The present invention provides a GLAST knockout mouse lacking the function of an endogenous glutamate transporter GLAST gene, which shows: 1) an intraocular pressure within the normal range; and 2) a reduction in the number of cells in the retinal ganglions when compared with a wild-type normal mouse. Owing to the ocular properties, this knockout mouse is useful as a model for normal tension glaucoma. By using this knockout mouse, a compound useful for the treatment of normal tension glaucoma can be screened.

Description of the Invention

The inventor has improved normal knockout mice (GLAST knockout mice), which conventionally exist and are deficient in the function of a glutamate transporter gene and as a result, could obtain improved GLAST knockout mice with the markedly reduced number of retinal ganglion cells due to degenerative loss of the cells, although the intraocular pressure is within the normal range. This knockout mouse was found to be useful as a model mouse for normal tension glaucoma.

Therefore, the present invention provides a GLAST knockout mouse deficient in the function of an endogenous GLAST gene, as a model for normal tension glaucoma and more particularly, a GLAST knockout mouse, in which 1) the intraocular pressure is within the normal range and 2) the number of cells in the retinal ganglions is reduced as compared to a wild-type mouse.

According to the present invention, the intraocular pressure of the GLAST knockout mouse is generally 21 mmHg or lower, for example, 10 to 21 mmHg. Also, the number of cells in the retinal ganglions is reduced by at least 20% in the GLAST knockout mouse, as compared to a wild-type mouse.

In the present invention, the genetic background of the GLAST knockout mouse is preferably the same or substantially the same as the genetic background of a C57BL J6 strain mouse, e.g., a C57BL/6J strain mouse.

Specifically, the present invention provides a GLAST knockout mouse carrying a neomycin-resistant gene inserted into the region of endogenous GLAST gene, for example, into the exon 6.

The present invention further provides use of such a GLAST knockout mouse as a model mouse for normal tension glaucoma.

In another aspect, the present invention provides a method of producing a GLAST knockout mouse deficient in the function of an endogenous GLAST gene. This production method comprises the following steps 1) to 6):

1) obtaining an ES cell from any mouse deficient in the function of one endogenous GLAST gene on the homologous chromosome,

2) obtaining a chimeric mouse carrying the ES cell using the cell obtained in step 1,

3) crossing the chimeric mouse obtained in step 2 with a normal C57BL/6 strain mouse to obtain a heterozygous knockout mouse,

4) crossing the heterozygous mouse obtained in step 3 with a normal C57BL/6 strain mouse to generate a heterozygous knockout mouse,

5) repeating the crossing described in step 4 at least a total of 5 times to generate a heterozygous knockout mouse thereby to bring the genetic background closer to the C57BL/6 strain mouse, and,

6) crossing the heterozygous knockout mice obtained in step 5 with each other to generate a homozygous or heterozygous GLAST knockout mouse.

In the production method of the invention, it is preferred to repeat the crossing described in step 4 at least a total of 9 times in step 5.

The present invention further includes the GLAST knockout mice produced by the production method of the invention, and the GLAST knockout mice thus produced can be used as model mice for normal tension glaucoma.

In yet another aspect, the present invention provides a method of using the GLAST knockout mice of the invention described above or the GLAST knockout mice produced by the production method of the invention described above, as model mice for normal tension glaucoma.

Therefore, the present invention provides a method of screening a compound useful for the prevention and/or treatment of normal tension glaucoma, which comprises using such GLAST knockout mice. More specifically, the screening method comprises:

1) administering a test compound to the GLAST knockout mouse of the invention,

2) administering a test compound to a wild-type mouse,

3) assessing the number or function of surviving optic nerve cells in each of the mice described above, prior to and after a given time period of the administration, and,

4) comparing the GLAST knockout mouse with the wild-type mouse in terms of the test results to determine effectiveness of the test compound.

