CN114868705A - Retinitis pigmentosa mouse model and construction method thereof - Google Patents

Abstract

The invention relates to a retinitis pigmentosa mouse disease model, which is characterized in that based on a RHO gene of a retinitis pigmentosa patient, a 75nt DNA fragment of the patient is knocked into a corresponding site in a mouse genome by using CRISPR/Cas9, and the fragment contains pathogenic mutation, so that the T17M gene knock-in mouse disease model is constructed, wherein the pathogenic mutation is named as c.C50T at a nucleic acid level, and the pathogenic mutation is named as p.T17M at a protein level. The retinitis pigmentosa mouse disease model provided by the invention has patient-specific mutant fragments, and provides powerful support for research and development of corresponding medicines and gene therapy methods.

Description

Translated fromChinese

一种视网膜色素变性小鼠模型及其构建方法A kind of retinitis pigmentosa mouse model and its construction method

技术领域technical field

本发明涉及基因工程领域，涉及一种视网膜色素变性小鼠模型及其构建方法。The invention relates to the field of genetic engineering, and relates to a retinitis pigmentosa mouse model and a construction method thereof.

背景技术Background technique

视网膜色素变性(retinitis pigmentosa，RP)是一种由于基因突变引起视网膜光感受器细胞渐进性凋亡的遗传性眼病。该病主要表现为视力进行性下降、视野缩窄、夜盲、眼底色素沉着以及视网膜电图(ERG)异常。据统计，全世界视网膜色素变性的群体发病率约为1/4000，已经成为主要的致盲疾病。按照遗传方式的不同分为三类，即常染色体显性遗传(autosomal dominant RP,ADRP),常染色体隐性遗传(autosomal recessive RP,ARRP)和性染色体连锁隐性遗传(X-linked RP,XLRP)。在常染色体显性遗传RP(autosomaldominant retinitis pigmentosa，ADRP)中，视紫红质(rhodopsin，RHO)基因突变率较高，大约占ADRP发病率的25％～30％。RHO基因位于3q22.1，由5个外显子构成，编码348个氨基酸。RHO基因突变引起视紫红质蛋白发生功能异常，最终导致感光细胞发生凋亡。因此这些突变位点也成为了基因治疗过程中的重要靶点。构建由于RHO基因突变所导致的ADRP疾病模型具有重要意义。Retinitis pigmentosa (RP) is an inherited eye disease caused by a genetic mutation that causes progressive apoptosis of retinal photoreceptor cells. The main manifestations of the disease are progressive decrease in visual acuity, constriction of visual field, night blindness, fundus pigmentation and abnormal electroretinogram (ERG). According to statistics, the incidence of retinitis pigmentosa in the world is about 1/4000, and it has become a major blindness disease. According to the different ways of inheritance, it is divided into three categories, namely autosomal dominant RP (ADRP), autosomal recessive RP (ARRP) and sex chromosome-linked recessive inheritance (X-linked RP, XLRP). ). In autosomal dominant RP (autosomal dominant retinitis pigmentosa, ADRP), the mutation rate of rhodopsin (RHO) gene is relatively high, accounting for about 25% to 30% of the incidence of ADRP. The RHO gene is located at 3q22.1 and consists of 5 exons, encoding 348 amino acids. RHO gene mutation causes abnormal function of rhodopsin protein, which eventually leads to apoptosis of photoreceptor cells. Therefore, these mutation sites have also become important targets in the process of gene therapy. It is of great significance to construct an ADRP disease model caused by RHO gene mutation.

因为小鼠基因组与人类基因组的差别，目前已报道的视网膜色素变性小鼠模型与病人的基因差别很大，这样导致现有的基于视网膜色素变性小鼠模型研发的基因编辑的方法无法直接用于病人临床治疗，这些严重影响了基因编辑用于视网膜色素变性疾病的治疗。Due to the difference between the mouse genome and the human genome, the genes of the reported mouse models of retinitis pigmentosa are very different from those of patients, so that the existing gene editing methods developed based on the mouse models of retinitis pigmentosa cannot be directly used for The clinical treatment of patients has seriously affected the use of gene editing in the treatment of retinitis pigmentosa.

