JP5938731B2 - Normal-tension glaucoma model non-human mammal - Google Patents
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- JP5938731B2 JP5938731B2 JP2013504766A JP2013504766A JP5938731B2 JP 5938731 B2 JP5938731 B2 JP 5938731B2 JP 2013504766 A JP2013504766 A JP 2013504766A JP 2013504766 A JP2013504766 A JP 2013504766A JP 5938731 B2 JP5938731 B2 JP 5938731B2
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Description
本発明は、正常眼圧緑内障モデル非ヒト哺乳動物などに関する。 The present invention relates to a normal-tension glaucoma model non-human mammal and the like.
緑内障は、何らかの原因で視神経が損傷し、視野に欠損を生じる進行性の疾患であり、そのまま放置しておくと、最終的には失明にいたる可能性が高い。 Glaucoma is a progressive disease in which the optic nerve is damaged for some reason and a defect occurs in the visual field. If it is left as it is, there is a high possibility that it will eventually lead to blindness.
緑内障の1つである高眼圧緑内障は、前房中の房水の排泄が悪くなり眼圧（眼球内の水圧）が上昇することにより視神経が圧迫されて委縮し、そのため視機能が障害を受け、視野が狭くなる疾患である。 High-intensity glaucoma, one of the glaucoma, is caused by the loss of aqueous humor in the anterior chamber and the increase in intraocular pressure (water pressure in the eyeball), which compresses and contracts the optic nerve, thus impairing visual function. This is a disease that narrows the field of vision.
正常眼圧緑内障は、その有病率の高さから、最近特に注目されている疾患である。日本人の緑内障患者は約400万人おり、そのうちの約3分の2が正常眼圧緑内障患者である。正常眼圧緑内障は、眼圧が正常(ヒトでは通常10〜21mmHg)であるにもかかわらず、高眼圧緑内障と同様の所見（視神経の委縮及び視野欠損）を呈する病態であり、具体的には、(a)神経の炎症をともなわないこと、(b)障害部が網膜神経節細胞(RGC: retinal ganglion cell)に限定的であること、(c)経時的に神経障害が進行すること、(d)視神経乳頭部に特徴的な所見(陥凹)を有すること、(e)眼圧は正常のまま維持されること、などの特徴を示す。正常眼圧緑内障は、進行が緩やかで自覚症状も少ないため、早期発見が難しく、また現在のところ、更に眼圧を下げること以外、決めてとなる治療法がない。 Normal-tension glaucoma is a disease that has received particular attention recently because of its high prevalence. There are about 4 million Japanese glaucoma patients, of which about two-thirds are normal-tension glaucoma patients. Normal-tension glaucoma is a pathological condition that presents the same findings (optic nerve atrophy and visual field loss) as high-tension glaucoma despite normal intraocular pressure (usually 10 to 21 mmHg in humans) Is (a) not accompanied by nerve inflammation, (b) the lesion is limited to retinal ganglion cells (RGC), (c) neuropathy progresses over time, (d) It has features such as having a characteristic finding (concave) in the optic nerve head, and (e) maintaining intraocular pressure as normal. Normal-tension glaucoma is difficult to detect early because it progresses slowly and has few subjective symptoms. At present, there is no definitive treatment other than lowering intraocular pressure.
正常眼圧緑内障モデル非ヒト哺乳動物が得られれば、正常眼圧緑内障の治療に有効な治療薬の開発のために、その治療方法の確立のために、そしてまた該疾患の原因や発生機序の解明のために、極めて有益であると期待される。 If a normal-tension glaucoma model non-human mammal is obtained, the development of therapeutic agents effective for the treatment of normal-tension glaucoma, the establishment of the treatment method, and the cause and mechanism of the disease It is expected to be extremely useful for elucidation of
ここで、正常眼圧緑内障モデルマウスとして、房水の排出路をレーザーで焼灼したもの(非特許文献1)、前房中にビーズを入れたもの(非特許文献2)、房水の排出静脈を凝固させたもの(非特許文献3)、などが知られている。しかしながら、いずれのモデルも、眼圧の調整が困難であること、炎症を伴う場合があること、発生機序に矛盾があること、などの問題があった。 Here, as a normal-tension glaucoma model mouse, the drainage of the aqueous humor was ablated with a laser (Non-patent Document 1), the bead was placed in the anterior chamber (Non-patent Document 2), the drainage vein of the aqueous humor A coagulated product (Non-patent Document 3) is known. However, all the models have problems such as difficulty in adjusting intraocular pressure, sometimes accompanied by inflammation, and inconsistency in the generation mechanism.
また、GLAST遺伝子を欠損させた正常眼圧緑内障モデルマウスも知られている(非特許文献4、特許文献1)。GLASTは、神経情報伝達物質である代謝型グルタミン酸の回収機構として知られているトランスポーターである。 In addition, normal-tension glaucoma model mice lacking the GLAST gene are also known (Non-Patent Document 4, Patent Document 1). GLAST is a transporter known as a mechanism for recovering metabotropic glutamate, which is a neurotransmitter.
正常眼圧緑内障モデルマウスとしては、症状の進行が緩やかであることなどを含め、実際のヒト正常眼圧緑内障と同様の発症メカニズムで同様の症状のものが求められている。 Normal-tension glaucoma model mice are required to have the same symptoms with the same onset mechanism as that of actual human normal-tension glaucoma, including the slow progression of symptoms.
上記状況において、正常眼圧緑内障モデルとして使用し得る新たな非ヒト哺乳動物などが求められていた。 In the above situation, a new non-human mammal that can be used as a normal-tension glaucoma model has been demanded.
本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、c-Srcタンパク質のCdk5リン酸化部位であるSerをAspに置換する変異をSrc遺伝子に導入した遺伝子改変マウスが正常眼圧緑内障モデルとして使用し得ることを見出した。そして、さらに検討を重ねた結果、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that a genetically modified mouse in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Asp is introduced into the Src gene. It was found that it can be used as a pressure glaucoma model. As a result of further studies, the present invention has been completed.
すなわち、本発明は、以下に示す、遺伝子改変非ヒト哺乳動物、及び当該非ヒト哺乳動物を用いた、正常眼圧緑内障の予防及び／又は治療剤のスクリーニング方法などを提供する。
[1] c-Srcタンパク質のCdk5リン酸化部位であるSerをAsp又はGluに置換する変異をSrc遺伝子に導入した遺伝子改変非ヒト哺乳動物。
[2] 前記Serが、c-Srcタンパク質のN末端から74番目のアミノ酸残基である、上記[1]記載の非ヒト哺乳動物。
[3] 前記SerがAspと置換された、上記[1]又は[2]記載の非ヒト哺乳動物。
[4] 前記非ヒト哺乳動物が、変異型Src遺伝子をホモ接合で有する、上記[1]〜[3]のいずれか1項記載の非ヒト哺乳動物。
[5] 前記非ヒト哺乳動物が次の条件：
1)その眼圧が正常範囲にあり、かつ
2)その網膜神経節の細胞数が、野生型非ヒト哺乳動物に比べて減少している、
を満たしている、上記[1]〜[4]のいずれか1項記載の非ヒト哺乳動物。
[6] 前記非ヒト哺乳動物がマウスである、上記[1]〜[5]のいずれか1項記載の非ヒト哺乳動物。
[7] 前記非ヒト哺乳動物が正常眼圧緑内障モデルとして用いられる、上記[1]〜[6]のいずれか1項記載の非ヒト哺乳動物。
[8] 上記[1]〜[7]のいずれか1項記載の非ヒト哺乳動物を用いた、正常眼圧緑内障の予防及び／又は治療剤のスクリーニング方法。
[9] 前記方法が、
1)上記[1]〜[7]のいずれか1項記載の非ヒト哺乳動物及び野生型非ヒト哺乳動物に候補物質を投与すること、
2)前記各非ヒト哺乳動物において、投与前、及び投与してから一定期間後に、生存する視神経細胞の数量を検査すること、及び
3)前記各非ヒト哺乳動物の検査結果を比較して、候補物質の有効性を評価すること、
を含む、上記[8]記載の方法。
[9-1] 前記方法が、
1)上記[1]〜[7]のいずれか1項記載の非ヒト哺乳動物及び対照非ヒト哺乳動物に候補物質を投与すること、
2)前記各非ヒト哺乳動物において、投与前、及び投与してから一定期間後に、生存する視神経細胞の数量を検査すること、及び
3)前記各非ヒト哺乳動物の検査結果を比較して、候補物質の有効性を評価すること、
を含む、上記[8]記載の方法。
[10] 前記生存する視神経細胞の数量の検査が、網膜神経節の神経細胞数を計数することである、上記[9]記載の方法。
[10-1] 前記生存する視神経細胞の数量の検査が、網膜神経節の神経細胞数を計数することである、上記[9-1]記載の方法。
[11] 前記対照非ヒト動物が、野生型非ヒト動物及び/又はc-Srcタンパク質のCdk5リン酸化部位であるSerをAlaに置換する変異をSrc遺伝子に導入した遺伝子改変非ヒト哺乳動物である、上記[9-1]又は[10-1]記載の方法。
[11-1] 対照非ヒト動物における前記Serが、c-Srcタンパク質のN末端から74番目のアミノ酸残基である、上記[11]記載の方法。
[11-3] 対照遺伝子改変非ヒト哺乳動物が、変異型Src遺伝子をホモ接合で有する、上記[11]又は[11-1]記載の方法。
[12] c-Srcタンパク質のCdk5リン酸化部位であるSerをAlaに置換する変異をSrc遺伝子に導入した遺伝子改変非ヒト哺乳動物。
[12-1] 前記Serが、c-Srcタンパク質のN末端から74番目のアミノ酸残基である、上記[12]記載の非ヒト哺乳動物。
[12-2] 前記非ヒト哺乳動物が、変異型Src遺伝子をホモ接合で有する、上記[12]又は[12-1]記載の非ヒト哺乳動物。That is, the present invention provides the following genetically modified non-human mammals and methods for screening agents for preventing and / or treating normal tension glaucoma using the non-human mammals.
[1] A genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Asp or Glu is introduced into the Src gene.
[2] The non-human mammal according to [1] above, wherein Ser is the 74th amino acid residue from the N-terminus of the c-Src protein.