According to the screening method of the invention, the number of nerve cells in the retinal ganglions is counted to assess the number of surviving optic neurons or the function of the optic neurons and in addition thereto, the assessment is conducted preferably by measurements of electroretinograms or visual evoked potentials (Porciatti et al., Vision Res., 39, 3071-3081, 1999), behavioral analysis such as the Visual Cliff test (Ma, L. et al., Neuron 36, 623-634, 2002), etc. in combination.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, mouse glutamate transporter GLAST (Glutamate/Aspartate Transporter) refers to a protein encoded by DNA strand having the base sequence represented by SEQ ID NO: 1 and having the amino acid sequence represented by SEQ ID NO: 2 (Tanaka, K., Neurosci. Lett., 159, 1803-186, 1993)). This protein is also termed GluT-1 in rat (Tanaka, K., Neurosci. Res. 16, 149-153, 1993; Storck, T., et al., Proc. Natl. Acad. Sci. USA, 89, 10955-10959, 1992). Both transporters are so-called counterparts.

The genomic structure of mouse GLAST has already been clarified and the details are described in Hagiwara, T., et al., Genomics, 33, 508-515, 1996. The structure of this gene is outlined in Table 1 and FIG. 1 (see Original Patent).

However, depending upon mouse strains, the mouse GLAST may undergo mutation in the encoded nucleotide sequence and amino acid sequence described above and its genomic sequence within the range to maintain the function, for instance, may undergo substitutions, deletions, additions or insertions of bases or amino acid residues. In the present invention, the mouse GLAST also includes these mutants such as a mutant with substitutions, deletions, additions or insertions of, e.g., 1 to 10, preferably 1 to 5 bases in the encoded nucleotide sequence described above, a mutant with substitutions, deletions, additions or insertions of, e.g., 1 to 10, preferably 1 to 5 amino acids in the amino acid sequence described above, etc.

Table 1 (see Original Patent) shows the sequencing of exon/intron splice site for GLAST gene. The nucleotide sequences for exons are represented by capitals and for introns by small letters.

In the present invention, the deficiency in the function of the glutamate transporter GLAST gene means that within the region of one or two endogenous GLAST genes present in one or two GLAST loci, functional GLAST remains unexpressed or expression of the GLAST gene is constantly suppressed, either by introducing a mutation into the region encoding its structure, e.g., into the exon, or by introducing a mutation into the region associated with expression of the GLAST gene, e.g., into the promoter or intron region. In any case, the term refers to such a state that one or two endogenous GLAST genes are substantially dysfunctional in vivo. In the present invention, therefore, the GLAST knockout mouse includes a homozygote deficient in the function of two endogenous GLAST genes and a heterozygote deficient in the function of one endogenous GLAST gene. In view of the effect of deficiency in the function of the gene, homozygous mice are preferred.

Such a deficiency in the function of the gene can be achieved by publicly known methods for generating knockout mice, e.g., a gene targeting strategy. Introduction of the mutation described above may also be substitutions of bases or deletions of bases in the GLAST gene region, or insertions of bases into the region.

In the present invention, the term "the same or substantially the same genetic background" is used to mean that all genotypes other than the targeted genotype (GLAST genotype) are the same by 99% or more. Specifically, this means that in EXAMPLES, F1 GLAST heterozygous knockout mice are backcrossed to normal C57B/L6 mice for at least 9 generations and the genes derived from the 129 strain have reached 1% or less than all genes.

1. GLAST Knockout Mouse of the Invention

The present invention provides a GLAST knockout mouse as a model mouse with normal tension glaucoma, which is deficient in the function of one or two endogenous GLAST genes on the homologous chromosome. Specifically, the present invention provides a GLAST knockout mouse, in which 1) the intraocular pressure is within the normal range and 2) the number of cells in the retinal ganglions is reduced as compared to a wild-type mouse. More particularly, the mouse preferably has the same or substantially the same genetic background as that of a C57BL/6 strain mouse, e.g., a C57BL/6J strain mouse.

The intraocular pressure of a wild-type normal mouse is generally 10 to 21 mmHg. The intraocular pressure of the knockout mouse of the present invention also lies within the normal range. The intraocular pressure may be outside the range above, but does not reach such a range as termed high pressure, e.g., 30 mmHg or higher. The intraocular pressure in mice can be determined by using, e.g., an electronic tonometer.

Furthermore, in the knockout mouse of the present invention, the number of nerve cells in the retinal ganglions is reduced by at least 20%, preferably at least about 50%, as compared to the number in normal mouse. The reduction in the number of nerve cells in the retinal ganglions can be microscopically determined by conventional histochemical means, for example, by hematoxylin/eosin staining using a section.

