WO2019109861A1 - 一种视网膜色素变性疾病动物模型的构建方法及应用 - Google Patents

一种视网膜色素变性疾病动物模型的构建方法及应用 Download PDF

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WO2019109861A1
WO2019109861A1 PCT/CN2018/118431 CN2018118431W WO2019109861A1 WO 2019109861 A1 WO2019109861 A1 WO 2019109861A1 CN 2018118431 W CN2018118431 W CN 2018118431W WO 2019109861 A1 WO2019109861 A1 WO 2019109861A1
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retinitis pigmentosa
animal model
hkdc1
animal
gene
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French (fr)
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朱献军
杨正林
张琳
杨业明
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四川省人民医院
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Definitions

  • the present disclosure relates to the field of medical engineering technology, and in particular to a method and application for constructing an animal model of a retinitis pigmentosa disease.
  • Retinitis Pigmentosa is a group of highly heterogeneous degenerative photoreceptor cytopathic and dying ophthalmic diseases.
  • the initial onset usually affects rod cells to cause dark vision loss and peripheral visual field damage.
  • the incidence of RP in China is about 1/4000.
  • RP patients can reach millions of people, which brings a heavy burden to families and society. More unfortunately, there is currently no systematic and systematic study on the etiology and pathological mechanism of the disease. A considerable number of RP gene pathogenic mechanisms are still unclear, which poses obstacles to targeted development of therapeutic interventions.
  • the root cause of the weakness of RP prevention and treatment measures is the lack of in-depth systematic research on the etiology and pathological mechanism of the disease.
  • the main reasons for this dilemma are as follows: 1) The clinical phenotype is heterogeneous. The clinical phenotype of the disease varies greatly. In severe cases, it begins to develop in adolescence and is rapidly blinded, while mild patients can still retain some visual functions until middle age. This feature of RP poses a huge challenge to traditional diagnostic methods. 2) There are many pathogenic genes and diverse genetic models. More than 70 RP genes are now known, which can explain about 60% of RP cases, but nearly 40% of cases are still unexplained.
  • the genetic pattern of the disease is diverse, including autosomal dominant, recessive inheritance, X-linked inheritance, mitochondrial inheritance and other genetic models. Therefore, the search for new disease-causing genes and the identification of their pathogenic mechanisms are the primary issues for the study of the disease. 3)
  • the pathogenic molecular mechanism is complex. It has been found that the cell biochemical processes involved in RP pathogenic genes are very complex, such as photoreduction, cytoskeleton, RNA splicing, ubiquitination and other biochemical processes, which makes the study of pathogenic molecular mechanisms difficult. It has brought many difficulties to the treatment intervention.
  • the objects of the present disclosure include, but are not limited to, providing a method for constructing an animal model of retinitis pigmentosa disease, by which an animal model of retinitis pigmentosa disease can be obtained, which can be used for researchers to systematically study retinitis pigmentosa disease and for retina Drug screening for pigmented degenerative diseases.
  • Another object of the present disclosure includes, but is not limited to, the use of an animal model of retinitis pigmentosa disease obtained by the above construction method in the study of retinitis pigmentosa disease.
  • Another object of the present disclosure includes, but is not limited to, providing a nucleic acid molecule for use in constructing an animal model of retinitis pigmentosa.
  • a method for constructing an animal model of retinitis pigmentosa disease comprising: knocking out a target sequence on a genome of a target animal, wherein the target sequence is a Hkdc1 gene, and a first constructed animal in which the Hkdc1 gene is knocked out is obtained.
  • An animal model of a retinitis pigmentosa disease obtained by the above-described method for constructing an animal model of retinitis pigmentosa disease, for screening for a medicament for treating a retinitis pigmentosa disease.
  • An animal model of retinitis pigmentosa disease obtained by the above-described method for constructing an animal model of retinitis pigmentosa disease is used for studying retinitis pigmentosa diseases.
  • a nucleic acid molecule for constructing an animal model of retinitis pigmentosa disease which is a gRNA, and the base sequence of the target site to which the gRNA is directed is shown in SEQ ID NO: 1.
  • the method for constructing an animal model of retinitis pigmentosa degeneration can obtain typical characteristics of retinitis pigmentosa such as impaired retinal dysfunction, progressive apoptosis of photoreceptor cells, etc. by knocking out the Hkdc1 gene on the genome of the target animal. Characteristics, the animal model can be used for the research of retinitis pigmentosa degeneration and screening for drugs for treating retinitis pigmentosa diseases, and its application prospect is broad.
  • FIG. 1 is a schematic diagram of a design of a CRISPR targeting sequence according to an embodiment of the present disclosure
  • Hkdc1 knockout mice is a genotype identification map of Hkdc1 knockout mice according to an embodiment of the present disclosure; (Mut: NM_145419.1, c.93_99delCTCGGATG, wherein WT represents wild type, Hom represents homozygous genotype, and Het represents gene Knock out the heterozygous genotype);
  • FIG. 3 is a WB result provided by an embodiment of the present disclosure.
  • Figure 4 is a result of IHC staining provided by an embodiment of the present disclosure (in the figure: WT is a wild type mouse, and Mut/Mut is a homozygous Hkdc1 knockout mouse);
  • Figure 6 shows the results of HE staining in retina of 11-month-old and 17-month-old mice
  • a and B in the figure represent the results of hematoxylin-eosin staining (H&E staining) of 11-month-old wild-type and mutant retinal sections, respectively
  • C represents the statistical results of 11-month-old photoreceptor cell counts
  • D, E represent the results of 17-month-old wild-type and mutant retinal sections with hematoxylin-eosin staining (H&E staining)
  • F represents 17-month-old photoreceptor cells Column number statistics).
  • HKDC1 Hexokinae Domain Containing 1
  • RP Hexokinae Domain Containing 1
  • This disclosure is the first to discover that the Hkdc1 gene is involved in retinitis pigmentosa.
  • an animal model with typical features of retinitis pigmentosa has been constructed, providing researchers with research and screening for retinitis pigmentation for retinitis pigmentosa.
  • Animal models of drugs for degenerative diseases have strongly promoted scientific research and provided the basis for further in-depth and systematic research on the pathogenesis of retinitis pigmentosa.