[3] The non-human mammal according to [1] or [2] above, wherein Ser is substituted with Asp.
[4] The non-human mammal according to any one of [1] to [3] above, wherein the non-human mammal has a mutant Src gene in a homozygote.
[5] The non-human mammal has the following conditions:
1) The intraocular pressure is in the normal range, and
2) The number of cells in the retinal ganglion is reduced compared to wild type non-human mammals,
The non-human mammal according to any one of [1] to [4] above, wherein
[6] The non-human mammal according to any one of [1] to [5] above, wherein the non-human mammal is a mouse.
[7] The non-human mammal according to any one of [1] to [6] above, wherein the non-human mammal is used as a normal-tension glaucoma model.
[8] A screening method for a prophylactic and / or therapeutic agent for normal-tension glaucoma using the non-human mammal according to any one of [1] to [7] above.
[9] The method comprises:
1) administering a candidate substance to the non-human mammal and the wild-type non-human mammal according to any one of the above [1] to [7],
2) In each non-human mammal, examining the number of optic nerve cells surviving before administration and after a certain period of time after administration; and
3) comparing the test results of each of the non-human mammals to evaluate the effectiveness of the candidate substance,
The method according to [8] above, comprising:
[9-1]
1) administering a candidate substance to the non-human mammal according to any one of [1] to [7] above and a control non-human mammal;
2) In each non-human mammal, examining the number of optic nerve cells surviving before administration and after a certain period of time after administration; and
3) comparing the test results of each of the non-human mammals to evaluate the effectiveness of the candidate substance,
The method according to [8] above, comprising:
[10] The method according to [9] above, wherein the examination of the number of viable optic nerve cells is counting the number of nerve cells in the retinal ganglion.
[10-1] The method according to [9-1] above, wherein the examination of the number of viable optic nerve cells is counting the number of nerve cells in the retinal ganglion.
[11] The control non-human animal is a wild-type non-human animal and / or a genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, is replaced with Ala. The method according to [9-1] or [10-1] above.
[11-1] The method according to [11] above, wherein the Ser in the control non-human animal is the 74th amino acid residue from the N-terminus of the c-Src protein.
[11-3] The method according to [11] or [11-1] above, wherein the control gene-modified non-human mammal has the mutant Src gene in a homozygote.
[12] A genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Ala is introduced into the Src gene.
[12-1] The non-human mammal according to [12] above, wherein the Ser is the 74th amino acid residue from the N-terminus of the c-Src protein.
[12-2] The non-human mammal according to [12] or [12-1] above, wherein the non-human mammal has a mutant Src gene in homozygosity.
本発明によれば、正常眼圧緑内障モデルとして使用し得る新たな非ヒト哺乳動物が提供される。本発明の好ましい態様の非ヒト哺乳動物は、症状の進行が緩やかであるなど、実際のヒト正常眼圧緑内障と同様の症状を示す。 According to the present invention, a new non-human mammal that can be used as a normal-tension glaucoma model is provided. The preferred embodiment of the non-human mammal of the present invention exhibits symptoms similar to those of actual human normal-tension glaucoma, such as a slow progression of symptoms.
以下、本発明について詳細に説明する。本発明の範囲はこれらの説明に拘束されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜変更し実施し得る。なお、本明細書に記載した全ての文献及び刊行物は、その目的にかかわらず参照によりその全体を本明細書に組み込むものとする。 Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these explanations, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention. It should be noted that all documents and publications described in this specification are incorporated herein by reference in their entirety regardless of their purposes.
1. 本発明の概要
本発明は、c-Srcタンパク質のCdk5リン酸化部位であるSerをAsp又はGluに置換する変異をSrc遺伝子に導入した遺伝子改変非ヒト哺乳動物を提供する。このような本発明の非ヒト哺乳動物では、変異型Src遺伝子が発現することでc-Srcタンパク質のCdk5リン酸化部位が恒常的にリン酸化された状態を疑似している。 1. Summary of the Invention The present invention provides a genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Asp or Glu is introduced into the Src gene. In such a non-human mammal of the present invention, the mutant Src gene is expressed to simulate a state in which the Cdk5 phosphorylation site of the c-Src protein is constantly phosphorylated.
後述の実施例に示したように、本発明のいくつかの態様の非ヒト哺乳動物は、(a)神経の炎症をともなわないこと(表1)、(b)障害部が網膜神経節細胞(RGG: retinal ganglion cell)に限定的であること(表1)、(c)経時的に神経障害が進行すること(図1、表1)、(d)眼圧は正常のまま維持されること(図3、表1)、などのヒトの正常眼圧緑内障の症状と同様の特徴を示す。
このため、本発明の好ましい態様の非ヒト哺乳動物は、正常眼圧緑内障モデルとして使用することができる。As shown in the examples below, the non-human mammal of some aspects of the present invention is (a) not accompanied by nerve inflammation (Table 1), (b) the damaged part is a retinal ganglion cell ( RGG: retinal ganglion cell) (Table 1), (c) Neuropathy progresses over time (Figure 1, Table 1), (d) Intraocular pressure remains normal (FIG. 3, Table 1), etc., showing the same characteristics as the symptoms of normal tension glaucoma in humans.
For this reason, the non-human mammal of the preferable aspect of this invention can be used as a normal-tension glaucoma model.
本発明のいくつかの態様の非ヒト哺乳動物で、ヒトの正常眼圧緑内障の症状と同様の特徴を示す理由として、次の仮説が考えられる。ただし、本発明は、以下の仮説に拘束されるものではない。
神経シナプスにおける主たる興奮性神経伝達物質はグルタミン酸である。何らかの原因で過剰に細胞外に放出されたグルタミン酸は、網膜神経節細胞のNMDAレセプター等と結合し、細胞内へのCaイオンの流入を促進する。この結果、網膜神経節細胞の細胞死が誘発される。がん原遺伝子SrcのCdk5によってリン酸化される部位に上記のような点変異を導入することにより、網膜神経節細胞のNMDAレセプターの正常な制御が崩れてCaイオンの流入が促進され、網膜神経節細胞の細胞死が誘発されることにより緑内障が発症すると考えられる。The following hypothesis can be considered as the reason why the non-human mammal of some embodiments of the present invention exhibits characteristics similar to those of normal-tension glaucoma in humans. However, the present invention is not limited to the following hypothesis.
The main excitatory neurotransmitter in the neural synapse is glutamate. Glutamate released excessively for some reason binds to NMDA receptors of retinal ganglion cells and promotes the inflow of Ca ions into the cells. As a result, cell death of retinal ganglion cells is induced. By introducing the above point mutation at the site of proto-oncogene Src phosphorylated by Cdk5, normal control of NMDA receptors in retinal ganglion cells is disrupted, and Ca influx is promoted. It is thought that glaucoma develops when cell death of node cells is induced.
さらに、本発明の好ましい態様の非ヒト哺乳動物を用いることで、正常眼圧緑内障の予防及び／又は治療剤をスクリーニングすることができる。 Furthermore, a prophylactic and / or therapeutic agent for normal tension glaucoma can be screened by using the non-human mammal of a preferred embodiment of the present invention.
2. 変異型Src遺伝子
c-Srcタンパク質は、がん原遺伝子であるSrc遺伝子にコードされている膜結合型タンパク質であり、通常の細胞でチロシンキナーゼとして機能する。例えば、マウスのc-Srcタンパク質は、配列番号：1に示す塩基配列を有するDNA鎖によってコードされ(NCBI Accession No. NM_009271.3)、配列番号：2に示すアミノ酸配列を有するタンパク質である(NCBI Accession No. NP_033297.2)。また、ラットについては、c-Srcタンパク質をコードするDNA配列は、NCBI Accession No. NM_031977.1に示され、c-Srcタンパク質のアミノ酸配列は、NCBI Accession No. NP_114183.1に示されている。 2. Mutant Src gene
c-Src protein is a membrane-bound protein encoded by Src gene, which is a proto-oncogene, and functions as a tyrosine kinase in normal cells. For example, the mouse c-Src protein is a protein encoded by a DNA chain having the base sequence shown in SEQ ID NO: 1 (NCBI Accession No. NM_009271.3) and having the amino acid sequence shown in SEQ ID NO: 2 (NCBI Accession No. NP_033297.2). For rats, the DNA sequence encoding c-Src protein is shown in NCBI Accession No. NM_031977.1, and the amino acid sequence of c-Src protein is shown in NCBI Accession No. NP_114183.1.
本発明の変異型Src遺伝子は、上記c-Srcタンパク質のCdk5リン酸化部位であるSerが、Asp又はGluに変異した遺伝子である。c-Srcタンパク質のCdk5リン酸化部位であるSerは、公知の方法で同定することができ、例えば、Shenoy, S. et al., “Purified maturation promoting factor phosphorylates pp60c-src at the sites phosphorylated during fibroblast mitosis”Cell 57, pp763-774、Kato,G. and Maeda, S., “Neuron-specific Cdk5 kinase is responsible for mitosis-independent phosphorylation of c-Src at Ser75 in human Y79 retinoblastoma cells”J. Biochem. 126, pp957-961 などに記載の方法又はこれに準ずる方法により同定することができる。
c-Srcタンパク質のCdk5リン酸化部位であるSerは、例えば、マウスではN末端から74番目のアミノ酸残基、すなわち、配列番号:2のアミノ酸配列では、N末端から74番目のアミノ酸残基である。このアミノ酸残基は、配列番号:1の塩基配列では、5'末端から604番目〜606番目のコドンに対応する。ここで、マウスc-Srcタンパク質におけるN末端から74番目のアミノ酸残基のSerは、ヒトの対応する75番目のSerでCdk5リン酸化部位であることが初めて明らかにされたことから一般に「Ser75」と呼ばれており、本明細書中でも「Ser75」という場合がある。
また、c-Srcタンパク質のCdk5リン酸化部位であるSerは、ラットでは、N末端から75番目のアミノ酸残基である。The mutant Src gene of the present invention is a gene in which Ser that is a Cdk5 phosphorylation site of the c-Src protein is mutated to Asp or Glu. Ser, which is a Cdk5 phosphorylation site of c-Src protein, can be identified by a known method. For example, Shenoy, S. et al., “Purified maturation promoting factor phosphorylates pp60 c-src at the sites phosphorylated during fibroblast mitosis ”Cell 57, pp763-774, Kato, G. and Maeda, S.,“ Neuron-specific Cdk5 kinase is responsible for mitosis-independent phosphorylation of c-Src at Ser75 in human Y79 retinoblastoma cells ”J. Biochem. 126, It can be identified by the method described in pp957-961 or the like or a method analogous thereto.