Further in the knockout mouse of the present invention, a reduced b-wave, which is a sort of potential change in the retina by photic stimulation, is also noted, when compared with a wild-type normal mouse. The b-wave reflects an action potential in the inner retinal layer containing Muller cells in which GLAST exists, suggesting that a mechanism regulating glutamate levels by GLAST would have an important role also in visual transmission (Harada, T., et al., Proc. Natl. Acad. Sci. USA, 95, 4663-4666, 1998).

This reduction in the number of nerve cells in the retinal ganglions can be regarded as neurodegeneration or neuronal apoptosis. In general, optic disc atrophy or deficits of the retinal nerve fibers are observed in glaucoma including normal tension glaucoma. Currently, the final clinical picture of glaucoma is thought to be retinal ganglion cell death.

Thus, taking into account these ocular properties of the GLAST knockout mouse of the present invention, namely, the intraocular pressure within the normal range and the reduction in the number of cells in the retinal ganglions, the mouse can be used as a model mouse for normal tension glaucoma.

2. Production of GLAST Knockout Mouse of the Invention

In a second aspect, the present invention provides a method of producing the GLAST knockout mouse of the present invention. This method comprises the following steps 1) to 6):

1) obtaining an ES cell from any mouse, which is deficient in the function of one endogenous GLAST gene on the homologous chromosome,

2) obtaining a chimeric mouse carrying the ES cell using the cell obtained in step 1,

3) crossing the chimeric mouse obtained in step 2 with a wild-type C57BL/6 strain mouse to generate a heterozygous knockout mouse,

4) crossing the heterozygous mouse obtained in step 3 with a wild-type C57BL/6 strain mouse to generate a heterozygous knockout mouse,

5) repeating the crossing described in step 4 at least a total of 5 times to generate a heterozygous knockout mouse thereby to bring the genetic background closer to the C57BL/6 strain mouse, and,

6) crossing the heterozygous knockout mice obtained in step 5 with each other to generate a homozygous or heterozygous GLAST knockout mouse.

In step 5 described above, the crossing is repeated preferably at least 9 times in total.

Basically, publicly known methods for producing knockout mice, e.g., gene targeting strategy, gene trapping strategy, etc., can be used to produce the GLAST knockout mouse of the present invention available as a model for normal tension glaucoma. Basic methods for producing knockout mice are not particularly limited, as far as the GLAST gene can be disrupted and mice whose surviving and reproductive potentials are not lost are obtained. For the methods of producing knockout mice, reference can be made to, e.g., "Jikken-Igaku-Bessatsu, Kaitei-Idenshi-Kogaku Handbook (Handbook of Genetic Engineering), 3rd revised version", edited by Masami Muramatsu and Tadashi Yamamoto (published by Yodosha Co., Ltd., 1996) or "Jikken-Igaku-Bessatsu, The Protocol Series: Latest Technology of Gene Targeting", edited by Takeshi Yagi (published by Yodosha Co., Ltd., 2000), which can be applied to carry out the present invention.

First, the steps 1 through 4 described above can be performed in accordance with conventional methods, e.g., the gene targeting strategy. These steps are disclosed also in Japanese Patent Laid-Open Application No. 10-33087 by the research group of the present inventors and Watase, K. et al, Eur. J. Neurosci., 10, 976-988, 1998.

Heretofore, homozygous or heterozygous GLAST knockout mice obtained by mating female and male of the heterozygous GLAST knockout mice obtained in step 4 described above are already disclosed but in this type of GLAST knockout mice, no significant change was noted in the number of cells in the retinal ganglions, when compared with wild-type normal mice; a decrease in the number of cells in the retinal ganglions was observed only after transient ischemia was achieved by instilling sterile saline into the eye of the GLAST knockout at 150 cm H.sub.2O pressure for 60 minutes (Harada, T., et al., Proc. Natl. Acad. Sci. USA, 95, 4663-4666, 1998).

For these reasons, such GLAST knockout mice hitherto known cannot be used as a model for normal tension glaucoma.

According to the production method of the present invention, such conventional GLAST knockout mice are further improved, whereby the GLAST knockout mouse of the present invention with normal tension glaucoma described above can be produced.

Hereinafter, the method for producing the knockout mouse of the present invention will be described below, taking as an example the gene targeting strategy which is a standard method for producing a knockout mouse.