  • the present disclosure provides a method for constructing an animal model of a retinitis pigmentosa disease, comprising: knocking out a target sequence on a genome of a target animal, wherein the target sequence is a Hkdc1 gene, and the first of the Hkdc1 gene knockout is obtained. Built animals.
  • the above sequence of interest is an exon sequence on the Hkdc1 gene.
  • the exon sequence is a second exon sequence.
  • Hkdc1 gene There are a plurality of exons on the Hkdc1 gene, and one or more of the knockout genes may be inactivated, abnormally expressed, and knocked out, and the first constructed animal in which the Hkdc1 gene is knocked out is obtained.
  • the foregoing construction method further includes:
  • the first constructed animal in which the Hkdc1 gene was knocked out was mated with a wild type animal of the same type to obtain a heterozygous Hkdc1 knockout animal;
  • Homozygous Hkdc1 knockout animals exhibit typical features of retinitis pigmentosa, such as impaired retinal function and progressive apoptosis of photoreceptors, and can be used as animal models of retinitis pigmentosa. This animal model can be used in the study of retinitis pigmentosa degeneration and in the screening of drugs for the treatment of retinitis pigmentosa.
  • the target animal is selected from the group consisting of a mouse, a rat, a dog, a monkey, and a donkey.
  • the Hkdc1 gene knockout first constructed animal is obtained by mixing a gRNA directed against the Hkdc1 gene sequence with a Cas9 endonuclease, and injecting it into a target animal fertilized egg, After development, the first constructed animal of the above Hkdc1 gene knockout was obtained.
  • the base sequence of the target site of the gRNA described above is set forth in SEQ ID NO: 1.
  • the CRISPR/Cas9 gene knockout system has been developed in recent years as a gene editing technology derived from the bacterial acquired immune system. It has been extensively applied to the study of various model organisms through manual modification. CRISPR-Cas9 technology has specific DNA recognition ability. Cas9 endonuclease cleaves double-stranded DNA under the guidance of ribonucleic acid gRNA, causing genomic double-strand breaks, using non-specific recombination of cell genome repair to produce repair errors. (insertion or deletion), which may cause a frameshift mutation and cause loss of gene function, achieving the purpose of gene knockout.
  • the base sequence of the gRNA target site can be designed according to the sequence of interest to be knocked out, which is not limited to the base sequence shown in SEQ ID NO: 1, and other sequences are used as targets.
  • the locus to knock out the Hkdc1 gene is also within the scope of protection of the present disclosure.
  • the target site shown in SEQ ID NO: 1 is located in exon 2 of the mouse Hkdc1 gene, as shown in FIG.
  • the Hkdc1 gene can also be knocked out using the Cre-loxP knockout technique, which is also within the scope of the present disclosure.
  • the present disclosure provides an animal model of a retinitis pigmentosa disease obtained by the method for constructing an animal model of a retinitis pigmentosa disease obtained by the above-described construction method, for screening for a medicament for treating a retinitis pigmentosa disease.
  • the present disclosure provides an animal model of a retinitis pigmentosa disease obtained by the method for constructing an animal model of a retinitis pigmentosa disease obtained by the above-described construction method, for use in the study of a retinitis pigmentosa disease.
  • the study is a pathogenesis or pathogenesis mechanism of retinitis pigmentosa disease.
  • the present disclosure provides an animal model of retinitis pigmentosa disease, which is constructed by the method of constructing an animal model of retinitis pigmentosa disease as described above.
  • the present disclosure provides an animal model of a retinitis pigmentosa disease, wherein the Hkdc1 gene sequence on the genome is knocked out.
  • the exon sequence of the Hkdc1 gene on the genome of the animal model of retinitis pigmentosa disease is knocked out.
  • the animal model is selected from any one of the group consisting of a mouse, a rat, a dog, a monkey, and a donkey.
  • the animal model is a mouse, and the second exon sequence of the Hkdc1 gene on the genome of the animal model is knocked out.
  • the base sequence of the Hkdc1 gene of the animal model has a deletion mutation at positions 93-99 compared to the wild type mouse, and the deleted base sequence is :CTCTCGG.
  • the present disclosure provides a method of screening for a medicament for preventing or treating a retinitis pigmentosa disease, comprising: applying an animal model to be retinitis pigmentosa disease as described above for preventing or treating retinitis pigmentosa Candidate reagent for disease.
  • Phenomenon (1) Compared with a retinitis pigmentosa disease in which the candidate agent is not applied, the retinitis pigmentosa disease to which the candidate agent is applied exhibits a decrease in the amount of rhodopsin accumulated in the inner cell and the cell body of the photoreceptor;
  • Phenomenon (2) The retinitis pigmentosa disease to which the candidate agent is applied exhibits an increase in retinal thickness compared to a retinitis pigmentosa disease to which the candidate agent is not applied.
  • the retinal rhodopsin protein trafficking in homozygous Hkdc1 knockout (KO) mice was abnormal: accumulation in the photoreceptor cells and cell bodies (Fig. 5). Then, if a candidate agent is applied, the rhodopsin protein accumulated in the photoreceptor cells and the cell body begins to decrease or even disappear, it can be proved that the candidate agent has an effect on the treatment of retinitis pigmentosa disease, and can be used as a preventive Or a drug that treats retinitis pigmentosa.
  • the thickness of the retina of the homozygous Hkdc1 knockout mice is significantly thinner: the thickness of the outer nuclear layer is significantly reduced (Fig. 6). Then, if a certain candidate agent is applied and the thickness of the retina is significantly thickened, it can be proved that the candidate agent is effective for treating retinitis pigmentosa degeneration and can be used as a drug for preventing or treating retinitis pigmentosa.
  • the present disclosure provides a nucleic acid molecule for constructing an animal model of a retinitis pigmentosa disease, which is a gRNA, the target sequence of the gRNA is located on the Hkdc1 gene of the target animal;
  • the target sequence is located in an exon of the Hkdc1 gene of the target animal;
  • the target animal is selected from any one of a mouse, a rat, a dog, a monkey, and a donkey.
  • the target animal is a mouse
  • the target sequence of the gRNA is set forth in SEQ ID NO: 1.