Ser, which is the Cdk5 phosphorylation site of c-Src protein, is, for example, the 74th amino acid residue from the N-terminal in mice, that is, the 74th amino acid residue from the N-terminal in the amino acid sequence of SEQ ID NO: 2. . This amino acid residue corresponds to codons 604 to 606 from the 5 ′ end in the nucleotide sequence of SEQ ID NO: 1. Here, the Ser at the 74th amino acid residue from the N-terminus in the mouse c-Src protein was first revealed to be the Cdk5 phosphorylation site at the corresponding 75th Ser in humans. And is sometimes referred to as “Ser75” in this specification.
In addition, Ser, the Cdk5 phosphorylation site of the c-Src protein, is the 75th amino acid residue from the N-terminus in rats.
本発明では、c-Srcタンパク質のCdk5リン酸化部位であるSerは、Serのリン酸化状態を疑似できるアミノ酸と置換している。そのようなアミノ酸は、Asp又はGluであり、好ましくはAspである。
具体的には、発明の変異型Src遺伝子では、Src遺伝子の前記Serをコードするコドン「AGC」、「AGT」、「TCA」、「TCC」、「TCG」又は「TCT」が、Aspをコードするコドン「GAC」又は「GAT」、あるいはGluをコードするコドン「GAA」又は「GAG」に変異している。本発明のいくつかの態様では、Src遺伝子の前記Serをコードするコドン「TCC」が、Aspをコードするコドン「GAC」又は「GAT」に、好ましくは、コドン「GAC」に変異している。In the present invention, Ser, which is a Cdk5 phosphorylation site of c-Src protein, is substituted with an amino acid that can simulate the phosphorylation state of Ser. Such an amino acid is Asp or Glu, preferably Asp.
Specifically, in the mutant Src gene of the invention, the codons “AGC”, “AGT”, “TCA”, “TCC”, “TCG” or “TCT” encoding the Ser of the Src gene encode Asp. The codons “GAC” or “GAT”, or the codons “GAA” or “GAG” encoding Glu. In some embodiments of the invention, the codon “TCC” encoding Ser of the Src gene is mutated to codon “GAC” or “GAT” encoding Asp, preferably codon “GAC”.
本発明の変異型Src遺伝子は、更にはそのゲノム配列が、その機能が維持される範囲内で上記Cdk5リン酸化部位のアミノ酸残基以外にも変異していること、例えば塩基やアミノ酸残基の置換、欠失、付加、又は挿入を受けていることもあり、本発明では、その様な変異体、例えば上記のコード核酸配列において、上記Cdk5リン酸化部位以外の部位で、例えば1〜10個、好ましくは1〜5個の塩基が置換、欠失、付加、又は挿入している変異体、あるいは上記アミノ酸配列において、上記Cdk5リン酸化部位以外の部位で、例えば1〜10個、好ましくは1〜5個のアミノ酸が置換、欠失、付加、又は挿入している変異体なども、本発明の変異型Src遺伝子に含まれる。 The mutant Src gene of the present invention is further mutated in addition to the amino acid residue of the Cdk5 phosphorylation site within the range in which the function of the mutant Src gene is maintained. The present invention may have undergone substitution, deletion, addition, or insertion. In the present invention, in such a mutant, for example, the above-described coding nucleic acid sequence, at a site other than the Cdk5 phosphorylation site, for example, 1 to 10 Preferably 1 to 5 nucleotides substituted, deleted, added or inserted, or in the amino acid sequence other than the Cdk5 phosphorylation site, for example 1 to 10, preferably 1 Mutants in which ˜5 amino acids are substituted, deleted, added, or inserted are also included in the mutant Src gene of the present invention.
本発明において、Src遺伝子に変異が導入された遺伝子改変非ヒト哺乳動物は、1つの内在性Src遺伝子に変異が導入されているヘテロ接合型、及び2つの内在性Src遺伝子に変異が導入されているホモ接合型を包含するが、本発明では、ホモ接合型がより好ましい。 In the present invention, the genetically modified non-human mammal in which a mutation is introduced into the Src gene is heterozygous in which the mutation is introduced into one endogenous Src gene, and the mutation is introduced into two endogenous Src genes. In the present invention, the homozygous type is more preferable.
この様な内在性Src遺伝子への変異の導入を、公知の遺伝子改変非ヒト哺乳動物の作製方法、例えば、ジーンターゲッティング法などにより達成することができる。 Such introduction of mutations into the endogenous Src gene can be achieved by a known method for producing a genetically modified non-human mammal, such as a gene targeting method.
3．本発明の非ヒト哺乳動物
本発明の非ヒト哺乳動物は、c-Srcタンパク質のCdk5リン酸化部位であるSerをAsp又はGluに置換する変異をSrc遺伝子に導入した遺伝子改変非ヒト哺乳動物である。本発明の非ヒト哺乳動物は、相同染色体上の1又は2つの内在性Src遺伝子が本発明の変異型Src遺伝子と置換されている。すなわち、本発明の非ヒト哺乳動物は、本発明の変異型Src遺伝子のヘテロ接合体又はホモ接合体であり、好ましくは、本発明の変異型Src遺伝子のホモ接合体である。 3． Non-human mammal of the present invention The non-human mammal of the present invention is a genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Asp or Glu is introduced into the Src gene. . In the non-human mammal of the present invention, one or two endogenous Src genes on the homologous chromosome are replaced with the mutant Src gene of the present invention. That is, the non-human mammal of the present invention is a heterozygote or homozygote of the mutant Src gene of the present invention, preferably a homozygote of the mutant Src gene of the present invention.
本発明の変異型Src遺伝子を有する非ヒト哺乳動物は、Src遺伝子を有するヒト以外の哺乳動物であれば、いかなる哺乳動物でもよい。そのような哺乳動物としては、例えば、ウシ、ミニブタ、ブタ、ヒツジ、ヤギ、ウサギ、イヌ、ネコ、モルモット、ハムスター、マウス、ラット、サル等が用いられる。非ヒト哺乳動物のなかでも、病態動物モデル系の作製の面から個体発生及び生物サイクルが比較的短く、また繁殖が容易なげっ歯動物、とりわけマウス又はラットが好ましく、マウスがより好ましい。 The non-human mammal having the mutant Src gene of the present invention may be any mammal as long as it is a mammal other than a human having the Src gene. Examples of such mammals include cattle, minipigs, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, monkeys, and the like. Among non-human mammals, rodents, particularly mice or rats, which have relatively short ontogeny and biological cycle and are easy to breed, are preferable from the viewpoint of producing a pathological animal model system, and mice are more preferable.
本発明のいくつかの態様の非ヒト哺乳動物は、1)その眼圧が正常範囲にあり、かつ2)その網膜神経節の細胞数が、野生型非ヒト哺乳動物に比べて減少している。「野生型非ヒト哺乳動物」は、例えば、緑内障を患っていない非ヒト哺乳動物であり、好ましくは、変異型Src遺伝子のヘテロ接合体マウスの交配により得られた野生型（正常型）Src遺伝子を有する緑内障を患っていない非ヒト哺乳動物である。 The non-human mammal of some embodiments of the present invention has 1) its intraocular pressure in the normal range, and 2) its retinal ganglion cell count is reduced compared to a wild-type non-human mammal. . The “wild-type non-human mammal” is, for example, a non-human mammal that does not suffer from glaucoma, and preferably a wild-type (normal) Src gene obtained by mating heterozygous mice of a mutant Src gene A non-human mammal having no glaucoma.
「その眼圧が正常範囲」とは、本発明の非ヒト哺乳動物の眼圧の測定値が、野生型非ヒト哺乳動物の眼圧の正常値の範囲内に収まることを意味する。例えば、野生型マウスの眼圧の正常値は、通常、10〜21 mmHgであり、本発明のマウスでもまた、その眼圧は正常範囲内にあるのが好ましい。ただし、個体により、上記範囲を逸脱することもあるが、高眼圧といわれる範囲、例えば30mmHg以上にまで達するものではない。非ヒト哺乳動物の眼圧は、例えば電子眼圧計を用いて測定することができる。 “The intraocular pressure is in a normal range” means that the measured value of the intraocular pressure of the non-human mammal of the present invention falls within the normal value of the intraocular pressure of the wild-type non-human mammal. For example, the normal value of intraocular pressure of a wild type mouse is usually 10 to 21 mmHg, and the intraocular pressure of the mouse of the present invention is preferably within the normal range. However, although it may deviate from the above range depending on the individual, it does not reach a range called high intraocular pressure, for example, 30 mmHg or more. The intraocular pressure of the non-human mammal can be measured using, for example, an electronic tonometer.
また、好ましくは、本発明の非ヒト哺乳動物では、その網膜神経節の神経細胞数が、野生型非ヒト哺乳動物に比べて減少しており、例えば、野生型非ヒト哺乳動物に比べて、好ましくは少なくとも20%ほど、より好ましくは少なくとも40%ほど、さらに好ましくは50%ほど減少している。網膜神経節の神経細胞数の減少は、慣用的な組織化学的な手法により、例えば切片を用いたヘマトキシリン/エオシン染色により、顕微鏡下に測定することができる。 Preferably, in the non-human mammal of the present invention, the number of neurons in the retinal ganglion is reduced compared to a wild-type non-human mammal, for example, compared to a wild-type non-human mammal, Preferably, it is reduced by at least 20%, more preferably by at least 40%, and even more preferably by 50%. The decrease in the number of neurons in the retinal ganglion can be measured under a microscope by a conventional histochemical method, for example, by hematoxylin / eosin staining using a section.