Step 1

(1) Preparation of Targeting Vector

According to the gene targeting strategy, in order to disrupt the GLAST locus on the chromosome in mouse ES cells, a targeting vector is used to introduce a mutation into the locus.

In order to render the function of GLAST gene defective, bases are deleted, point mutation is introduced or other genes are inserted into any part of the GLAST gene, e.g., one or more exon regions. Generally in order to select the endogenous GLAST gene-disrupted ES cells more readily, it is preferred to insert a selection marker gene.

As such a gene, a marker gene for positive selection, for example, a neomycin (neo)-resistant gene can be employed. This neomycin-resistant gene enables to screen the objective gene by using the neomycin analog G418. Also, a marker gene for negative selection can also be used to screen and remove the objective gene. Examples of such genes used include thymidine kinase (tk) gene (using ganciclovir, FIAU, etc. as a screening marker, a non-homologous recombinant is screened and removed by the sensitivity thereto) and diphtheria toxin A fragment (DT-A) gene (a non-homologous recombinant is screened and removed by diphtheria toxin expressed by DT-A). Alternatively, a combination of the foregoing can also be used for positive/negative selection. It is preferred to insert, e.g., neomycin-resistant gene and diphtheria toxin A fragment gene (Yagi, Nada, Watanabe, et al., Analytical Biochemistry, 214, 77-86, 1993), or neomycin-resistant gene and thymidine kinase gene (Mansour, Thomas and Capacchi, Nature, 336, 348-352, 1988).

In the gene sequence, a site to introduce a mutation, for example, a site to insert the marker gene described above is not particularly limited, so far as it is a site that function of the gene is lost, but the site is usually an exon site.

The genomic structure (restriction enzyme map and each exon-intron splice point) of GLAT gene are already known (Hagiwara, T., et al., Genomics, 33, 508-515, 1996), which structure is outlined in FIG. 1 and Table 1 (see Original Patent). The mouse GLAST gene comprises 10 exons. It is preferred to insert a marker gene into any one of the exons so as to cause deficiency of the gene.

In order to disrupt the function of the gene as described above, homologous recombination with the target gene is available so that a targeting vector (DNA for homologous recombination) capable of introducing a mutation into the target gene can be prepared by conventional DNA recombinant techniques, for example, PCR or site-specific mutagenesis, based on the GLAST-encoding nucleotide sequence (SEQ ID NO: 1) and information of genomic sequence of the GLAST gene (Hagiwara, T., et al., Genomics, 33, 508-515, 1996).

For example, a DNA molecule containing the entire gene or its fragment is isolated in a conventional manner from the mouse strain, from which ES cells used are derived. The DNA molecule may be a DNA molecule containing the entire GLAST gene or further containing the 5' upstream region and/or the 3' downstream region of the gene, in addition to the entire gene.

Next, a modified DNA molecule is prepared from the resulting DNA molecule by introducing a desired mutation into the site corresponding to the mutation site in the gene, e.g., by introducing the marker gene described above. Modification of the base sequence can be made by conventional recombinant DNA techniques such as ligation of DNA molecules amplified by PCR, site-specific mutation, etc. In constructing such a targeting vector, plasmid vectors commercially available for targeting vector construction may also be used.

(2) Introduction of Targeting Vector into ES Cells and Homologous Recombination with Endogenous GLAST Gene

The thus obtained targeting vector is introduced into mouse embryonic stem cells (ES cells) to perform homologous recombination. Introduction of the targeting vector into ES cells can be made by conventional DNA transfection techniques, e.g., electroporation, lipofection, etc. In the targeting vector-transfected cells, homologous recombination occurs between the GLAST gene on the chromosome and the counterpart on the targeting vector so that the modified base sequence in the targeting vector, e.g., a marker gene is introduced into the endogenous gene. As a result, the ES cells are deficient in the function of endogenous GLAST gene and at the same time contains, e.g., the marker gene. The cells where the targeting vector is introduced are then screened by the screening function of, e.g., the marker gene, or in a conventional manner such as southern blotting for confirming homologous recombination, PCR, etc. to obtain ES cells deficient in the function of GLAST gene (hereinafter referred to as recombinant ES cells). By such homologous recombination, usually ES cells with disrupted GLAST gene only on one homologous chromosome are obtained.