  • the present disclosure also provides a nucleic acid molecule for constructing an animal model of retinitis pigmentosa disease, which is a gRNA, the base sequence of which is represented by SEQ ID NO: 1.
  • the nucleic acid molecule of SEQ ID NO: 1 is a target site, and the site is used as a target, and the Hkdc1 gene partial sequence of a target animal such as a mouse can be knocked out by the CRISPR-Cas9 technique to obtain an animal model of retinitis pigmentosa disease.
  • the present disclosure also provides a method of constructing an animal model of retinitis pigmentosa degeneration comprising: inhibiting or silencing Hkdc1 gene expression in a target animal.
  • an animal model of retinitis pigmentosa disease can be obtained by inhibiting or silencing the Hkdc1 gene expression of a target animal by a suitable method, and such suitable methods may be, for example, knocking out the Hkdc1 gene at a gene level, using siRNA inhibits transcription of Hkdc1 gene at the RNA level or inhibits translation of mRNA of transcriptional maturation, or Hkdc1 antibody neutralizing Hkdc1 protein at the protein level, and these methods can inactivate or lack Hkdc1 protein in a target animal. Further, an animal model of retinitis pigmentosa disease can be obtained. Therefore, as long as the method of inhibiting or silencing the expression of the Hkdc1 gene of the target animal falls within the protection scope of the present disclosure, the animal model obtained by these methods is also the protection scope of the present disclosure.
  • the target animal is selected from any one of a mouse, a rat, a dog, a monkey, and a donkey.
  • mouse Hkdc1 gene was knocked out by the CRISPR-Cas9 technology to construct an animal model of retinitis pigmentosa disease, as follows.
  • the gRNA target site sequence was designed for the second exon region of the mouse Hkdc1 gene as follows:
  • RNA was synthesized in vitro, and microinjected into the mouse fertilized egg together with Cas9 endonuclease, and after development, a founder of the gene mutation was obtained.
  • FIG. 1 A schematic diagram of the structure of the gRNA sequence targeting the Hkdc1 gene is shown in FIG.
  • Fragments near the position of the CRISPR target were sequenced using mouse genomic DNA to determine that the first constructor mouse had a frameshift mutation that affected the reading frame.
  • the tube was taken out, cooled to room temperature, and 100 ⁇ l of a neutralizing solution (40 mM Tris-HCl, pH 5.5) was added thereto, and centrifuged at 10,000 g for 2 minutes, and then the supernatant was taken for mouse genotype identification.
  • a neutralizing solution 40 mM Tris-HCl, pH 5.5
  • the PCR reaction system was configured according to the following system:
  • the base sequence of the Hkdc1-Forward primer is as follows:
  • the base sequence of the Hkdc1-Reverse primer is as follows:
  • the PCR reaction was carried out according to the following reaction conditions:
  • the amplified PCR fragment needs to be purified by using FastAP (exo-enzyme, reaction buffer and ddH2O) to remove primers and other interfering fragments.
  • FastAP exo-enzyme, reaction buffer and ddH2O
  • the specific purification system is:
  • reaction conditions are:
  • the purified fragment subjected to the above steps is then used in the sequencing reaction.
  • the specific sequencing reaction system is:
  • the sequencing reaction conditions are as follows:
  • the founder with mutation was mated with wild-type mice to obtain a heterozygous mouse (+/-) F1 generation with gene mutation, and DNA sequencing was performed to confirm that the target gene was shifted as shown in Figure 2.
  • the mutation of the code causes the target protein to be inactivated, and the Hkdc1 gene knockout is achieved.
  • HKDC1 protein in the retina of homozygous mutant mice was verified by Western blot (WB) and immunohistochemistry (Immunohistochemistry), respectively.
  • the WB method is as follows:
  • mice ie Hkdc1 knockout homozygous mouse (-/-)) retinal tissue, placed in a 1.5ml centrifuge tube, and added 200 ⁇ l protein lysate RIPA;
  • the immunohistochemical method is as follows:
  • the eyeballs were quickly taken and placed in 4% PFA. After fixing for 15 minutes on ice, a hole was cut on the cornea and then fixed on ice. After 2 h, the PBS buffer was washed 3 times, then the eyeball was dehydrated in a 30% sucrose solution for 2 h, then the cornea and crystals were cut off under a dissecting microscope, OCT was embedded and quickly frozen in a -80 ° C refrigerator. After about 10 minutes, the OCT-embedded eyeballs were taken out and placed in a frozen slicer at -25 ° C for about 30 min to be sliced. The slice thickness was 12 ⁇ m.
  • the higher quality film is placed in an oven at 37 ° C for 30 min, then the immunohistochemical pen is circled in the place with retinal tissue, washed three times with PBS to remove OCT, and then 5% of NDS (containing 0.25% Triton) The cells were blocked for 2 h, and the primary antibody was incubated at 4 ° C overnight. The next day, after PBS was washed twice, the corresponding fluorescent secondary antibody was incubated, then washed twice with PBS, mounted, and observed.
  • the gRNA sequence designed by the present disclosure it is possible to guide Cas9 to rapidly, efficiently and specifically knock out the Hkdc1 gene of a mouse, and to construct a Hkdc1 +/- mouse model (heterozygous) of Hkdc1 knockout using the disclosed method and The Hkdc1-/- mouse model (homozygous), both of which can be used for subsequent functional studies.
  • the characteristics of the retinal degenerative disease exhibited by the Hkdc1 knockout mouse obtained in Example 1 are specifically described below by way of a test example.
  • Hkdc1 gene knockout was homozygous at 9 months of age.
  • Rhodopsin is the main photoreceptor protein, which is extremely critical for visual formation. Its mislocalization leads to apoptosis of photoreceptor cells. Therefore, it is known that Hkdc1 gene plays an important role in retinal degenerative diseases.
  • H&E staining method The retinas of 11-month-old and 17-month-old mice were stained with paraffin sections and hematoxylin-eosin staining (H&E staining method) as follows:
  • Hkdc1 knockout mouse model constructed by the method of the present disclosure has typical retinal degenerative disease characteristics and can be used for retinal degenerative diseases research.
  • the nucleotide sequence specifically knocking out the Hkdc1 gene by the CRISPR-Cas9 technology can guide the rapid, efficient and specific knockout of the Hkdc1 gene of the mouse, and obtain the retinitis pigmentosa degenerative disease.