また、好ましくは、本発明の非ヒト哺乳動物及び野生型非ヒト哺乳動物から採取した網膜神経節の神経細胞をそれぞれ培養した時に、本発明の非ヒト哺乳動物の培養細胞生存率が、野生型非ヒト哺乳動物に比べて、低下しており、例えば、野生型非ヒト哺乳動物に比べて、好ましくは少なくとも20%ほど、より好ましくは少なくとも40%ほど、さらに好ましくは50%ほど低下している。 Preferably, when the retinal ganglion nerve cells collected from the non-human mammal and wild-type non-human mammal of the present invention are cultured, the cultured cell viability of the non-human mammal of the present invention is wild-type. Reduced compared to non-human mammals, for example, preferably reduced by at least about 20%, more preferably at least about 40%, and even more preferably about 50% compared to wild-type non-human mammals .
この網膜神経節の神経細胞数の減少及び培養細胞生存率の低下は、神経変性又は神経細胞死ととらえることができる。一般に、正常眼圧緑内障を含む緑内障では、視神経乳頭部の萎縮や網膜神経繊維の欠損が認められており、現在では眼圧依存性にせよ非依存性にせよ緑内障の最終病像は、網膜神経節細胞死であると考えられている。 This decrease in the number of neurons in the retinal ganglion and the decrease in cell viability can be regarded as neurodegeneration or neuronal cell death. In general, glaucoma, including normal-tension glaucoma, has atrophy of the optic nerve head and deficits in retinal nerve fibers.Currently, the final image of glaucoma, whether dependent on intraocular pressure or independent, is It is thought to be node cell death.
従って、本発明の好ましい態様の非ヒト哺乳動物は、その眼の性状、すなわち眼圧が正常範囲にあること、及び網膜神経節の細胞数が減少していること、また、培養細胞生存率が低下していることを考慮すると、正常眼圧緑内障のモデル非ヒト哺乳動物として使用することができる。 Therefore, the non-human mammal of a preferred embodiment of the present invention has an eye property, that is, that the intraocular pressure is in a normal range, the number of cells in the retinal ganglion is decreased, and the cultured cell viability In consideration of the decrease, it can be used as a model non-human mammal of normal pressure glaucoma.
4. 本発明の非ヒト哺乳動物の作製
本発明は、本発明の非ヒト哺乳動物の作製方法を提供する。本発明の非ヒト哺乳動物は、例えば、標準的な方法で作製することができる。
当該方法は、
(a)遺伝子のターゲティングにより相同染色体上の内在性Src遺伝子を本発明の変異型Src遺伝子に変異させたES細胞を作製すること、
(b)ES細胞を用いて、キメラ非ヒト哺乳動物を作製すること、
(c)キメラ非ヒト哺乳動物を野生型非ヒト哺乳動物と交配してヘテロ接合体非ヒト哺乳動物を作製すること、
(d)ヘテロ接合体非ヒト哺乳動物同士を交配して、ホモ接合体非ヒト哺乳動物を作製すること、を含む。
以下、上記各工程について詳細に説明する。 4. Production of non-human mammal of the present invention The present invention provides a method for producing the non-human mammal of the present invention. The non-human mammal of the present invention can be produced, for example, by a standard method.
The method is
(a) producing an ES cell in which an endogenous Src gene on a homologous chromosome is mutated to the mutant Src gene of the present invention by gene targeting;
(b) producing a chimeric non-human mammal using ES cells;
(c) crossing a chimeric non-human mammal with a wild-type non-human mammal to produce a heterozygous non-human mammal;
(d) crossing heterozygous non-human mammals to produce homozygous non-human mammals.
Hereafter, each said process is demonstrated in detail.
工程(a): 遺伝子ターゲティング
遺伝子ターゲティング法では、ES細胞内の染色体上のSrc遺伝子を本発明の変異型Src遺伝子に変異させるために、ターゲティングベクターを用いる。
Src遺伝子を本発明の変異型Src遺伝子に変異させるためには、Src遺伝子中のCdkリン酸化部位であるSerをコードする部分に、例えば、点変異を導入する。点変異の導入は、公知の部位特異的突然変異誘発法(例えば、Deng, W.P. et al., “Site-directed mutagenesis of virtually any plasmid by eliminating a unique site”, Anal. Biochem. 200, pp81-88に記載の方法、文献Ling, M.M. and Robinson, B.H., “Approaches to DNA mutagenesis: An overview”, Anal. Biochem. 254, pp157-178に記載の方法、又はそられに準じる方法等)により行うことができる。点変異の導入は、市販のキット、例えば、MutanTM-super Express Km（宝酒造（株））、MutanTM-K（宝酒造（株））等を用いて行うこともできる。 Step (a): In the gene targeting gene targeting method, a targeting vector is used to mutate the Src gene on the chromosome in the ES cell to the mutant Src gene of the present invention.
In order to mutate the Src gene into the mutant Src gene of the present invention, for example, a point mutation is introduced into a portion encoding Ser, which is a Cdk phosphorylation site in the Src gene. Point mutations can be introduced by known site-directed mutagenesis methods (eg, Deng, WP et al., “Site-directed mutagenesis of virtually any plasmid by using a unique site”, Anal. Biochem. 200, pp81-88. The method described in the literature, Ling, MM and Robinson, BH, “Approaches to DNA mutagenesis: An overview”, Anal. Biochem. 254, pp157-178, or a method according to the method) it can. Point mutations can also be introduced using commercially available kits such as Mutan ™ -super Express Km (Takara Shuzo), Mutan ™ -K (Takara Shuzo).
また、ターゲティングベクターは、相同組換えを起こさせた後の組換え体のスクリーニングが容易となるように構築することが好ましい。例えば、ポジティブ−ネガティブ選択をするために、ベクターに薬剤耐性遺伝子又は毒素遺伝子などの選択マーカーを連結することができる。ポジティブ−ネガティブ選別法は、当分野において周知である。すなわち、ポジティブ選別は、選択マーカー遺伝子が組み込まれなかった細胞は、薬剤を含む培養液で培養すると、耐性遺伝子を含まないために死ぬことを利用するものであり、ネガティブ選別法は、組み込みがランダムに起こった細胞では、ネガティブ選別用遺伝子が発現するために、細胞が死ぬことを利用するものである。その結果、相同組換えを起こした細胞のみが生き残り、選別される。 In addition, the targeting vector is preferably constructed so as to facilitate screening of recombinants after homologous recombination has occurred. For example, a selection marker such as a drug resistance gene or a toxin gene can be linked to the vector for positive-negative selection. Positive-negative selection methods are well known in the art. In other words, positive selection utilizes the fact that cells that have not incorporated the selectable marker gene die when they are cultured in a culture medium containing a drug because they do not contain a resistance gene, and the negative selection method uses random integration. In the cells that occurred in the above, the fact that the cells die is used to express the negative selection gene. As a result, only cells that have undergone homologous recombination survive and are selected.
選択マーカー遺伝子は、ポジティブ選択用として例えばネオマイシン耐性遺伝子(neo)、ハイグロマイシンBホスホトランスフェラーゼ遺伝子などを使用することができ、ネガティブ選択用として例えばヘルペスウイルスチミジンキナーゼ遺伝子(HSV-tk)、ジフテリア毒素A遺伝子などを使用することができる。 As the selection marker gene, for example, neomycin resistance gene (neo), hygromycin B phosphotransferase gene and the like can be used for positive selection, and for example, herpesvirus thymidine kinase gene (HSV-tk), diphtheria toxin A Genes and the like can be used.
上記の方法により作製したターゲティングベクターを用いて、相同組換えを行う。現在確立されている遺伝子ターゲティング法では、本発明の変異型Src遺伝子を有する非ヒト哺乳動物を作製する場合には、ES細胞を使用することが望ましい。ES細胞として、マウスの場合には、CCE、E14、J1、TT2、D3、BL/6-III等を適宜選択して使用することができる。 Homologous recombination is performed using the targeting vector prepared by the above method. In the currently established gene targeting method, it is desirable to use ES cells when producing a non-human mammal having the mutant Src gene of the present invention. In the case of mice, ES cells such as CCE, E14, J1, TT2, D3, BL / 6-III and the like can be appropriately selected and used as ES cells.
相同組換えを起こさせるために、上記ターゲティングベクターを細胞中に導入する。ターゲティングベクターを細胞に導入する方法としては、エレクトロポレーション法、リン酸カルシウム法、DEAE−デキストラン法、リポソーム法などを使用することができる。効率、作業の容易性などを考慮して、エレクトロポレーション法を用いることが好ましい。そして細胞中の目的とするゲノムDNA配列を、ターゲティングベクターの相同組換えによって置換する。 In order to cause homologous recombination, the targeting vector is introduced into cells. As a method for introducing a targeting vector into a cell, an electroporation method, a calcium phosphate method, a DEAE-dextran method, a liposome method, or the like can be used. The electroporation method is preferably used in consideration of efficiency, ease of work, and the like. Then, the target genomic DNA sequence in the cell is replaced by homologous recombination of the targeting vector.
その後、選択マーカー遺伝子は、除去するようにしてもよい。選択マーカーの除去は、Askew GR, Doetschman T, Lingrel JB. Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy. Mol Cell Biol. 1993;13(7):4115-24.、Wu H, Liu X, Jaenisch R. Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells. Proc Natl Acad Sci U S A. 1994;91(7):2819-23.などに記載のdouble replacement/tag-and-exchange procedure； Giese KP, Fedorov NB, Filipkowski RK, Silva AJ. Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning. Science. 1998;279(5352):870-3.などに記載のCre/loxP procedure； Horie K, Maeda S, Nishiguchi S, Gottesman ME, Shimada K. A replacement vector used to introduce subtle mutations into mouse genes. Gene. 1995;166(2):197-204.などに記載のreplacement and excision procedure； などにより行うことができる。 Thereafter, the selectable marker gene may be removed. Selective marker removal is done by Askew GR, Doetschman T, Lingrel JB. Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy. Mol Cell Biol. 1993; 13 (7): 4115-24 ., Wu H, Liu X, Jaenisch R. Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells.Proc Natl Acad Sci US A. 1994; 91 (7): Double replacement / tag-and-exchange procedure as described in 2819-23; Giese KP, Fedorov NB, Filipkowski RK, Silva AJ. Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning. Science. 1998; 279 (5352): 870-3., Etc .; Horie K, Maeda S, Nishiguchi S, Gottesman ME, Shimada K. A replacement vector used to introduce subtle mutations into mouse genes. Gene. 1995; 166 (2): It can be performed by the replacement and excision procedure described in 197-204.
