, such as kidney, small intestine, retina and testis, is unlikely to be due to a position effect at the transgene integration site, since the hair cell restricted pattern of expression was found in the two independent YAC transgenic mice that we obtained. The transgenic YAC contains the 5 kb sequence upstream of the Myo7a exon 1 (a non-coding exon) and the entire Myo7a genomic sequence. Therefore, we conclude that (i) these sequences are sufficient to control the hair cell expression of a gene and (ii) the locus control regions or enhancers responsible for Myo7a expression in other organs lie outside the YAC transgene sequences. Such a large dispersion of the regulatory elements that control gene expression in different tissues is not uncommon (26,27).
Sequences regulating MYO7A expression in the pigment cell epithelium of the retina have been analysed in the ARPE-19 cell line. A promoter region has been reported in the last 160 bp of the first intron (25). Therefore, although our primer extension experiments only detected a transcription initiation site upstream of the first exon in the mouse inner ear, we generated transgenic mice for constructs containing only the first intron or a part of the first intron. None of the seven mice obtained expressed GFP in either the inner ear or the retina, thus suggesting that an intronic transcription initiation site, if functional in vivo, is not used at least in mouse. The nested deletions of the promoter sequence then generated, showed that the 118 bp sequence located upstream of the transcription initiation site still directs, in the presence of intron 1, GFP expression in hair cells. Interestingly, this sequence presents 84% homology with the human sequence. However, a reduction in the number of GFP labelled hair cells, parallel to the reduction in the length of the promoter sequence, was observed. This indicates that enhancer sequences were deleted and/or that the mouse genomic DNA sequences surrounding the insertion of the transgene impair its full expression. Arguing in favour of the last proposal, the insertion of the insulator increases the number of fluorescent hair cells (Fig. 3D and E). The persistence of the hair cell GFP expression with only an 118 bp promoter sequence led us to investigate the contribution of intron 1. Whereas the construct containing a 2 kb promoter sequence plus intron 1, M(–2063 to +1776), had roughly the same expression pattern as the YAC transgene (M3H11) in the four independent transgenic mice generated, none of the four mice which carry the same transgene but without intron 1, M(ins –2063 to +75), expressed GFP in hair cells. This demonstrates the presence of a strong enhancer in intron 1. Comparative analysis of mouse and human intron 1 sequences revealed seven conserved islands dispersed throughout the intron, which are candidate enhancer elements (Fig. 1A).
The absence of the detection of GFP in OHCs of the cochlear basal and middle turns in transgenic mice expressing GFP in all IHCs and vestibular cells is intriguing. It is worth noting that apical OHCs share several features with IHCs, in particular they both receive mainly afferent innervation. We cannot exclude the possibility that either the GFP protein or mRNA has a lifespan shorter in the more basal OHCs and/or that the endogeneous transcription rate of the myosin VIIA gene is lower in these cells. However, this result may indicate that other regulatory elements, not included in the YAC construct, control myo7a transcription in the majority of OHCs.
Early expression of Myo7a
The expression of the YAC transgene in the inner ear as early as E9.5 strictly parallels the endogenous myosin VIIA gene expression described previously (15). Both the transgene and Myo7a are expressed in the neuroblasts of the stato-acoustic ganglion and in the facing otic vesicle subregion from which they delaminate. Moreover, the expression of both genes is downregulated from E12 onward in the stato-acoustic ganglion, whereas they persist in the individualized hair cells. Myosin VIIA is synthesized in the mitotic cells of the presumptive sensory areas of the otic vesicle before hair and supporting cells can be morphologically distinguished (15). At this time, Myo7a expression extending to the stato-acoustic ganglion may be indicative of a common progenitor for neuroblasts and cells of the sensory areas expressing Myo7a. Evidence for an early determination of sensory versus non-sensory areas in the rudimentary otocyst, as early as E9, has already been provided (28). BMP4, which encodes a member of the transforming growth factor-ß gene family, and Lunatic fringe (Fng), which encodes an extracellular protein that regulates Notch signalling, also show an expression restricted to the sensory area in the early mouse otocyst (29). The presence of myosin VIIA from E9 in mitotic progenitors of the inner ear sensory epithelia suggests an early role for this protein. We have shown that myosin VIIA is anchored at cell–cell junctions by a transmembrane protein vezatin, where it likely controls the tension between the junction and the actin cytoskeleton and is thus involved in the establishment of the planar polarity of these cells (30). Such a control of adhesion forces between adjacent cells is likely to be required during the early steps of sensory area development.