发明内容SUMMARY OF THE INVENTION

有鉴于此，本发明的目的在于研发一种视网膜色素变性小鼠模型，该发明涉及的视网膜色素变性小鼠疾病模型在对应基因组位点带有病人特异的突变片段，为对应的药物和基因治疗方法的研发提供有力支撑。In view of this, the purpose of the present invention is to develop a mouse model of retinitis pigmentosa, the mouse disease model of retinitis pigmentosa involved in the invention has patient-specific mutation fragments at the corresponding genomic locus, which is the corresponding drug and gene therapy. The research and development of the method provides strong support.

为了实现上述发明目的，本发明研发一种视网膜色素变性小鼠疾病模型，以视网膜色素变性病人RHO基因为基础，利用CRISPR/Cas9将病人的75nt DNA片段敲入到小鼠基因组中对应位点，该片段含有致病突变，从而构建T17M基因敲入小鼠疾病模型，所述致病突变由核酸水平命名为c.C50T，所述致病突变由蛋白质水平命名为p.T17M。In order to achieve the above purpose of the invention, the present invention develops a retinitis pigmentosa mouse disease model, which is based on the RHO gene of the retinitis pigmentosa patient, and uses CRISPR/Cas9 to knock the patient's 75nt DNA fragment into the corresponding site in the mouse genome, This fragment contains a disease-causing mutation named c.C50T at the nucleic acid level and p.T17M at the protein level to construct a T17M knock-in mouse disease model.

优选的，突变型Rho基因对应的蛋白质序列如SEQ ID NO.1所示。Preferably, the protein sequence corresponding to the mutant Rho gene is shown in SEQ ID NO.1.

优选的，T17M基因敲入小鼠Rho部分基因序列如SEQ ID NO.3所示。Preferably, the partial Rho gene sequence of the T17M knock-in mouse is shown in SEQ ID NO.3.

优选的，上述一种视网膜色素变性小鼠模型的构建方法，用CRISPR/Cas9构建基因敲入小鼠包括如下步骤，Preferably, the above-mentioned method for constructing a mouse model of retinitis pigmentosa, using CRISPR/Cas9 to construct a gene knock-in mouse comprises the following steps:

(1)设计两条特定的sgRNA序列；(1) Design two specific sgRNA sequences;

(2)在小鼠Rho基因外显子1上引入错义突变RHO,p.T17M，设计基因敲入的修复模板，修复模板序列如SEQ ID NO.2；(2) Introduce the missense mutation RHO, p.T17M into the exon 1 of the mouse Rho gene, and design a repair template for gene knock-in. The repair template sequence is as shown in SEQ ID NO.2;

(3)采用显微注射的方式将体外转录的Cas9 mRNA，sgRNA和修复模板注射入C57BL/6J小鼠受精卵中，CRISPR/Cas9基因编辑系统在小鼠受精卵中进行基因切割，诱发同源重组修复，受精卵发育为胚胎；(3) The in vitro transcribed Cas9 mRNA, sgRNA and repair template were injected into C57BL/6J mouse zygotes by microinjection, and the CRISPR/Cas9 gene editing system performed gene cleavage in the mouse zygotes to induce homology Recombination repair, the fertilized egg develops into an embryo;

(4)基因编辑后的胚胎立即转入假孕母鼠子宫中，生产后得到基因敲入小鼠。(4) The gene-edited embryos were immediately transferred into the uterus of pseudopregnant female mice, and gene knock-in mice were obtained after delivery.

优选的，两条特定的sgRNA序列包括sgRNA1和sgRNA2，分别靶向基因组特定位点，画线序列为PAM，所述sgRNA1和sgRNA2的序列如下:Preferably, two specific sgRNA sequences comprise sgRNA1 and sgRNA2, target genome specific sites respectively, the underlined sequence is PAM, and the sequences of described sgRNA1 and sgRNA2 are as follows:

sgRNA1:CGGCTCTCGAGGCTGCCCCACGG；sgRNA1:CGGCTCTCGAGGCTGCCCCACGG ;

sgRNA2:CTTCTCCAACGTCACAGGCGTGG。sgRNA2: CTTCTCCAACGTCACAGGCGTGG .

优选的，CRISPR/Cas9基因编辑系统与基因组的靶位点进行结合，Cas9发挥切割活性，产生DNA双链断裂，从而诱发DNA损伤修复，细胞通过同源重组修复DNA，将修复模板定点敲入到小鼠Rho基因中。Preferably, the CRISPR/Cas9 gene editing system is combined with the target site of the genome, and Cas9 exerts cutting activity to generate DNA double-strand breaks, thereby inducing DNA damage repair, and cells repair DNA through homologous recombination. in the mouse Rho gene.