Src遺伝子がターゲティングされているか否かは、PCR法、サザンブロット法等により確認することができる。 Whether the Src gene is targeted or not can be confirmed by PCR, Southern blotting, or the like.
工程（b):キメラ非ヒト哺乳動物の作製
相同組換えの結果得られた組換えES細胞を、8細胞期又は胚盤胞の胚内に移植する。このES細胞移植胚を偽妊娠仮親の子宮内に移植して出産させることによりキメラ動物を作製する。
ES細胞を胚内に移植する方法として、マイクロインジェクション法、凝集法などの公知の手法を用いることができる。 Step (b): Production of chimeric non-human mammal Recombinant ES cells obtained as a result of homologous recombination are transplanted into the 8-cell stage or blastocyst embryo. This ES cell transplanted embryo is transplanted into the womb of a pseudopregnant foster parent to give birth to a chimeric animal.
As a method for transplanting ES cells into the embryo, known methods such as a microinjection method and an aggregation method can be used.
マウスの場合、まず、ホルモン剤により過排卵処理を施した雌マウスを、雄マウスと交配させる。その後、8細胞期胚を用いる場合には受精から2.5日目に、胚盤胞を用いる場合には受精から3.5日目に、それぞれ卵管又は子宮から初期発生胚を回収する。回収した胚に、相同組換えを行ったES細胞を注入し、キメラ胚を作製する。 In the case of a mouse, first, a female mouse subjected to superovulation treatment with a hormonal agent is mated with a male mouse. Thereafter, early embryos are collected from the oviduct or uterus on day 2.5 after fertilization when using 8-cell stage embryos, and on day 3.5 after fertilization when using blastocysts. ES cells that have undergone homologous recombination are injected into the collected embryos to produce chimeric embryos.
一方、仮親にするための偽妊娠雌マウスは、正常性周期の雌マウスを、精管結紮などにより去勢した雄マウスと交配することにより得ることができる。作出した偽妊娠マウスに対して、上述の方法により作製したキメラ胚を子宮内移植し、その後出産させることによりキメラマウスを作製することができる。 On the other hand, a pseudopregnant female mouse for use as a foster parent can be obtained by mating a female mouse having a normal cycle with a male mouse castrated by vagina ligation or the like. A chimeric mouse can be produced by transplanting the chimeric embryo produced by the above-mentioned method in utero to the produced pseudopregnant mouse and then giving birth.
マウス以外の非ヒト哺乳動物についても、上記と同様にして、キメラ非ヒト哺乳動物を作製することができる。 For non-human mammals other than mice, chimeric non-human mammals can be prepared in the same manner as described above.
工程(c):ヘテロ接合体非ヒト哺乳動物の作製
上記のようにして得たキメラ非ヒト哺乳動物の中から、ES細胞移植胚由来の雄キメラ非ヒト哺乳動物を選択する。マウスの場合、選択したES細胞移植胚由来の雄マウスが成熟した後、このマウスを純系マウス系統の雌マウスと交配させる。そして、誕生した子マウスに、ES細胞に由来するマウス（ES細胞に組み込まれたゲノムを有していたマウス）の被毛色が現れることにより、ES細胞がキメラマウスの生殖系列へ導入されたことを確認することができる。そして、胚内に移植された組換えES細胞が生殖系列に導入されたヘテロ接合体非ヒト哺乳動物を繁殖する。 Step (c): Production of heterozygous non-human mammal A male chimeric non-human mammal derived from an ES cell-transplanted embryo is selected from the chimeric non-human mammal obtained as described above. In the case of a mouse, after the male mouse derived from the selected ES cell transplanted embryo matures, this mouse is mated with a female mouse of a pure mouse strain. In addition, ES cells were introduced into the germ line of chimeric mice by the appearance of the coat color of the mouse derived from the ES cell (the mouse that had the genome incorporated into the ES cell) in the born mouse. Can be confirmed. Then, a heterozygous non-human mammal in which the recombinant ES cells transplanted into the embryo are introduced into the germ line is bred.
工程（d）：ホモ接合体非ヒト哺乳動物の作製
上記のようにして得たヘテロ接合体非ヒト哺乳動物同士を交配させて、ホモ接合体非ヒト哺乳動物を得ることができる。 Step (d): Production of homozygous non-human mammal A heterozygous non-human mammal can be obtained by mating the heterozygous non-human mammals obtained as described above.
工程（c）又は（d）においてヘテロ接合体又はホモ接合体非ヒト哺乳動物が得られたことの確認については、組織から染色体DNAを抽出しサザンブロット法やPCR法で行うことができる。さらに、組織からRNAを抽出し、ノーザンブロット解析により遺伝子の発現パターンを解析することもできる。本発明の変異Src遺伝子がコードするタンパク質に対する抗体を用いてウエスタンブロッティングを行なってもよい。 Confirmation that a heterozygous or homozygous non-human mammal has been obtained in step (c) or (d) can be carried out by extracting chromosomal DNA from the tissue and performing Southern blotting or PCR. In addition, RNA can be extracted from the tissue and the gene expression pattern can be analyzed by Northern blot analysis. Western blotting may be performed using an antibody against the protein encoded by the mutant Src gene of the present invention.
また、樹立した動物系統について、ヘテロ接合体非ヒト哺乳動物及びホモ接合体非ヒト哺乳動物の表現型を解析することもできる。表現型の解析は、肉眼的観察、解剖による内部の観察、各臓器の組織切片、X線撮影による観察、行動や記憶の観察、血液検査や血清生化学検査、眼圧検査、細隙灯検査、眼底検査などで行う。解析時期は、胎生期から成体までの任意の時期であってよく、特に限定されるものではない。 In addition, phenotypes of heterozygous non-human mammals and homozygous non-human mammals can be analyzed for the established animal lines. Phenotype analysis includes macroscopic observation, internal observation by dissection, tissue section of each organ, observation by X-ray photography, observation of behavior and memory, blood test, serum biochemical test, intraocular pressure test, slit lamp test This is done by examining the fundus. The analysis period may be any period from the embryonic period to the adult period, and is not particularly limited.
さらに、上記の通りに作製したホモ接合体又はヘテロ接合体において、その眼圧が正常範囲にあること、及び網膜神経節の神経細胞が減少していることを確認する。
本発明の非ヒト哺乳動物が、マウスの場合、その眼圧は、通常約21mmHg以下、例えば約10〜21mmHgである。用いたES細胞の起源であるマウスの系統、又はキメラ胚の作製に用いた正常胚の起源であるマウスの系統によって、この眼圧範囲は多少変動しうるが、それを考慮しても。30mmHg未満であるのが好ましい。眼圧の測定方法は、公知の慣用的な方法を用いて行うことができる。より具体的には、例えば、後述の実施例に記載の方法により行うことができる。Furthermore, in the homozygote or heterozygote prepared as described above, it is confirmed that the intraocular pressure is in the normal range and that the nerve cells of the retinal ganglion are reduced.
When the non-human mammal of the present invention is a mouse, its intraocular pressure is usually about 21 mmHg or less, for example, about 10 to 21 mmHg. This intraocular pressure range may vary somewhat depending on the strain of mouse that is the origin of the ES cells used, or the strain of the mouse that is the origin of the normal embryo that was used to generate the chimeric embryo. Preferably it is less than 30 mmHg. The intraocular pressure can be measured using a known and common method. More specifically, for example, it can be carried out by the method described in the examples described later.
本発明の非ヒト哺乳動物では、その網膜神経節の神経細胞数が、野生型マウスに比べて減少しており、例えば、少なくとも20%、より好ましくは少なくとも50%ほど減少している。網膜神経節細胞数の計測方法も、公知の慣用的な方法を用いて行うことができる。より具体的には、例えば、後述の実施例に記載の方法により行うことができる。 In the non-human mammal of the present invention, the number of nerve cells in the retinal ganglion is reduced as compared to the wild type mouse, for example, at least 20%, more preferably at least 50%. A method for measuring the number of retinal ganglion cells can also be performed using a known and conventional method. More specifically, for example, it can be carried out by the method described in the examples described later.
5.正常眼圧緑内障の予防及び／又は治療剤のスクリーニング方法
本発明の非ヒト哺乳動物は、正常眼圧緑内障を予防及び／又は治療剤、特には網膜神経節の神経細胞を含む、視神経細胞の死若しくは変性、又はその機能の低下を抑制するために有効な薬剤、あるいは視神経細胞又はその機能を回復させるために有効な薬剤をスクリーニングするために用いることができる。 5. Method for Screening Agent for Prevention and / or Treatment of Normal Tension Glaucoma The non-human mammal of the present invention is a optic nerve cell comprising a nerve agent for prevention and / or treatment of normal tension glaucoma, particularly retinal ganglion. It can be used for screening an agent effective for suppressing death or degeneration of cerebral dysfunction, or a decrease in its function, or an agent effective for restoring optic nerve cells or its function.