Mouse ES cells used are generally 129 ES cells already established. In addition, ES cells are established by publicly known methods (Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (Robertson, E. J., ed.), IRL press, Oxford, 1987) using C57BL/6 or BDF1 strain mice (F1 mice obtained by crossing C57BL6 with DBA/2). These ES cells may also be used. Preferably, 129-derived ES cells are used.

Steps 2 and 3

Production of Heterozygous GLAST Gene Knockout Mouse in the F1 Generation

Next, the resulting recombinant ES cells are developed to generate a chimeric mouse. For this purpose, the recombinant ES cells are injected into normal mouse embryos at the blastocyst stage, the 8-cell stage, etc. by microinjection or aggregation. The thus obtained chimeric embryos are transplanted to the uterine horn of a pseudopregnant female mouse. This transplanted mouse can be bred in a conventional manner and allowed to deliver the offspring of the chimeric mouse. Preferably, the recombinant ES cells are injected into the embryos of C57BL/6 strain mice.

In general, this chimeric mouse comprises cells derived from the recombinant ES cells and normal cells as its somatic cells and germ cells. By crossing the chimeric mouse with a wild-type mouse of a proper line, preferably a C57BL/6 strain mouse, e.g., a C57BL/6J strain mouse, heterozygous F1 offspring are obtained. Usually, a male chimeric mouse is mated with a female wild-type mouse to generate heterozygous offspring of the F1 generation. If the germ cells of the chimeric mouse used for the mating are derived from the recombinant ES cells described above, i.e., cells carrying the disrupted endogenous GLAST gene present on one of the homologous chromosomes, then desired heterozygous F1 mice deficient in the function of the gene can be obtained.

In the step described above, to generate the heterozygous F1 mice with high efficiency, for example, normal host embryonic cells derived from a mouse having a coat color different from the mouse of the recombinant ES cell origin are used in combination, e.g., in the preparation of chimeric embryos. By inspection of the coat color, a chimeric mouse showing a higher rate of the recombinant ES cells in vivo or a heterozygous F1 mouse can be easily screened.

It can be confirmed by analysis of DNA extracted from the tail using southern blotting or PCR whether a desired genotype is achieved in the F1 generation or not.

Steps 4, 5 and 6

(1) Obtaining of GLAST Gene Knockout Mouse of the Invention

In the present invention, it is preferred to bring the genetic background of the GLAST gene knockout mouse as closer as possible to the C57BL/6 strain mouse. For this purpose, the F1 heterozygous mouse produced as described above is further crossed with a C57BL/6 strain mouse, e.g., a C57BL/6J strain mouse and the delivered heterozygous mouse is again crossed with a C57BL/6 strain wild-type mouse. The crossing procedures are repeated normally at least 5 times in total, preferably at least 9 times and more preferably at least 15 times. Finally by crossing female and male of the resulting heterozygous mice with each other, the homozygous or heterozygous knockout mouse of the present invention deficient in the function of GLAST gene can be obtained. In view of the effect of deficiency in the function of glutamate transporter gene, homozygous mice are preferred.

Whether a desired genotype is achieved in the respective generations or not may be determined by conventional techniques, including southern blotting, PCR, base sequencing, etc., as described above.

Once the knockout mice of the present invention which can be produced as above are obtained in the combination of males and females, subsequently knockout mice having the same genotype can be readily obtained in the number as required, by appropriately breeding the offspring, depending on necessity.

(2) Analysis of the Retina and Intraocular Pressure in GLAST Knockout Mouse of the Invention

Finally, it is confirmed on the homozygous or heterozygous GLAST knockout mice produced as described above that the intraocular pressure is within the normal and nerve cells in the retinal ganglions are reduced. When these ocular properties cannot be confirmed, the crossing described in step 5 is repeated so that the knockout mouse of the present invention satisfying the properties above can be obtained.

The intraocular pressure of the GLAST knockout mouse of the present invention is generally about 21 mmHg or lower, for example, about 10-21 mmHg. This intraocular pressure range may be somewhat varied depending upon the strain of mice, from which the ES cells used are derived, or the strain of mice from which the normal embryos used to prepare chimeric embryos are originated. Even in view of the foregoing, the intraocular pressure should be not higher than 30 mmHg.

In the GLAST knockout mouse of the present invention, the number of nerve cells in the retinal ganglions is reduced by at least 20%, preferably at least about 50%, as compared to a wild-type mouse.