Among the large number of transcription factors expressed in the inner ear that could be involved in Myo7a expression, one has caught our attention, namely the basic-helix–loop–helix (bHLH) transcription factor Math1. The expression of Math1 is first detected at E12 in the sensory epithelia of the inner ear and its knockout leads to the complete absence of hair cells (31). The importance of this transcription factor in hair cell genesis is further supported by the observation that ectopic expression of Math1 in the inner ear induces the formation of extra hair cells that express myosin VIIA (32). Math1 has been shown to bind the E-box CAGGTG sequence with high affinity (33). Interestingly, this E-box is present in the Myo7a regulating sequences in the intron (conserved block 7, position 1058–1063). The absence of Math1 expression before E12 suggests that other transcription factor(s) control(s) the early expression of myosin VIIA. Therefore, the present observation led us to conclude that Myo7a gene expression proceeds in two steps: (i) from E9 to at least E12, its expression in mitotic cells of the sensory epithelia is likely to be independent of Math1; and (ii) from E12 onwards, its expression in post-mitotic cells is controlled by this proneural gene. The regulatory sequences defined in the present study open the way towards the identification of the transcription factors controlling early inner ear expression of the myosin VIIA gene.
Myo7a sequences controlling hair cell-specific expression exploited as a tool
At least three applications can be foreseen from this study on the Myo7a promoter. Firstly, the in vivo GFP labelling of inner ear sensory cells in the transgenic animals generated in this study should permit the purification of these cells by fluorescence activated cell sorter (FACS). In so doing, it will be possible to explore the transcriptome of the hair cells, which, due to the small number of these cells in the inner ear, have so far escaped characterization. Secondly, it can be used to target the expression of Cre recombinase in the hair cells. If the transient embryonic expression in the stato-acoustic ganglion is undesirable, inducible promoter sequences could be associated (34). In this regard, it is noteworthy that the promoter of the 
9 acetylcholine receptor gene, which has been proposed previously for a similar approach, drives expression of the reporter gene in the cochlear ganglion throughout life and in a variety of other tissues (35). Thirdly, the sequences defined here are short enough to be used to deliver a therapeutic gene product to sensory hair cells. The restoration of photoreceptor structure and function in retinal degeneration by gene therapy using the photoreceptor-specific Rhodopsin promoter has been reported in the mouse (36). Such an application could now be considered for some inner ear defects with this first description of a promoter specific to inner ear hair cells. Indeed, it drives specific expression of a transgene in IHCs, the genuine sensory cells, as well in OHCs (which amplify sound stimulation and tune the auditory response), situated at the apex of the cochlea, namely in the range of low frequencies that correspond to speech.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Identification of myosin VIIA sequences
The 5'-untranslated region of Myo7a was obtained by RACE-PCR amplification of cDNA sequences generated from vestibular mRNA (Marathon cDNA amplification kit, Clontech) using a first primer located in exon 3, 5'-CCTGACTTCAGGTCCATCCATACATAGTCC-3'; then a nested primer located in exon 2, 5'-ATAGTCCCCCTTCTGCAGGATAACCATGAC-3'.
The murine M3H11 YAC clone (ICRFy902H1193) was isolated by PCR screening of a mouse YAC library (18) using primers located in exon 3, 5'-GGGACTATGTATGGATGAC-3' and 5'-GTCTTCATCATCCACCACCTG-3'. This YAC was subcloned into phage 
GEM11 (Promega). The human 250B9 YAC clone was isolated from the CEPH library and subcloned (19). Murine and human DNA sequences extending 4 kb and encompassing exon 1, intron 1 and exon 2 (containing the translation initiation site) were determined (ABI 373 and 377 sequencers, Perkin-Elmer ABP division). Northern blot hybridizations were performed using a human multiple tissue northern blot (HMTN, Clontech) and a probe spanning the 3'-untranslated region of the CAPN5 gene.
Recombinant YAC construct
In a vector containing the enhanced green fluorescent protein gene (pEGFP1, Clontech), we inserted downstream of this gene the yeast histidinol-phosphate aminotransferase gene (HIS5). Upstream of EGFP, a 200 bp fragment of murine genomic sequence located upstream of the Myo7a ATG initiation site was subcloned. Downstream of HIS5, a 300 bp fragment located downstream of the Myo7A ATG initiation site was subcloned. This EGFP-HIS5 cassette was inserted by homologous recombination into YAC M3H11-H (37). The size of YAC M3H11-H was reduced by homologous recombination with a plasmid containing murine genomic sequences located 5 kb upstream of the ATG initiation site and a yeast auxotrophic marker, the -aminoadipate reductase gene (LYS2) (22). The YAC obtained, M3H11-R, was isolated by pulse-field gel electrophoresis and purified using agarase (New England Biolabs).
In transgenic mice, the integrity of the promoter was checked by PCR (Expand Long Template PCR system, Roche) using one primer located in exon 1 of Myo7a (position +238) (5'-GGGTTTTATCCACACCCTCCG-3') and the other located in the YAC arm (5'-CGCCCGATCTCAAGATTAC-3').