従って、本発明は、正常眼圧緑内障の予防及び／又は治療剤のスクリーニング方法であって、
1)本発明の非ヒト哺乳動物及び野生型非ヒト哺乳動物に候補物質を投与すること、
2)前記各非ヒト哺乳動物において、投与前、及び投与してから一定期間後に、生存する視神経細胞の数量を検査すること、及び
3)前記各非ヒト哺乳動物の検査結果を比較して、候補物質の有効性を評価すること、
を含んで成るスクリーニング方法を提供する。Therefore, the present invention is a screening method for a preventive and / or therapeutic agent for normal pressure glaucoma,
1) administering a candidate substance to the non-human mammal and wild-type non-human mammal of the present invention;
2) In each non-human mammal, examining the number of optic nerve cells surviving before administration and after a certain period of time after administration; and
3) comparing the test results of each of the non-human mammals to evaluate the effectiveness of the candidate substance,
A screening method comprising:
本発明の別のいくつかの態様は、正常眼圧緑内障の予防及び／又は治療剤のスクリーニング方法であって、
1)本発明の非ヒト哺乳動物及び対照非ヒト哺乳動物に候補物質を投与すること、
2)前記各非ヒト哺乳動物において、投与前、及び投与してから一定期間後に、生存する視神経細胞の数量を検査すること、及び
3)前記各非ヒト哺乳動物の検査結果を比較して、候補物質の有効性を評価すること、
を含んで成るスクリーニング方法を提供する。Another aspect of the present invention is a screening method for a prophylactic and / or therapeutic agent for normal-tension glaucoma,
1) administering a candidate substance to the non-human mammal of the present invention and a control non-human mammal;
2) In each non-human mammal, examining the number of optic nerve cells surviving before administration and after a certain period of time after administration; and
3) comparing the test results of each of the non-human mammals to evaluate the effectiveness of the candidate substance,
A screening method comprising:
前記対照非ヒト哺乳動物は、野生型非ヒト動物及び/又はc-Srcタンパク質のCdk5リン酸化部位であるSerをAlaに置換する変異をSrc遺伝子に導入した遺伝子改変非ヒト哺乳動物である。好ましくは、前記対照非ヒト哺乳動物は、c-Srcタンパク質のCdk5リン酸化部位であるSerをAlaに置換する変異をSrc遺伝子に導入した遺伝子改変非ヒト哺乳動物である。対照として前記遺伝子改変非ヒト哺乳動物を用いることで、Src遺伝子により特異的な正常眼圧緑内障の予防及び／又は治療剤をスクリーニングすることができる。
c-Srcタンパク質のCdk5リン酸化部位であるSerは、前記説明したのと同様である。前記対照非ヒト哺乳動物では、c-Srcタンパク質のCdk5リン酸化部位であるSerは、Serの非リン酸化状態を疑似できるアミノ酸と置換している。そのようなアミノ酸は、Alaである。
対照として用いられる前記遺伝子改変非ヒト哺乳動物は、前記本発明の非ヒト哺乳動物を作製する方法に準ずる方法により作製することができる。The control non-human mammal is a wild-type non-human animal and / or a genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Ala is introduced into the Src gene. Preferably, the control non-human mammal is a genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Ala is introduced into the Src gene. By using the genetically modified non-human mammal as a control, a prophylactic and / or therapeutic agent for normal tension glaucoma specific to the Src gene can be screened.
Ser which is a Cdk5 phosphorylation site of c-Src protein is the same as described above. In the control non-human mammal, Ser, which is the Cdk5 phosphorylation site of c-Src protein, is substituted with an amino acid that can mimic the non-phosphorylation state of Ser. Such an amino acid is Ala.
The genetically modified non-human mammal used as a control can be produced by a method according to the method for producing the non-human mammal of the present invention.
候補物質が、正常眼圧緑内障の予防及び／又は治療にとって有効であるか否かは、緑内障に特徴的な徴候を改善できるか否かを検査することのよって判定することができる。例えば、網膜神経節の神経細胞数を計数することにより、試験化合物の投与により、対照マウスに比べて、その数が少なくとも10％、好ましくは少なくとも20％、より好ましくは少なくとも30％回復していれば、その試験化合物は、医薬上有効であると判定してよい。 Whether or not a candidate substance is effective for the prevention and / or treatment of normal-tension glaucoma can be determined by examining whether or not the symptoms characteristic of glaucoma can be improved. For example, by counting the number of neurons in the retinal ganglion, administration of the test compound can restore that number by at least 10%, preferably at least 20%, more preferably at least 30% compared to control mice. For example, the test compound may be determined to be pharmaceutically effective.
本発明のいくつかの態様の非ヒト哺乳動物は、出生後、月齢と共に網膜神経節の細胞数が徐々に低下していくことが判明している（図1）。従って、上記スクリーニング方法の1つの態様として、1）出生後直ぐに、1群の本発明の非ヒト哺乳動物には定期的に候補物質を投与し、もう1群の本発明の非ヒト哺乳動物には候補物質を投与せず、2）各月齢で、網膜神経節の細胞数を測定し、そして3）両者の比較から、網膜神経節の細胞数の経時的な低下を抑制する候補物質を選別することもできる。 It has been found that the number of retinal ganglion cells gradually decreases with age in non-human mammals according to some embodiments of the present invention (FIG. 1). Therefore, as one embodiment of the above screening method, 1) a candidate substance is regularly administered to a group of non-human mammals of the present invention immediately after birth, and another group of non-human mammals of the present invention is administered. Do not administer the candidate substance, 2) measure the number of cells in the retinal ganglion at each age, and 3) select candidate substances that suppress the decrease in the number of retinal ganglion cells over time from the comparison of both You can also
候補物質としては、例えば、ペプチド、タンパク、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、細胞培養上清、植物抽出液、哺乳動物の組織抽出液、血漿などが挙げられ、これら化合物は新規な化合物であってもよいし、公知の化合物であってもよい。これら候補物質は塩を形成していてもよく、候補物質の塩としては、生理学的に許容される酸（例えば、有機酸又は無機酸など）や塩基（例えば、金属酸など）などとの塩が用いられ、とりわけ生理学的に許容される酸付加塩が好ましい。この様な塩としては、例えば、無機酸（例えば、塩酸、リン酸、臭化水素酸、硫酸など）との塩、或いは有機酸（例えば、酢酸、ギ酸、プロピオン酸、フマル酸、マレイン酸、コハク酸、酒石酸、クエン酸、リンゴ酸、蓚酸、安息香酸、メタンスルホン酸、ベンゼンスルホン酸など）との塩などが用いられる。また、候補物質は、所望のタンパク質を発現するための遺伝子ベクターであってもよい。 Candidate substances include, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts, plasma, etc. The compound may be a novel compound or a known compound. These candidate substances may form salts, and the salts of candidate substances include salts with physiologically acceptable acids (for example, organic acids or inorganic acids) and bases (for example, metal acids). And physiologically acceptable acid addition salts are particularly preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like. The candidate substance may be a gene vector for expressing a desired protein.
候補物質の投与経路も、候補物質の性質が許す限り、種々の方法を試みてよく、例えば点眼や経口投与によることもできる。投与期間や投与様式も、候補物質の効果を最大限にする様に選択される。この様な候補物質の種類や投与方法は、製薬分野又は医学分野における通常の方法に従ってもよい。 As for the route of administration of the candidate substance, various methods may be used as long as the nature of the candidate substance permits, for example, eye drops or oral administration. The administration period and administration mode are also selected so as to maximize the effect of the candidate substance. Such candidate substance types and administration methods may be in accordance with conventional methods in the pharmaceutical or medical fields.
更に、本発明の非ヒト哺乳動物を、他の種類の疾患モデル非ヒト哺乳動物と交配して、新たな疾患モデル非ヒト哺乳動物を作製することもできる。この様な、疾患モデル非ヒト哺乳動物の使用もまた本発明に含まれる。 Furthermore, the non-human mammal of the present invention can be crossed with other types of disease model non-human mammals to produce new disease model non-human mammals. Such use of a disease model non-human mammal is also included in the present invention.
以下、実施例により本発明をより詳細に説明するが、本発明は、これらに限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.
実施例1: SDマウス及びSAマウスの作製
(1) ES細胞(SD)及びES細胞(SA)の作製
Src遺伝子にCdk5リン酸化部位であるSer75をAspに置換する変異を導入したES細胞(「ES細胞(SD)」)、及び同リン酸化部位であるSer75をAlaに置換する変異を導入したES細胞(「ES細胞(SA)」)を次のように作製した。選択マーカー遺伝子や外来部位特異的組換え配列による内在遺伝子の発現や染色体構造への影響を排除するため、点変異のみを導入するためのベクターを作製した。即ち、HSV-tk遺伝子とネオマイシン耐性遺伝子が直列に結合した塩基配列の両端に、内在性Src遺伝子の一部の塩基配列を順向きに接続した。この点変異導入用ターゲティングベクターをエレクトロポレーション法でES細胞へ移入した。G418（ネオマイシン）に耐性となった細胞より、相同組換えによって一方のアレルに点変異が導入された細胞（相同組換え体）を以下のように選択した。即ち、サザンブロット法で相同組換え型のゲノムを有する細胞を選択後、PCR−アレル特異的プローブドットハイブリダイゼーション法により目的の点変異が導入された細胞を選択した。更に、分子内相同組換えでマーカー遺伝子を欠失してFIAU（ウラシル誘導体）耐性、G418感受性となった細胞を単離し、サザンブロット法及びPCR-アレル特異的プローブドットハイブリダイゼーション法でヘテロ接合体変異ES細胞を選択した。 Example 1: Production of SD and SA mice
(1) Preparation of ES cells (SD) and ES cells (SA)
ES cells introduced with a mutation that replaces Ser75 (Cdk5 phosphorylation site) with Asp in the Src gene ("ES cell (SD)"), and ES cells with a mutation that replaces Ser75 (Pd) phosphorylation site with Ala ("ES cell (SA)") was prepared as follows. In order to eliminate the influence of the selection marker gene and the exogenous site-specific recombination sequence on the expression of endogenous genes and the chromosome structure, a vector for introducing only point mutations was prepared. That is, a partial base sequence of the endogenous Src gene was forwardly connected to both ends of the base sequence in which the HSV-tk gene and the neomycin resistance gene were connected in series. This point mutation-introducing targeting vector was transferred to ES cells by electroporation. From the cells that became resistant to G418 (neomycin), cells in which a point mutation was introduced into one allele by homologous recombination (homologous recombinants) were selected as follows. That is, after selecting cells having a homologous recombination type genome by Southern blotting, cells into which the target point mutation was introduced were selected by PCR-allele specific probe dot hybridization. Furthermore, FIAU (uracil derivative) resistant and G418 sensitive cells were isolated by deletion of the marker gene by intramolecular homologous recombination, and heterozygotes were obtained by Southern blotting and PCR-allele specific probe dot hybridization. Mutant ES cells were selected.
(2) SDマウス(キメラ)及びSAマウス(キメラ)の作製
SDマウス(キメラ)及びSAマウス(キメラ)を次のように作製した。すなわち、発情期の雌のC57BL/6マウスと雄の同マウスを交配させ、膣栓形成したの雌マウスについて4日後に卵管から胚盤胞（3.5日胚）を回収した。この胚盤胞にヘテロ接合体変異ES細胞をマイクロマニュピレーターで注入し、あらかじめ準備した仮親の子宮に移植し仔を出産させた。仔の毛色によりES細胞の貢献率を推測した。(2) Production of SD mouse (chimera) and SA mouse (chimera)
SD mice (chimera) and SA mice (chimera) were prepared as follows. That is, female C57BL / 6 mice in estrus and male mice were mated, and blastocysts (3.5 day embryos) were collected from the oviduct 4 days later for female mice with vaginal plug formation. A heterozygous mutant ES cell was injected into the blastocyst with a micromanipulator and transplanted into a preparatory uterus prepared in advance to give birth. The contribution rate of ES cells was estimated by the color of the offspring.