Hereinafter, a method for measurement of the number of cells in the retinal ganglions and a method for measurement of the intraocular pressure will be described by way of examples but is not deemed to be limited thereto and any conventional publicly known methods may also be used. Reference may be made to, e.g., Harada, T., et al., Proc. Natl. Acad. Sci. USA, 95: 4663-4666, 1998 or Harada, C., et al., Neurosci. Lett., 292, 134-136, 2000.

In these measurements, the wild-type normal mice delivered simultaneously with the homozygous or heterozygous knockout mice of the present invention described above or mere normal (wild-type) C57BL/6 strain mice can be used as control mouse against these mice of the present invention.

In addition to the control mouse described above, the measurements are also applied, if necessary, to the homozygous or heterozygous GLAST knockout mice obtained by mating the F1 heterozygous knockout mice described above or their females and males, heterozygous knockout mice obtained during backcrossing, and so on.

Measurement of the Number of Cells in the Retinal Ganglions:

The number of cells in the mouse retinal ganglion can be measured by conventional histochemical means or retrograde labeling.

(a) Method using Pathological Section

1) Test mice are anesthetized to keep them still and perfused with 4% paraformaldehyde/PBS solution to fix.

2) Eye globes are enucleated and fixed at 4.degree. C. in the same solution for further 2 hours.

3) After the eye globes are embedded in paraffin, sections, e.g., 7 .mu.m thick sections of the retina are prepared.

4) The sections are stained with hematoxylin/eosin and the number of cells in the ganglion was counted under microscope on the cross-section containing optic nerves.

(b) Method using Retrograde Labeling

1) Following anesthetic sedation, the mouse is placed in a stereotaxic head frame.

2) After spraying ethanol under microscope, an incision is made with scissors along the midline of the head, exposing the skull.

3) After confirming sutures and blood vessels, holes for operation are drilled with a grinder and through the holes, e.g., fluorescent dye Fluoro-Gold (general name, aminostilbamidine; Molecular Probes, Inc.) or a carbocyanine fluorescent dye such as DiI (general name, 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate; Molecular Probes, Inc.) is injected into the superior colliculi.

4) After the skin is fastened with clips, an antihypnotic is intraperitoneally injected and the recovery is ensured.

5) Following normal breeding for 7 days after the operation, the treated mice are anesthetized with ether resulting in death and eye globes are enucleated. The anterior part of the eye is removed.

6) The posterior part of the eye including the retina is placed in a solution of 4% paraformaldehyde and fixed at 4.degree. C. for 20 minutes.

7) The retina is taken out and a whole-mount preparation is made.

8) After photographs are taken with a fluorescence microscope, the number of fluorescence-labeled cells in the retinal ganglions is counted.

Any anesthetic can be used in such a concentration range that mice are not dead, as far as it is an anesthetic available for ordinary animal tests. For example, a 1:1 mixture of ketamine (10 mg/ml)/medetomidine (1 mg/ml) (0.15-0.2 ml/mouse) may be employed. In this case, the animal can be awakened with atipamezole (5 mg/ml) (0.15-0.2 ml/mouse).

Measurement of Intraocular Pressure:

1) Mice are anesthetized with sedation in a conventional manner, as described above.

2) The intraocular pressure of sedated mice is measured with an electronic tonometer (e.g., TONOPEN XL, manufactured by Medtronic Solan, US).

To assess the visual function or the function of optic nerve cells, electroretinograms (ERG) may be measured. This is to measure an electric response obtained from the retina caused by photic stimulation and is a good indicator representing the activity of neuronal transduction of optic nerve cells in vivo. For more details, reference is made to "Gendai-No-Me-Kagaku (Modern Textbook of Ophthalmology)," revised 8th edition (edited by Takashi Tokoro, Atsushi Kanai, published by Kanehara & Co., Ltd.), "Shino-Kyoseigaku (Visual Orthotics), revised 2nd edition" (edited by Toshio Maruo, published by Kanehara & Co., Ltd.), or Non-Patent Literature 1 (Harada, T., et al. Proc. Natl. Acad. Sci. USA, 95: 4663-4666, 1998).

The measurement method is briefly explained below

1) Mice are anesthetized in a conventional manner and secured with a head holder to fix the position of each mouse.