  • the mouse model provides a reliable animal model for the study of Hkdc1 gene-related diseases, which has a wide range of applications.
  • the method for constructing an animal model of retinitis pigmentosa disease can construct an animal model of retinitis pigmentosa disease in the retina which specifically knocks out the HKDC1 gene, which model exhibits typical retinitis pigmentosa disease characteristics, and the model can The use of retinitis pigmentosa disease research or screening for drugs for the treatment or prevention of retinitis pigmentosa diseases provides a model basis for further understanding the pathogenesis of retinitis pigmentosa degeneration and screening for drugs for retinitis pigmentosa.

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Abstract

一种视网膜色素变性疾病动物模型的构建方法及应用，涉及医学工程技术领域。所述的视网膜色素变性疾病动物模型的构建方法，通过敲除目标动物的基因组上Hkdc1基因序列，获得网膜色素变性疾病动物模型，该动物模型表现出视网膜色素变性的典型特征如表现出视网膜功能受损、感光细胞渐进性凋亡等特征，该动物模型可用于视网膜色素变性疾病的研究和筛选治疗视网膜色素变性疾病的药物中。

Description

一种视网膜色素变性疾病动物模型的构建方法及应用
本申请要求于2017年12月04日提交中国专利局的申请号为201711265100.7、名称为“一种视网膜色素变性疾病动物模型的构建方法及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及医学工程技术领域，具体而言，涉及一种视网膜色素变性疾病动物模型的构建方法及应用。
背景技术
视网膜色素变性(Retinitis Pigmentosa，RP)是一组具较强异质性的退行性视网膜感光细胞病变、死亡的眼科疾病，初始发病通常首先影响视杆细胞导致暗视力下降，周边视野受损，逐步发展为管状视野，直至失明；眼底检查可见视网膜色素沉着。RP在我国的发病率约为1/4000，基于我国庞大的人口基数，RP患者可达数百万之众，给家庭和社会带来了沉重的负担。更加不幸的是，目前对该病的病因和病理机制缺乏深入系统的研究，相当多的RP基因致病机制尚不清楚，给针对性的发展治疗干预措施带来障碍。
RP防治措施的乏力的根本原因在于对该病的病因和病理机制缺乏深入系统的研究。造成这种困局的原因主要有以下几个方面：1)临床表型异质性强。该病的临床表型差异巨大，严重者在青少年时期即开始发病，并很快失明，而轻微患者一直到中年时仍能存留部分视觉功能。RP的这一特征给传统的诊断方法带来巨大挑战。2)致病基因多、遗传模式多样。现在已知的RP基因已超过了70个，可以解释约60％的RP病例，然而仍有将近40％的病例无法解释。同时，该病的遗传模式多样，包括常染色体显性、隐性遗传，X-连锁遗传、线粒体遗传等多种遗传模式。因此，寻找新的致病基因，并明确其致病机制是对于该疾病研究首要解决的问题。3)致病分子机制复杂。已经发现的RP致病基因参与到的细胞生化过程非常复杂，如光信号转导、细胞骨架、RNA剪接、泛素化降解等多种生化过程，这使得其致病分子机制的研究不易，也给治疗干预带来了诸多困难。
而目前尚缺乏视网膜色素变性疾病动物模型，给进一步深入系统研究视网膜色素变性疾病带来不便。
鉴于此，特提出本公开。
公开内容
本公开的目的包括但不限于提供一种视网膜色素变性疾病动物模型的构建方法，利用该构建方法可以得到视网膜色素变性疾病动物模型，可用于研究者深入系统研究视网膜色素变性疾病，以及用于视网膜色素变性疾病的药物筛选。
本公开的另一目的包括但不限于提供一种由上述构建方法得到的视网膜色素变性疾病动物模型在视网膜色素变性疾病研究中的应用。
本公开的另一目的包括但不限于提供一种用于构建视网膜色素变性疾病动物模型的核酸分子。
本公开是这样实现的：
一种视网膜色素变性疾病动物模型的构建方法，其包括：敲除目标动物的基因组上的目的序列，上述目的序列为Hkdc1基因，获得Hkdc1基因敲除的首建动物。
由上述的视网膜色素变性疾病动物模型的构建方法所得到的视网膜色素变性疾病动物模型在筛选治疗视网膜色素变性疾病的药物中的应用。
由上述的视网膜色素变性疾病动物模型的构建方法所得到的视网膜色素变性疾病动物模型在研究视网膜色素变性疾病中的应用。
一种用于构建视网膜色素变性疾病动物模型的核酸分子，其为gRNA，该gRNA针对的靶标位点的碱基序列如SEQ ID NO：1所示。
本公开具有以下有益效果：
本公开提供的视网膜色素变性疾病动物模型的构建方法，通过敲除目标动物基因组上的Hkdc1基因，可得到表现出视网膜色素变性的典型特征如表现出视网膜功能受损、感光细胞渐进性凋亡等特征，该动物模型可用于视网膜色素变性疾病的研究和筛选治疗视网膜色素变性疾病的药物中，其应用前景广阔。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本公开实施例提供的CRISPR靶向序列设计示意图；