(3) SDマウス(ヘテロ接合体)及びSAマウス(ヘテロ接合体)の作製
SDマウス(ヘテロ接合体)及びSAマウス(ヘテロ接合体)を次のように作製した。すなわち、50%以上の貢献率を呈した雄キメラマウスを雌のC57BL/6マウスと交配させ、生まれた仔の毛色（129マウス）によりES細胞がキメラマウスの生殖細胞に分化したことを確認した。更に、仔マウスの尾よりゲノムDNAを抽出後、PCR−アレル特異的プローブドットハイブリダイゼーション法により、Src遺伝子の一方のアレルに点変異をもつことを確認した。得られた生殖細胞のSrc遺伝子に目的の点変異を持つキメラマウスとC57BL/6マウスとを交配させ、生まれた仔の遺伝子型解析をPCR−アレル特異的プローブドットハイブリダイゼーション法で行い、ヘテロ接合体マウス株を樹立した。(3) Production of SD mice (heterozygotes) and SA mice (heterozygotes)
SD mice (heterozygotes) and SA mice (heterozygotes) were prepared as follows. That is, male chimeric mice with a contribution ratio of 50% or more were mated with female C57BL / 6 mice, and it was confirmed that ES cells differentiated into germ cells of chimeric mice by the color of the born pups (129 mice) . Furthermore, after extracting genomic DNA from the tail of a pup mouse, it was confirmed by PCR-allele-specific probe dot hybridization that one allele of the Src gene had a point mutation. The resulting germ cell Src gene is crossed with a C57BL / 6 mouse and a chimeric mouse having the desired point mutation, and then the genotype analysis of the born pup is performed by PCR-allele-specific probe dot hybridization. A body mouse strain was established.
(4) SDマウス(ホモ接合体)及びSAマウス(ホモ接合体)の作製
SDマウス(ホモ接合体)及びSAマウス(ホモ接合体)を次のように作製した。すなわち、ヘテロ接合体マウス株同士を交配させ、生まれた仔の遺伝子型解析をPCR−アレル特異的プローブドットハイブリダイゼーション法で行い、目的の点変異が導入されたホモ接合体マウス株を確立した。(4) Preparation of SD mice (homozygotes) and SA mice (homozygotes)
SD mice (homozygotes) and SA mice (homozygotes) were prepared as follows. That is, heterozygous mouse strains were mated with each other, and the genotype analysis of the born pups was performed by PCR-allele-specific probe dot hybridization to establish a homozygous mouse strain into which the target point mutation was introduced.
上記のようにして得られたSDマウス(ヘテロ接合体又はホモ接合体)では、c-Srcタンパク質のSer75がAspに置換されており、この置換により、Ser75の恒常的なリン酸化状態を疑似している。一方、SAマウス(ヘテロ接合体又はホモ接合体)では、c-Srcタンパク質のSer75がAlaに置換されており、この置換によりSer75でリン酸化が恒常的に起こらない。 In the SD mouse (heterozygous or homozygous) obtained as described above, Ser75 of c-Src protein is substituted with Asp, and this substitution simulates the constant phosphorylation state of Ser75. ing. On the other hand, in SA mice (heterozygote or homozygote), Ser75 of c-Src protein is substituted with Ala, and phosphorylation does not occur constantly at Ser75 due to this substitution.
実施例2: 視神経の炎症、及び視神経の障害部分
実施例1で得られたSDマウス(ホモ接合体)及びSAマウス(ホモ接合体)について、視神経の炎症、及び視神経の障害部分を次のように観察した。
7か月、11か月、17か月、23か月齢時の各マウスから眼球を摘出し、光学顕微鏡にて観察し、炎症性細胞の浸潤や、出血、浮腫などの炎症性所見のないことを組織学的に確認した。 Example 2: Inflammation of optic nerve and damaged part of optic nerve About SD mouse (homozygote) and SA mouse (homozygote) obtained in Example 1, inflammation of optic nerve and damaged part of optic nerve are as follows: Observed.
The eyeballs are removed from each mouse at 7 months, 11 months, 17 months, and 23 months of age and observed with an optical microscope, and there is no inflammatory findings such as infiltration of inflammatory cells, bleeding, or edema Was confirmed histologically.
その結果を下記の表1にまとめた。表1に示したように、SDマウス(ホモ接合体)では、視神経の炎症が見られず、視神経の障害部分は網膜神経細胞のみであった。一方、対照モデルのSAマウス(ホモ接合体)では、視神経の炎症が見られず、視神経の障害もなかった。 The results are summarized in Table 1 below. As shown in Table 1, in the SD mouse (homozygote), inflammation of the optic nerve was not observed, and the damaged part of the optic nerve was only retinal neurons. On the other hand, the control model SA mice (homozygotes) showed no optic nerve inflammation and no optic nerve damage.
実施例3: 網膜標本細胞数の測定
野生型正常マウス、並びに実施例1で得られたSDマウス(ヘテロ接合体又はホモ接合体)及びSAマウス(ヘテロ接合体又はホモ接合体)について、網膜標本細胞数を次のように測定した。
各マウスから眼球を摘出し、網膜切片標本を作製した。視神経からの距離を各測定眼一定にして、遺伝子型をマスクした検者が網膜神経節細胞数を測定した。測定のばらつきや誤差をなくすために1つの眼球から、すべてのサンプルで同様な位置が選択されるように複数の測定点を選択した。 Example 3: Measurement of the number of retinal specimen cells About wild-type normal mice and SD mice (heterozygotes or homozygotes) and SA mice (heterozygotes or homozygotes) obtained in Example 1, retinal specimens The cell number was measured as follows.
Eyeballs were removed from each mouse to prepare retinal slice specimens. The examiner who masked the genotype measured the number of retinal ganglion cells with the distance from the optic nerve kept constant for each eye. In order to eliminate measurement variations and errors, multiple measurement points were selected from one eye so that similar positions were selected for all samples.
その結果を、図1及び2、並びに下記表1に示す。
図1及び表1に示したように、SDマウス(ヘテロ接合体又はホモ接合体)では、6〜7月齢時には、網膜神経節細胞(RGG)の数は野生型正常マウスと比較してほとんど変わらないが、16〜23月齢時には、RGGの数が野生型正常マウスと比較して有意に低下していた。平均値で比較すると、SDマウス(ヘテロ接合体)及びSDマウス(ホモ接合体)では、野生型マウスと比較して、それぞれ、RGGの数の平均値が約20％及び20から40％減少していた。このことから、SDマウス(ヘテロ接合体又はホモ接合体)では、経時的に緑内障の症状が進行していることが分かる。
一方、図2及び表1に示したように、対照モデルのSAマウス(ヘテロ接合体又はホモ接合体)では、6〜7月齢時でも17〜23月齢時でも、RGGの数は野生型正常マウスと比較してほとんど変わらなかった。The results are shown in FIGS. 1 and 2 and Table 1 below.
As shown in Figure 1 and Table 1, in SD mice (heterozygotes or homozygotes), the number of retinal ganglion cells (RGG) is almost the same compared to wild-type normal mice at 6-7 months of age. However, at 16-23 months of age, the number of RGGs was significantly reduced compared to wild type normal mice. When compared by mean, SD mice (heterozygotes) and SD mice (homozygotes) have an average decrease in RGG numbers of about 20% and 20-40%, respectively, compared to wild-type mice. It was. This shows that in SD mice (heterozygotes or homozygotes), the symptoms of glaucoma have progressed over time.
On the other hand, as shown in FIG. 2 and Table 1, in the control model SA mice (heterozygotes or homozygotes), the number of RGGs was normal in wild-type mice at 6 to 7 months and at 17 to 23 months Compared with almost no change.
実施例4: 眼圧の測定
野生型正常マウス、並びに実施例1で得られたSDマウス(ヘテロ接合体又はホモ接合体)及びSAマウス(ヘテロ接合体又はホモ接合体)について、７か月、18か月齢時において眼圧を次のように測定した。マウスの眼圧が最も正確に測定可能であるリバウンド式の眼圧計を用いた。測定前にマウスには全身麻酔薬を腹腔内に注射して十分に麻酔が導入され眼圧測定に支障がない状態として測定した。測定においては測定器の自己信頼性判断結果が良好な値だけを採用し、3回繰り返した後、平均値を採用した。 Example 4: Measurement of intraocular pressure For wild-type normal mice and SD mice (heterozygotes or homozygotes) and SA mice (heterozygotes or homozygotes) obtained in Example 1, At 18 months of age, intraocular pressure was measured as follows. A rebound tonometer that can measure the intraocular pressure of the mouse most accurately was used. Prior to the measurement, mice were injected with a general anesthetic into the abdominal cavity, and the anesthesia was sufficiently introduced to measure the intraocular pressure measurement. In the measurement, only the value with a good self-reliability judgment result of the measuring instrument was adopted, and after repeating three times, the average value was adopted.
その結果を、図3及び表1に示す。図3及び表1に示したように、SDマウス(ヘテロ接合体又はホモ接合体)の眼圧は、野生型正常マウスと比較してほとんど変わらず、正常値の範囲内であった。また、表1に示したように、SAマウス(ヘテロ接合体又はホモ接合体)の眼圧も、正常値の範囲内であった。 The results are shown in FIG. As shown in FIG. 3 and Table 1, the intraocular pressure of SD mice (heterozygotes or homozygotes) was almost the same as that of wild-type normal mice, and was within the range of normal values. Further, as shown in Table 1, the intraocular pressure of SA mice (heterozygotes or homozygotes) was also within the range of normal values.