2) The pupils are dilated by administration of 0.5% phenylephrine and 0.5% tropicamide.

3) A carbon fiber electrode is placed on the corneal surface and a reference electrode is attached subcutaneously on the forehead.

4) Mice undergo dark adaptation for 30 minutes.

5) Test flashes of 10 .mu.s duration are given with an intensity of 0.6 or 1.2 J for retinal stimulation, using the photostimulator (SLS-3100, Nihon Kohden, Japan) were presented (SLS-3100, Nihon Kohden).

6) A bandpass frequency is set at 50 to 1000 Hz and 1 to 1000 Hz on the amplifier (MEB-5304, Nihon Kohden), and the potentials generated (in the case where each oscillatory potential OP and the a-wave and b-wave are measured, respectively.) are amplified.

7) The two responses are averaged and recorded.

8) The a-wave, b-wave and oscillatory potentials are analyzed as data.

3. Method of Screening a Compound useful for the Prevention/Treatment of Normal Tension Glaucoma

The knockout mouse of the present invention can be used for screening a compound useful for the prevention/treatment of normal tension glaucoma, especially a compound useful for preventing the death or degeneration of optic nerve cells including retinal ganglions, or for preventing diminished activity of the function, or a compound useful for recovering optic nerve cells or their function.

Therefore, the present invention provides a method of screening a compound useful for the prevention/treatment of normal tension glaucoma and comprises:

1) administering a test compound to the homozygous or heterozygous GLAST knockout mouse of the present invention,

2) administering a test compound to a wild-type normal mouse,

3) assessing the number or function of surviving optic nerve cells in each of the mice described above, prior to and after a given time period of the administration, and,

4) comparing the GLAST knockout mouse with the wild-type mouse in terms of the test results to determine effectiveness of the test compound.

Whether a test compound is useful for the prevention/treatment of normal tension glaucoma or not can be determined by examining whether the compound can improve a sign characteristic of glaucoma. For example, after a test compound is administered, the number of nerve cells in the retinal ganglions is counted; when the number is recovered by at least 10%, preferably by at least 20% and more preferably by at least 30%, as compared to the control mouse, the test compound can be judged to be medically effective. Furthermore, after a test compound is administered, the retinal potentials are measured; when the amplitude of, e.g., b-wave or oscillation potential is recovered by at least 10%, preferably by at least 20% and more preferably by at least 30%, as compared to the control mouse, the test compound can be judged to be medically effective.

In the heterozygous knockout mouse of the present invention, the number of cells in the retinal ganglions is found to be gradually reduced with the age of weeks after birth (FIG. 4 (see Original Patent)). Accordingly, in one embodiment of the screening method described above, a compound which prevents a reduction of the number of cells in the retinal ganglions with passage of time can also be screened by 1) regularly administering a test compound to a group of heterozygous knockout mice immediately after birth but administering no test compound to another group of heterozygous knockout mice, 2) counting the number of cells in the retinal ganglions at the age of respective weeks, and 3) comparing the two groups.

The test compound which can be used includes, in addition to naturally occurring and synthetic compounds, optional compounds such as animal and plant extracts, fermentation products, peptides, proteins, nucleic acid molecules, etc. The test compound may be a gene vector for expressing a desired protein. For administration of the test compound, a variety of routes may be attempted as far as properties of the test compound permit, and the compound may be administered, e.g., as eye drops or by oral administration. Dosing period or dosing mode can be chosen so as to maximize the effect of a test compound. Kind and dosing of such a test compound may also be in accordance with conventional methods in the pharmaceutical field or medical field.

Furthermore, the GLAST knockout mouse of the present invention can be crossed with other strain of knockout mouse or other strain of disease model mouse to generate a novel disease model mouse. The present invention also includes such use of the GLAST knockout mouse of the present invention.
 

Claim 1 of 7 Claims

1. A GLAST knockout mouse deficient in the function of an endogenous GLAST gene, wherein said mouse has the same genome, except for the targeted endogeneous GLAST gene, as that of a C57BL/6 mouse strain, and wherein, when ischemic load is not applied: 1) the intraocular pressure is not greater than 21 mmHg, and, 2) the number of cells in the retinal ganglions is 20% to 50% less than that of a wild-type C57BL/6 strain mouse.

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