图2为本公开实施例提供的Hkdc1敲除小鼠基因型鉴定图；(Mut:NM_145419.1，c.93_99delCTCGGATG，图中WT代表野生型，Hom代表基因敲除纯合基因型，Het代表基因敲除杂合基因型)；
图3为本公开实施例提供的WB结果；
图4为本公开实施例提供的IHC染色结果(图中：WT为野生型小鼠，Mut/Mut为纯合型Hkdc1基因敲除小鼠)；
图5为本公开实施例提供的KO小鼠视网膜的视紫红质蛋白错误定位于感光细胞的内节和胞体的结果(小鼠年龄：9个月；箭头指示错误定位的视紫红质蛋白)；
图6为11个月龄和17个月龄小鼠视网膜HE染色结果(图中A，B分别代表11个月龄野生型和突变型视网膜切片苏木精-伊红染色结果(H&E染色)；C代表11个月龄感光细胞列数统计结果；D，E分别代表17个月龄野生型和突变型视网膜切片苏木精-伊红染色结果(H&E染色)；F代表17个月龄感光细胞列数统计结果)。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本公开实施例的一种视网膜色素变性疾病动物模型的构建方法及应用进行具体说明。
己糖激酶结构域蛋白1(Hexokinae Domain Containing 1，HKDC1)，为己糖激酶家族成员之一，可以磷酸化葡萄糖，促进体内葡萄糖代谢。通过全基因组关联实验表明HKDC1与孕期高血糖症有关，通过生物信息学分析表明HKDC1在肺癌组织中高表达，可能是新的肿瘤治疗靶点。且发明人前期研究表明，Hkdc1基因突变与RP有关，对探索RP的致病机制有极大帮助。因此，深入研究HKDC1对多种疾病的治疗及病因探讨潜力巨大。
目前对HKDC1基因的研究还处于初期阶段，其在体内的具体作用机制尚不清楚，限制了其开发应用。
本公开首次发现Hkdc1基因与视网膜色素变性相关，通过敲除Hkdc1基因，构建出了具有视网膜色素变性的典型特征的动物模型，为研究者提供了用于视网膜 色素变性疾病的研究和筛选治疗视网膜色素变性疾病的药物的动物模型，有力地促进了科学研究，为进一步深入和系统研究视网膜色素变性疾病致病机理提供基础。
基于此，一方面，本公开提供了一种视网膜色素变性疾病动物模型的构建方法，其包括：敲除目标动物的基因组上的目的序列，上述目的序列为Hkdc1基因，获得Hkdc1基因敲除的首建动物。
可选地，在本公开的一些实施方案中，上述目的序列为Hkdc1基因上的外显子序列。
可选地，在本公开的一些实施方案中，上述外显子序列为第二外显子序列。
Hkdc1基因上有多个外显子，敲除其中的一个或多个均可以是实现该基因的失活，表达异常，实现敲除目的，得到Hkdc1基因敲除的首建动物。
可选地，在本公开的一些实施方案中，上述构建方法还包括：
将上述Hkdc1基因敲除的首建动物与同类的野生型动物交配，获得杂合子Hkdc1基因敲除动物；
将上述杂合子Hkdc1基因敲除动物自交，获得纯合子Hkdc1基因敲除动物。
纯合子Hkdc1基因敲除动物表现出典型的视网膜色素变性特征，例如出视网膜功能受损、感光细胞渐进性凋亡等特征，可作为视网膜色素变性疾病动物模型。该动物模型可用于视网膜色素变性疾病的研究和筛选治疗视网膜色素变性疾病的药物中。
可选地，在本公开的一些实施方案中，上述目标动物选自小鼠、大鼠、狗、猴以及猿中的任意一种。
可选地，在本公开的一些实施方案中，上述Hkdc1基因敲除的首建动物通过如下方法获得：将针对Hkdc1基因序列的gRNA与Cas9核酸内切酶混合，注射至目标动物授精卵，经发育成熟后，获得上述Hkdc1基因敲除的首建动物。
可选地，在本公开的一些实施方案中，上述gRNA的靶标位点的碱基序列如SEQ ID NO:1所示。
CRISPR/Cas9基因敲除系统是近年来发展起来的一种来源于细菌获得性免疫系统的基因编辑技术，经过人工的改造，现已被广泛运用于多种模式生物的研究。CRISPR-Cas9技术具有特异性DNA识别能力，Cas9核酸内切酶在向导核糖核酸gRNA引导下切割双链DNA，造成基因组双链断裂，利用细胞基因组修复的不稳定性产生非特异重组来产生修复错误(插入或者缺失)，从而可能产生移码突变而造成基因功能的丧失，实现基因敲除的目的。
当然，在其他的实施例中，gRNA靶标位点的碱基序列可以根据待敲除的目的序列进行设计，其并不限于SEQ ID NO:1所示的碱基序列，以其他的序列作为靶标位点以敲除Hkdc1基因也属于本公开的保护范围。
SEQ ID NO:1所示的靶标位点位于小鼠Hkdc1基因的第二号外显子，如图1所示。
另外，在其他的实施例中，也可以采用Cre-loxP敲除技术敲除Hkdc1基因，其也属于本公开的保护范围。
另一方面，本公开提供了由上述构建方法得到的视网膜色素变性疾病动物模型的构建方法所得到的视网膜色素变性疾病动物模型在筛选治疗视网膜色素变性疾病的药物中的应用。
另一方面，本公开提供了由上述构建方法得到的视网膜色素变性疾病动物模型的构建方法所得到的视网膜色素变性疾病动物模型在研究视网膜色素变性疾病中的应用。
可选地，在本公开的一些实施方案中，所述研究为视网膜色素变性疾病发病机理或致病机制研究。
另一方面，本公开提供了一种视网膜色素变性疾病动物模型，其由如上所述的视网膜色素变性疾病动物模型的构建方法所构建得到。
另一方面，本公开提供了一种视网膜色素变性疾病动物模型，其基因组上的Hkdc1基因序列被敲除。
可选地，在本公开的一些实施方案中，所述视网膜色素变性疾病动物模型的基因组上的Hkdc1基因的外显子序列被敲除。
可选地，在本公开的一些实施方案中，所述动物模型选自小鼠、大鼠、狗、猴以及猿中的任意一种。
可选地，在本公开的一些实施方案中，所述动物模型为小鼠，所述动物模型的基因组上的Hkdc1基因的第2外显子序列被敲除。