优选的，设计195nt的修复模板，所述修复模板包含75nt的患者DNA序列和致病突变RHO,p.T17M，通过与野生型小鼠Rho基因序列进行对比，所述修复模板包含BstXI酶切位点5’-CCANNNNNNTGG-3’，而野生型小鼠Rho基因序列中无，因此该酶切位点可用作基因型鉴定。Preferably, a repair template of 195nt is designed, and the repair template includes a 75nt patient DNA sequence and a pathogenic mutation RHO, p.T17M, and compared with the wild-type mouse Rho gene sequence, the repair template includes a BstXI restriction site The point 5'-CCANNNNNNTGG-3' is absent in the wild-type mouse Rho gene sequence, so this restriction site can be used for genotype identification.

优选的，所述修复模板序列如SEQ ID NO.2所示；Preferably, the repair template sequence is shown in SEQ ID NO.2;

T17M基因敲入小鼠Rho部分基因序列如SEQ ID NO.3所示。The partial Rho gene sequence of T17M knock-in mouse is shown in SEQ ID NO.3.

优选的，小鼠鉴定的步骤包括：Preferably, the step of mouse identification includes:

假孕母鼠产仔后，即为F0代小鼠；After the pseudo-pregnant female mice give birth, they are the F0 generation mice;

取F0代小鼠的尾巴和脚趾，提取全基因组，进行PCR扩增和测序，F0代阳性小鼠与野生型小鼠进行杂交，获得F1代小鼠，取小鼠的尾巴和脚趾，提取全基因组，PCR扩增后利用BstXI酶切验证或者进行基因测序鉴定。The tail and toes of the F0 generation mice were taken, the whole genome was extracted, PCR amplification and sequencing were performed, the F0 generation positive mice were crossed with wild-type mice to obtain F1 generation mice, the tails and toes of the mice were taken, and the whole genome was extracted. The genome was verified by BstXI digestion after PCR amplification or identified by gene sequencing.

本发明中，T17M基因敲入小鼠视网膜的结构和功能都出现明显异常，这与临床病人表征相同。因此该发明为视网膜色素变性临床治疗性药物和基因治疗方法的研发提供有力支撑。In the present invention, the structure and function of the retina of the T17M gene knock-in mouse are obviously abnormal, which is the same as that of the clinical patient. Therefore, the invention provides strong support for the research and development of clinical therapeutic drugs and gene therapy methods for retinitis pigmentosa.

附图说明Description of drawings

图1为一种视网膜色素变性小鼠模型的构建图；Fig. 1 is a construction diagram of a retinitis pigmentosa mouse model;

图2为T17M基因敲入小鼠鉴定结果图；Figure 2 is a diagram showing the identification results of T17M gene knock-in mice;

图3为T17M基因敲入小鼠视网膜结构(OCT和免疫组化)鉴定结果；Figure 3 shows the identification results of retinal structure (OCT and immunohistochemistry) of T17M gene knock-in mice;

图4为T17M基因敲入小鼠视网膜功能(ERG)鉴定结果；Figure 4 is the identification result of retinal function (ERG) of T17M gene knock-in mice;

图5为T17M基因敲入小鼠眼底和血管造影图。Figure 5 is the fundus and angiogram of T17M gene knock-in mice.

具体实施方式Detailed ways

为更进一步阐述本发明为实现预定发明目的所采取的技术手段及功效，以下结合附图及较佳实施例，对依据本发明的具体实施方式、结构、特征及其功效，详细说明如后。In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific embodiments, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

本发明的参照图1至5所示，本发明所述的一种视网膜色素变性转基因小鼠模型，以视网膜色素变性病人RHO基因为基础，利用CRISPR/Cas9将病人的75DNA片段敲入到小鼠基因组中，该片段含有RHO,p.T17M致病突变，从而构建T17M基因敲入小鼠疾病模型。其中T17M是RHO基因上的一个突变位点。Referring to Figures 1 to 5 of the present invention, a retinitis pigmentosa transgenic mouse model of the present invention is based on the RHO gene of a patient with retinitis pigmentosa, using CRISPR/Cas9 to knock the patient's 75 DNA fragment into the mouse In the genome, this fragment contains the RHO, p.T17M pathogenic mutation, thereby constructing a T17M knock-in mouse disease model. Among them, T17M is a mutation site on the RHO gene.