実施例5: 培養細胞生存率
野生型正常マウス、並びに実施例1で得られたSDマウス(ホモ接合体)及びSAマウス(ホモ接合体)について、網膜神経節細胞の単離、単離した細胞の培養、及び培養細胞生存率の測定を、次のように行った。
先ず、野生型マウス、SDマウス(ホモ接合体型)及びSAマウス(ホモ接合体型)の各々から、網膜神経節細胞を次のようにして単離した。具体的には、Barres BA, Silverstein BE, Corey DP, Chun LLY. Immunological, ｍorphological, and elecrtrophysiological variation among retinal ganglion calls purified by panning. Neuron. 1988; 1: 791-803.に記載の免疫学的手法であるtwo-step panning法を用いた。すなわち、マウス網膜神経節細胞（RGC）と交差反応をする細胞の除去のために第一段階ではマクロファージ抗体を用い第2段階においてRGCの特異的抗体であるThy1.2抗体を用いてRGCを単離した。 Example 5: Survival rate of cultured cells Wild type normal mice, and SD mice (homozygotes) and SA mice (homozygotes) obtained in Example 1, isolated retinal ganglion cells, isolated cells The culture and cell viability were measured as follows.
First, retinal ganglion cells were isolated from wild type mice, SD mice (homozygote) and SA mice (homozygote) as follows. Specifically, the immunological method described in Barres BA, Silverstein BE, Corey DP, Chun LLY. Immunological, morphological, and elecrtrophysiological variation among retinal ganglion calls purified by panning. Neuron. 1988; 1: 791-803. A two-step panning method was used. That is, in order to remove cells that cross-react with mouse retinal ganglion cells (RGC), macrophage antibody is used in the first stage and RGC specific antibody is used in the second stage using Thy1.2 antibody, which is a specific antibody of RGC. Released.
次に、単離した細胞について、Barres BA, Silverstein BE, Corey DP, Chun LLY. Immunological, Morphological, and elecrtrophysiological variation among retinal ganglion calls purified by panning. Neuron. 1988; 1: 791-803.、Kashiwagi F, Kashiwagi K, Iizuka Y, Tsukahara S. Effects of brain-derived neurotrophic factor and neurotrophin-4 on isolated cultured retinal ganglion cells: evaluation by flow cytometry. Invest Ophthalmol Vis Sci. 2000 Jul;41(8):2373-7.、及びKashiwagi K, Iizuka Y, Araie M, Suzuki Y, Tsukahara S. Effects of retinal glial cells on isolated rat retinal ganglion cells. Invest Ophthalmol Vis Sci.2001 Oct;42(11):2686-94.に記載の神経細胞培養のために開発した無血清培地を用いて培養を行った。 Next, for isolated cells, Barres BA, Silverstein BE, Corey DP, Chun LLY.Immunological, Morphological, and elecrtrophysiological variation among retinal ganglion calls purified by panning. Neuron. 1988; 1: 791-803., Kashiwagi F, Kashiwagi K, Iizuka Y, Tsukahara S. Effects of brain-derived neurotrophic factor and neurotrophin-4 on isolated cultured retinal ganglion cells: evaluation by flow cytometry.Invest Ophthalmol Vis Sci. 2000 Jul; 41 (8): 2373-7. And Kashiwagi K, Iizuka Y, Araie M, Suzuki Y, Tsukahara S. Effects of retinal glial cells on isolated rat retinal ganglion cells.Neurocytes described in Invest Ophthalmol Vis Sci. 2001 Oct; 42 (11): 2686-94. Culture was performed using a serum-free medium developed for the culture.
そして、培養細胞の生存率を次のように測定した。生存細胞、死亡した細胞の鑑別のためにLive-dead assayを用いた(Kashiwagi F, Kashiwagi K, Iizuka Y, Tsukahara S. Effects of brain-derived neurotrophic factor and neurotrophin-4 on isolated cultured retinal ganglion cells: evaluation by flow cytometry. Invest Ophthalmol Vis Sci. 2000 Jul;41(8):2373-7.、及びKashiwagi K, Iizuka Y, Araie M, Suzuki Y, Tsukahara S. Effects of retinal glial cells on isolated rat retinal ganglion cells. Invest Ophthalmol Vis Sci.2001 Oct;42(11):2686-94.)。これにより生存細胞、死亡細胞はそれぞれことなる蛍光染色を示すようになる。Assayで発色した細胞は、蛍光顕微鏡下で細胞条件をマスクして評価し生細胞、死細胞の評価を行い生存率を定量的に計測した。 And the survival rate of the cultured cell was measured as follows. Live-dead assay was used to differentiate between viable and dead cells (Kashiwagi F, Kashiwagi K, Iizuka Y, Tsukahara S. Effects of brain-derived neurotrophic factor and neurotrophin-4 on isolated cultured retinal ganglion cells: evaluation by Flow cytometry.Invest Ophthalmol Vis Sci. 2000 Jul; 41 (8): 2373-7. and Kashiwagi K, Iizuka Y, Araie M, Suzuki Y, Tsukahara S. Effects of retinal glial cells on isolated rat retinal ganglion cells. Invest Ophthalmol Vis Sci. 2001 Oct; 42 (11): 2686-94.). As a result, viable cells and dead cells show different fluorescent staining. Cells colored by the assay were evaluated by masking the cell conditions under a fluorescence microscope, and live cells and dead cells were evaluated, and the survival rate was quantitatively measured.
その結果を図4及び図5、並びに表1に示す。図4及び表1に示すようにSDマウス(ホモ接合体)では、野生型マウスと比較して、培養細胞生存率が有意に減少していた。平均値で比較すると、SDマウス(ホモ接合体)では、野生型マウスと比較して、培養細胞生存率の平均値が約50%減少していた。一方、図5に示すようにSAマウス(ホモ接合体)では、野生型マウスと比較して、培養細胞生存率が有意に上昇していた。 The results are shown in FIGS. 4 and 5 and Table 1. As shown in FIG. 4 and Table 1, the survival rate of cultured cells was significantly decreased in SD mice (homozygotes) compared to wild type mice. In comparison with the average value, the SD cell (homozygote) had a mean cell viability decrease of about 50% compared to the wild type mouse. On the other hand, as shown in FIG. 5, the survival rate of cultured cells was significantly increased in SA mice (homozygotes) compared to wild-type mice.
上記実施例の結果を、下記表1にまとめた。 The results of the above examples are summarized in Table 1 below.
以上の実施例で示したように、SDマウスは、神経の炎症をともなわないこと、障害部分が網膜神経節細胞に限定的であること、経時的に神経障害が進行すること、眼圧は正常なまま維持されることなどの条件を満たしており、ヒト正常眼圧緑内障と同様の症状を示す。よって、SDマウスは、正常眼圧緑内障モデルとして使用し得る。したがって、本発明のいくつかの態様の非ヒト哺乳動物は、正常眼圧緑内障モデル非ヒト哺乳動物として使用し得ることが分かる。 As shown in the above examples, SD mice are not accompanied by nerve inflammation, the restricted part is limited to retinal ganglion cells, neuropathy progresses over time, normal intraocular pressure It satisfies the conditions such as being maintained as it is, and exhibits symptoms similar to those of human normal-tension glaucoma. Therefore, the SD mouse can be used as a normal-tension glaucoma model. Therefore, it can be seen that the non-human mammal of some aspects of the present invention can be used as a normal-tension glaucoma model non-human mammal.
一方、対照モデルとして用いたSAマウスは、正常眼圧緑内障の原因となる網膜神経節細胞の障害がおこらず、正常であった。SAマウスでは、癌原遺伝子c-SrcのCdk5リン酸化部位のリン酸化が恒常的に抑えられている。よって、癌原遺伝子c-SrcのCdk5リン酸化部位のリン酸化を抑制する薬剤は、正常眼圧緑内障の原因となる網膜神経節細胞の障害を抑制し得る。したがって、本発明のいくつかの態様の非ヒト哺乳動物は、正常眼圧緑内障の予防及び／又は治療剤のスクリーニング方法に使用し得る。 On the other hand, SA mice used as a control model were normal with no retinal ganglion cell damage causing normal pressure glaucoma. In SA mice, phosphorylation of the Cdk5 phosphorylation site of the proto-oncogene c-Src is constantly suppressed. Therefore, a drug that suppresses phosphorylation of the Cdk5 phosphorylation site of the proto-oncogene c-Src can suppress damage to retinal ganglion cells that cause normal pressure glaucoma. Therefore, the non-human mammal of some aspects of the present invention can be used in a screening method for a prophylactic and / or therapeutic agent for normal tension glaucoma.
[配列番号：1]マウスc-Srcタンパク質をコードする塩基配列である。
[配列番号：2]マウスc-Srcタンパク質のアミノ酸配列である。[SEQ ID NO: 1] is a nucleotide sequence encoding mouse c-Src protein.
[SEQ ID NO: 2] is an amino acid sequence of mouse c-Src protein.
Claims (17)
1)その眼圧が正常範囲にあり、かつ
2)その網膜神経節の細胞数が、野生型非ヒト哺乳動物に比べて減少している、
を満たしている、請求項1〜4のいずれか1項記載の使用。 The non-human mammal has the following conditions:
1) The intraocular pressure is in the normal range, and
2) The number of cells in the retinal ganglion is reduced compared to wild type non-human mammals,
The use according to any one of claims 1 to 4, wherein:
1)その眼圧が正常範囲にあり、かつ1) The intraocular pressure is in the normal range, and
2)その網膜神経節の細胞数が、野生型非ヒト哺乳動物に比べて減少している、2) The number of cells in the retinal ganglion is reduced compared to wild type non-human mammals,
を満たしている、請求項8〜11のいずれか1項記載の方法。The method according to any one of claims 8 to 11, wherein:
1)前記非ヒト哺乳動物及び対照非ヒト哺乳動物に候補物質を投与すること、
2)前記各非ヒト哺乳動物において、投与前、及び投与してから一定期間後に、生存する視神経細胞の数量を検査すること、及び
3)前記各非ヒト哺乳動物の検査結果を比較して、候補物質の有効性を評価すること、
を含む、請求項8〜14のいずれか1項記載の方法。 The method comprises
1) administering a candidate substance to said non-human mammal and control non-human mammal,
2) In each non-human mammal, examining the number of optic nerve cells surviving before administration and after a certain period of time after administration; and
3) comparing the test results of each of the non-human mammals to evaluate the effectiveness of the candidate substance,
15. The method according to any one of claims 8 to 14 , comprising:
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