可选地，在本公开的一些实施方案中，相较于与野生型小鼠，所述动物模型的的Hkdc1基因的cDNA序列第93-99位碱基具有缺失突变，缺失的碱基序列为：CTCTCGG。
另一方面，本公开提供了一种筛选用于预防或治疗视网膜色素变性疾病药物的方法，其包括：向如上所述的视网膜色素变性疾病动物模型施加待筛选的用于预防或治疗视网膜色素变性疾病的候选试剂。
可选地，在本公开的一些实施方案中，如果施加所述候选试剂后，检测到所述 视网膜色素变性疾病动物模型出现以下现象中的一个或一个以上，则指示该候选试剂可以作为用于预防或治疗视网膜色素变性疾病的药物：
现象(1)：相较于未施加该候选试剂的视网膜色素变性疾病，施加了该候选试剂的视网膜色素变性疾病表现出：感光细胞内节和胞体累积的视紫红质蛋白减少；
现象(2)：相较于未施加该候选试剂的视网膜色素变性疾病，施加了该候选试剂的视网膜色素变性疾病表现出：视网膜厚度增加。
由本公开试验例的结果可以看出，纯合的Hkdc1基因敲除(KO)小鼠的视网膜视紫红质蛋白运输异常：在感光细胞内节和胞体积累(图5)。那么，如果施加了某候选试剂后，其在感光细胞内节和胞体积累的视紫红质(Rhodopsin)蛋白开始减少甚至消失，则可以证明该候选试剂对治疗视网膜色素变性疾病有效果，可以作为预防或治疗视网膜色素变性疾病的药物。
此外，由本公开试验例的结果可以看出，纯合的Hkdc1基因敲除小鼠的视网膜厚度明显变薄：外核层厚度明显减少(图6)。那么，如果施加了某候选试剂后，其视网膜厚度明显变厚，则可以证明该候选试剂对治疗视网膜色素变性疾病有效果，可以作为预防或治疗视网膜色素变性疾病的药物。
另一方面，本公开提供了一种用于构建视网膜色素变性疾病动物模型的核酸分子，其为gRNA，所述gRNA的靶序列位于目标动物的Hkdc1基因上；
优选的，所述靶序列位于目标动物的Hkdc1基因的外显子；
优选的，所述目标动物选自小鼠、大鼠、狗、猴以及猿中的任意一种。
在本公开的一些实施方案中，所述目标动物为小鼠，所述gRNA的靶序列如SEQ ID NO：1所示。再一方面，本公开还提供了一种用于构建视网膜色素变性疾病动物模型的核酸分子，其为gRNA，其靶标位点的碱基序列如SEQ ID NO：1所示。
SEQ ID NO：1的核酸分子为靶标位点，以该位点作为靶点，可以借助CRISPR-Cas9技术以敲除目标动物例如小鼠的Hkdc1基因部分序列，以获得视网膜色素变性疾病动物模型。
另一方面，本公开还提供了另一种视网膜色素变性疾病动物模型的构建方法，其包括：抑制或沉默目标动物的Hkdc1基因表达。
根据本公开的实验例结果显示，只要通过合适的方法抑制或沉默目标动物的Hkdc1基因表达既可以得到视网膜色素变性疾病动物模型，这些合适的方法可以是例如在基因水平上敲除Hkdc1基因、使用siRNA在RNA水平上抑制Hkdc1基因的转录或抑制转录成熟的mRNA的翻译、或者在蛋白水平上使用Hkdc1抗体中和 Hkdc1蛋白等方法，这些方法均可以使目标动物体内的Hkdc1蛋白失活或缺乏，进而可得到视网膜色素变性疾病动物模型。因此，只要是抑制或沉默目标动物的Hkdc1基因表达的方法均属于本公开的保护范围，通过这些方法得到的动物模型也是本公开的保护范围。
优选的，所述目标动物选自小鼠、大鼠、狗、猴以及猿中的任意一种。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例
本实施例采用CRISPR-Cas9技术敲除小鼠Hkdc1基因构建视网膜色素变性疾病动物模型，具体如下。
1、设计靶向敲除HHkdc1基因的gRNA序列，获得F0代Hkdc1基因敲除小鼠
针对小鼠Hkdc1基因的第二个外显子区域设计gRNA靶标位点序列，如下：
5’-CCTGTATCACATGCGGCTCTCGG-3’(SEQ ID NO.1)。
体外合成RNA，与Cas9核酸内切酶一起显微注射到小鼠受精卵中，经发育成熟后，得到发生基因突变的首建者小鼠(founder)。
gRNA序列靶向Hkdc1基因的结构示意图如图1所示。
2、测序验证Hkdc1基因敲除结果
利用小鼠的基因组DNA，对本CRISPR靶点位置附近片段进行测序，确定首建者小鼠带有影响阅读框的移码突变。
具体如下：
2.1、提取扩增用样品
①剪步骤1得到的首建者小鼠小鼠尾梢少许组织样本，置于干净的1.5ml离心管中；
②在离心管中加入100μl裂解液(40mM NaOH，0.2mM EDTA溶液)，并在金属浴100℃加热1h；
③取出离心管，冷却至室温后，加入100μl的中和液(40mM Tris-HCl，pH5.5)，10000g离心2min后，取上清用于小鼠基因型鉴定。
2.2、小鼠基因型鉴定
①PCR扩增：按照如下体系配置PCR反应体系
Figure PCTCN2018118431-appb-000001
Figure PCTCN2018118431-appb-000002
其中，
Hkdc1-Forward引物碱基序列如下：
5’-TGGTTAGGAACACATAGAACAAA-3’；
Hkdc1-Reverse引物碱基序列如下：
5’-CTAAGGGGCTGGTATGGGAAT-3’。
按照如下反应条件进行PCR反应：
Figure PCTCN2018118431-appb-000003
②纯化：
扩增出的PCR片段需要使用FastAP(已加入外切酶、反应buffer和ddH2O)纯化以去除引物及其他干扰片段，具体的纯化体系为：
PCR片段：               4.5μL；
FastAP：                1.5μL。
反应条件为：
Figure PCTCN2018118431-appb-000004
③测序
经过上述步骤的纯化片段随后用于测序反应。具体的测序反应体系为：
Figure PCTCN2018118431-appb-000005