Recombinant plasmid constructs
Various sizes of promoter fragments of human or murine origin were amplified by PCR and inserted upstream of EGFP into the pEGFP1 vector.
The primers listed below were used to generate murine promoter sequences. The primers are named according to the position of their 5' end on the genomic sequences; base number 1 refers to the transcription initiation site (Fig. 1). Restriction sites inserted in the primers are underlined.
Mouse constructs. The reverse primer +1776, containing sequences immediately upstream of the ATG translation initiation site of Myo7a and introducing a SacII site, 5'-ATCCGCGGCTTCTACGTCTGCACAC-3', was used in combination with the following forward primers introducing a SalI site: primer –2063, 5'-ATGTCGACCTAGAGGGATCTGTCTGTTTC-3'; primer –1011, 5'-ATGTCGACCTTGGACCGTGGTCTCAC-3'; primer –507, 5'-ATGTCGACCAGCACACTCAAGACTCC-3'; and primer –118, 5'-ATGTCGACCTTGGGCAACCTCTAGACG-3'.
Primer –2063 was also used in combination with the reverse primer +75, 5'-ATCCGCGGCCTTGGGCTACTCTGTTTC-3', containing sequences encompassing exon 1 (transcription start) (see below).
Human construct. The reverse primer +2369, containing sequences immediately upstream of the ATG initiation site of MYO7A and introducing a SalI site, 5'-ATGTCGACGGTCCCAAGTCAGGAG-3', was used in combination with a forward primer introducing a XhoI site, primer –2109, 5'-ATCTCGAGCTTTCCTGTGTCCCTCT-3'.
Insertion of insulator sequences. A plasmid containing a tandem repeat of insulator sequences (DNA sequence at the 5' end of the chicken ß-globin locus) was generously provided by F. Recillas-Targa and G. Felsenfeld (24). An EcoRI–SalI fragment containing these sequences was isolated and inserted upstream of the Myo7a genomic sequences.
Transgenic mouse production
DNA fragments for microinjection were isolated with ELUTIP-D columns (Schleicher and Schuell). Chimeric fusion genes were micro-injected in the absence of plasmid sequences into the pronuclei of fertilized one-cell eggs from C57BL6/SJL. For PCR genotyping, a GFP 5' primer, 5'-CGACGTAAACGGCCACAAGTTC-3', and 3' primer, 5'-TCGTCCATGCCGAGAGTGATC-3', were used. All the positive transgenic lines obtained were stable for at least five generations. The M3H11 line has been tested positively for more than 15 generations.
Immunohistofluorescence
Transgenic mouse embryos and inner ears were fixed by immersion in 4% paraformaldehyde (pH 7.4) for 1–4 h at 4°C. After three PBS rinses, they were immersed in 20% sucrose-PBS for 
12 h at 4°C and then frozen in OCT embedding medium (Miles). Immunolabelling of cryostat sections (8–12 µm) was performed with anti-myosin VIIA polyclonal IgG (1/2000) as described by El-Amraoui et al. (14). Fluorescent observations of immunostaining and GFP were made using a photonic fluorescent or confocal microscope.
Sequence analysis
The percentage identity plot of the human and mouse genomic sequences was obtained using DNA Block Aligner (DBA) (21) and Winsconsin Sequence Analysis Package (GCG 9.1). The neural promoter prediction program (NNPP, http://www.fruitfly.org/seq_tools/promoter.html) was used to scan the genomic interval between the second Capn5/CAPN5 polyadenylation signal and exon 1 of MYO7A/Myo7a.
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ACKNOWLEDGEMENTS |
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Special thanks go to V. Guyot and S.E.A.T (C.N.R.S, Villejuif) for mouse egg injections, J. Levilliers, J.P. Hardelin and I. Zwaenepoel for critical comments on the manuscript, A. El-Amraoui and M. Leibovici for cryosectioning, tissue dissection and labelling lessons, E.Verpy for providing the vestibular cDNA library, M. Holley for the UB/UE cell line, C. Fairhead and E. Heard for helping with yeast transgenesis protocols, I. Poras (Genethon) for screening the YAC library and S. Blanchard for sequencing. The confocal microscope was purchased with a donation from Marcel and Liliane Pollack. This work was supported by EC grant QLG2-CT-1999-00988, Fondation R. and G. Strittmatter (France), C. and J-P. Bernais donation (France) and A. and M. Suchert and Forschung contra Blindheit-Initiative Usher Syndrome (Germany). B.B. is a recipient from MENRT.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +33 1 45 68 88 90; Fax: +33 1 45 67 69 78; Email: cpetit@pasteur.fr
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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