Figure PCTCN2018118431-appb-000006
测序反应条件如下：
Figure PCTCN2018118431-appb-000007
测序反应结束后，每孔中加入50μL的70％酒精，然后12，000×g离心30min使DNA扩增片段沉淀于96孔板底，随后打开盖子，缓缓倒掉上清液体，然后倒扣96孔板，1，000×g离心1min，敞口避光放置约30min以使酒精彻底挥发干净，然后加入10μL ddH 2O以重新溶解DNA片段，最后上机测序。
测序得到的移码突变结果见图2。
Mut:NM_145419.1，c.93_99delCTCTCGG；可见，相较于野生型小鼠Hkdc1基因的cDNA序列，Hkdc1基因敲除小鼠的Hkdc1基因的cDNA序列的第93-99位碱基共7个碱基被删除，删除的序列为CTCTCGG。
3、获得F1代Hkdc1基因敲除小鼠
将带有突变的首建者小鼠(founder)与野生型小鼠交配，得到基因突变的杂合子小鼠(+/-)F1代，并进行DNA测序，确认目标基因发生了图2的移码突变从而目标蛋白被失活，实现Hkdc1基因敲除。
4、获得F2代Hkdc1基因敲除小鼠
将基因型相同的杂合子小鼠(+/-)相互交配，得到Hkdc1基因敲除的纯合子小鼠(-/-)F2代。结果得到了在第二个外显子上缺失7个碱基对(Mut:del ctctcgg)的纯合敲除小鼠(测序结果如图2纯合子所示)。
5、检测HKDC1蛋白情况
分别通过免疫印迹(western blot，WB)和免疫组织化学(Immunohistochemistry)验证在纯合突变小鼠视网膜中，HKDC1蛋白的表达。
其中，WB方法具体如下：
①分别分离野生型和突变型小鼠(即Hkdc1基因敲除的纯合子小鼠(-/-))视网膜组织，置于1.5ml离心管，并加入200μl蛋白裂解液RIPA；
②超声破碎视网膜组织后，在冰上裂解20min；
③4℃，16000g离心10min后，取上清转移至另一干净离心管，加入50μl的蛋白上样液，混匀后95℃加热5min；
④待样本冷却后，分别取20μl，160V电压进行聚丙烯酰胺凝胶电泳(SDS-PAGE)以分离蛋白；
⑤SDS-PAGE结束后，根据需要，裁剪适当大小的硝酸纤维素膜，按顺序铺上滤纸、胶、硝酸纤维素膜及滤纸，并赶去气泡，转膜槽放入冰水浴中，采用恒流0.28A的电流进行转膜，转膜2h；
⑥转膜完毕后，纯水冲洗硝酸纤维素膜一遍，晾干并标记。然后用8％的脱脂牛奶封闭2h；
⑦封闭完成后，加入一定量的按一定比例(按照抗体使用说明书)稀释于封闭液的一抗，4℃孵育过夜；
⑧回收一抗，1×TBST缓冲液洗膜4次，每次10min，根据一抗来源，选择合适二抗，用1×TBST稀释辣根过氧化氢酶(HRP)标记的二抗，室温于摇床上孵育2h；
⑨二抗孵育结束后，用1×TBST洗膜3次，每次10min，用Thermo的ELC发光试剂盒检测蛋白，所用仪器为Bio-Rad的化学发光凝胶成像系统。结果如图3所示。图3结果显示突变型小鼠视网膜组织中未表达HKDC1蛋白。
免疫组织化学方法具体如下：
小鼠断颈处死后，快速取眼球，并放入4％的PFA中，冰上固定15min后，在角膜上剪一个口子，然后继续冰上固定。2h后，PBS缓冲液冲洗3遍，然后将眼球置于30％蔗糖溶液中脱水2h，然后解剖镜下剪去角膜及晶体，OCT包埋并迅速置于-80℃冰箱冷冻。大约10min后，取出OCT包埋的眼球，置于冰冻切片机-25℃平衡约30min后即可切片。切片厚度为12μm。
切片完成后，选取质量较高的片子于37℃烘箱放置30min，然后免疫组化笔在有视网膜组织的地方画圈，PBS洗三遍以去除OCT，然后5％的NDS(含有0.25％Triton)封闭通透2h，孵育一抗，4℃过夜。第二天，PBS清洗两遍后，孵育相应的荧光二抗，然后再用PBS清洗两遍，封片，观察。
结果如图4所示。图4结果显示突变型小鼠视网膜组织中未观测到荧光，说明HKDC1蛋白未表达。
综合图3和图4结果，可知，在纯合突变小鼠视网膜中，HKDC1蛋白不再表达。
因此，利用本公开设计的gRNA序列，能够引导Cas9快速、高效、特异性敲除小鼠的Hkdc1基因，利用本公开方法可以构建Hkdc1基因敲除的Hkdc1+/-小鼠模型(杂合型)和Hkdc1-/-小鼠模型(纯合型)，二者均可用于后续的功能性研究。
试验例
以下通过试验例具体说明由实施例1得到Hkdc1基因敲除小鼠表现出的视网膜变性疾病特征。
利用实施例1构建Hkdc1基因敲除小鼠模型进行视网膜变性疾病研究
1、对敲除型小鼠的视网膜进行了冰冻切片、使用免疫组织化学染色(免疫组织化学方法同实施例1)分析，发现在9个月大小时，纯合的Hkdc1基因敲除(KO)小鼠的视网膜视紫红质(Rhodopsin)蛋白运输异常：在感光细胞内节和胞体积累(图5)。视紫红质是主要的光感蛋白，对视觉形成极其关键，其错误定位会导致感光细胞凋亡，因此，可知Hkdc1基因在视网膜变性疾病中发挥重要作用。
2、H&E染色结果显示KO小鼠(纯合的Hkdc1基因敲除小鼠)视网膜感光细胞渐进性凋亡
对11个月龄和17个月龄小鼠的视网膜进行石蜡切片、苏木精-伊红染色法(H&E染色方法)染色，具体操作如下：
1)快速取小鼠眼球组织，并置于固定液中固定24h；
2)石蜡包埋，切片，厚度为4μm；
3)切片常规用二甲苯脱蜡，经多级乙醇至水洗：二甲苯(I)5min→二甲苯(Ⅱ)5min→100％乙醇2min→95％的乙醇1min→80％乙醇1min→75％乙醇1min→蒸馏水洗2min；
4)苏木素染色5分钟，自来水冲洗；
5)盐酸乙醇分化30秒；
6)自来水浸泡15分钟；
7)置伊红液2分钟；
8)常规脱水，透明，封片：95％乙醇(I)1min→95％乙醇(Ⅱ)1min→100％乙醇(I)1min→100％乙醇(Ⅱ)1min→二甲苯石碳酸(3：1)1min→二甲苯(I)1min→二甲苯(Ⅱ)1min→中性树脂封固。
9)显微镜下拍照。
如图6所示，结果发现在11个月时，WT(野生)和KO小鼠的视网膜厚度无明显改变，然而，在17个月龄时，KO小鼠的视网膜厚度明显变薄：外核层厚度明显减少(图6)。
可见，利用本公开方法构建的Hkdc1基因敲除小鼠模型，其具有典型的视网膜变性疾病特征，可以用于视网膜变性疾病研究。
综上，本公开实施例提供的通过CRISPR-Cas9技术特异性靶向敲除Hkdc1基因的核苷酸序列，能够引导Cas9快速、高效、特异性敲除小鼠的Hkdc1基因，得到视网膜色素变性疾病小鼠模型，为Hkdc1基因相关疾病的研究提供了可靠的动物模型，其具有广阔的应用广阔。
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性：本公开提供的视网膜色素变性疾病动物模型的构建方法可以构建出视网膜中特异敲除HKDC1基因的视网膜色素变性疾病动物模型，该模型表现出典型的视网膜色素变性疾病特征，该模型可以用视网膜色素变性疾病研究或用于筛选出治疗或预防视网膜色素变性疾病的药物等领域，为进一步了解视网膜色素变性疾病的发病机制、筛选视网膜色素变性疾病药物提供了模型基础。

Claims (20)

  1. 一种视网膜色素变性疾病动物模型的构建方法，其特征在于，其包括：敲除目标动物的基因组上的目的序列，所述目的序列为Hkdc1基因，获得Hkdc1基因敲除的首建动物。
  2. 根据权利要求1所述的视网膜色素变性疾病动物模型的构建方法，其特征在于，所述目的序列为Hkdc1基因上的外显子序列。
  3. 根据权利要求2所述的视网膜色素变性疾病动物模型的构建方法，其特征在于，所述外显子序列为第二外显子序列。
  4. 根据权利要求1-3中任一项所述的视网膜色素变性疾病动物模型的构建方法，其特征在于，所述构建方法还包括：
    将所述Hkdc1基因敲除的首建动物与同类的野生型动物交配，获得杂合子Hkdc1基因敲除动物；
    将所述杂合子Hkdc1基因敲除动物自交，获得纯合子Hkdc1基因敲除动物。
  5. 根据权利要求1-4中任一项所述的视网膜色素变性疾病动物模型的构建方法，其特征在于，所述目标动物选自小鼠、大鼠、狗、猴以及猿中的任意一种。
  6. 根据权利要求1-5中任一项所述的视网膜色素变性疾病动物模型的构建方法，其特征在于，所述Hkdc1基因敲除的首建动物通过如下方法获得：将针对Hkdc1基因序列的gRNA与Cas9核酸内切酶混合，注射至目标动物授精卵，经发育成熟后，获得所述Hkdc1基因敲除的首建动物。
  7. 根据权利要求6中任一项所述的视网膜色素变性疾病动物模型的构建方法，其特征在于，所述gRNA针对的靶标位点的碱基序列如SEQ ID NO:1所示。
  8. 由权利要求1-7中任一项所述的视网膜色素变性疾病动物模型的构建方法所得到的视网膜色素变性疾病动物模型在筛选治疗视网膜色素变性疾病的药物中的应用。
  9. 由权利要求1-7中任一项所述的视网膜色素变性疾病动物模型的构建方法所得到的视网膜色素变性疾病动物模型在研究视网膜色素变性疾病中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述研究为视网膜色素变性疾病发病机理或致病机制研究。
  11. 一种视网膜色素变性疾病动物模型，其特征在于，其由权利要求1-7中任一项所述的视网膜色素变性疾病动物模型的构建方法所构建得到。
  12. 一种视网膜色素变性疾病动物模型，其特征在于，其基因组上的Hkdc1基 因序列被敲除。
  13. 根据权利要求12所述的视网膜色素变性疾病动物模型，其特征在于，所述视网膜色素变性疾病动物模型的基因组上的Hkdc1基因的外显子序列被敲除。
  14. 根据权利要求13所述的视网膜色素变性疾病动物模型，其特征在于，所述动物模型选自小鼠、大鼠、狗、猴以及猿中的任意一种。
  15. 根据权利要求14所述的视网膜色素变性疾病动物模型，其特征在于，所述动物模型为小鼠，所述动物模型的基因组上的Hkdc1基因的第2外显子序列被敲除。
  16. 根据权利要求15所述的视网膜色素变性疾病动物模型，其特征在于，相较于与野生型小鼠，所述动物模型的Hkdc1基因的cDNA序列的第93-99位碱基具有缺失突变，缺失的碱基序列为：CTCTCGG。
  17. 一种筛选用于预防或治疗视网膜色素变性疾病药物的方法，其特征在于，其包括：向权利要求11-16任一项所述的视网膜色素变性疾病动物模型施加待筛选的用于预防或治疗视网膜色素变性疾病的候选试剂；
    如果施加所述候选试剂后，检测到所述视网膜色素变性疾病动物模型出现以下现象中的一个或一个以上，则指示该候选试剂可以作为用于预防或治疗视网膜色素变性疾病的药物：
    现象(1)：相较于未施加该候选试剂的视网膜色素变性疾病，施加了该候选试剂的视网膜色素变性疾病表现出：感光细胞内节和胞体累积的视紫红质蛋白减少；
    现象(2)：相较于未施加该候选试剂的视网膜色素变性疾病，施加了该候选试剂的视网膜色素变性疾病表现出：视网膜厚度增加。
  18. 一种用于构建视网膜色素变性疾病动物模型的核酸分子，其特征在于，其为gRNA，所述gRNA的靶序列位于目标动物的Hkdc1基因上；
    优选的，所述靶序列位于目标动物的Hkdc1基因的外显子；
    优选的，所述目标动物选自小鼠、大鼠、狗、猴以及猿中的任意一种。
  19. 根据权利要求18所述的用于构建视网膜色素变性疾病动物模型的核酸分子，其特征在于，所述目标动物为小鼠，所述gRNA的靶序列如SEQ ID NO：1所示。
  20. 一种视网膜色素变性疾病动物模型的构建方法，其特征在于，其包括：抑制或沉默目标动物的Hkdc1基因表达。
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