T17M基因敲入小鼠模型Rho基因对应的蛋白质序列如SEQ ID NO.1所示：The protein sequence corresponding to the Rho gene of the T17M knock-in mouse model is shown in SEQ ID NO.1:

MNGTEGPNFYVPFSNAMGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVVFTWIMALACAAPPLVGWSRYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEVTRMVIIMVIFFLICWLPYASVAFYIFTHQGSNFGPIFMTLPAFFAKSSSIYNPVIYIMLNKQFRNCMLTTLCCGKNPLGDDDASATASKTETSQVAPA*.突变氨基酸下标有下划线。MNGTEGPNFYVPFSNAM GVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVVFTWIMALACAAPPLVGWSRYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEVTRMVIIMVIFFLICWLPYASVAFYIFTHQGSNFGPIFMTLPAFFAKSSSIYNPVIYIMLNKQFRNCMLTTLCCGKNPLGDDDASATASKTETSQVAPA*.突变氨基酸下标有下划线。

上述一种视网膜色素变性转基因小鼠模型的构建方法，包括如下步骤：The above-mentioned method for constructing a transgenic mouse model of retinitis pigmentosa comprises the following steps:

(1)设计两条特定的sgRNA序列。(1) Design two specific sgRNA sequences.

两条特定的sgRNA序列包括sgRNA1和sgRNA2，分别靶向基因组特定位点，CRISPR/Cas9的sgRNA1和sgRNA2的序列如下:Two specific sgRNA sequences, including sgRNA1 and sgRNA2, target specific sites in the genome, respectively. The sequences of sgRNA1 and sgRNA2 of CRISPR/Cas9 are as follows:

sgRNA1:CGGCTCTCGAGGCTGCCCCACGG；sgRNA1:CGGCTCTCGAGGCTGCCCCCACGG;

sgRNA2:CTTCTCCAACGTCACAGGCGTGG。sgRNA2: CTTCTCCAACGTCAACAGGCGTGG.

(2)在小鼠Rho基因外显子1上引入错义突变(RHO,p.T17M)，设计基因敲入的修复模板序列，(2) Introduce a missense mutation (RHO, p.T17M) in the exon 1 of the mouse Rho gene, and design a repair template sequence for gene knock-in,

所述修复模板序列如SEQ ID NO.2所示：The repair template sequence is shown in SEQ ID NO.2:

5’-GGGAGCCGTCAGTGGCTGAGCTCGCCAAGCAGCCTTGGTCTCTGTCTACGAAGAGCCCGTGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCGATGGGCGTGGTGCGGAGCCCCTTCGAGCAGCCGCAGTACTACCTGGCGGAACCATGGCAGTTC-3’，在小鼠Rho基因外显子1上引入错义突变(RHO,p.T17M)，该突变位点标有下划线，通过与野生型小鼠Rho基因序列对比分析，发现该修复模板包含BstXI酶切位点5’-CCANNNNNNTGG-3’，而野生型小鼠Rho基因序列中没有，因此该酶切位点可用来做基因型鉴定。5'-GGGAGCCGTCAGTGGCTGAGCTCGCCAAGCAGCCTTGGTCTCTGTCTACGAAGAGCCCGTGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCGATGGGCGTGGTGCGGAGCCCCTTCGAGCAGCCGCAGTACTACCTGGCGGAACCATGGCAGTTC-3'，在小鼠Rho基因外显子1上引入错义突变(RHO,p.T17M)，该突变位点标有下划线，通过与野生型小鼠Rho基因序列对比分析， It was found that the repair template contained the BstXI restriction site 5'-CCANNNNNNTGG-3', which was not found in the wild-type mouse Rho gene sequence, so this restriction site could be used for genotype identification.

(3)T7启动子序列通过PCR扩增插入到Cas9(利用的SpCas9,编码序列来自https://www.addgene.org/42230/)的编码区以及sgRNA序列之前，用作体外转录模板。采用显微注射的方式将体外转录的Cas9 mRNA，sgRNA和修复模板注射入C57BL/6J受精卵中，CRISPR/Cas9系统显微注射进小鼠受精卵中进行基因切割，诱发同源重组修复。(3) The T7 promoter sequence was inserted into the coding region of Cas9 (SpCas9 used, coding sequence from https://www.addgene.org/42230/) and the sgRNA sequence by PCR amplification, and used as an in vitro transcription template. The in vitro transcribed Cas9 mRNA, sgRNA and repair template were injected into C57BL/6J zygotes by microinjection, and the CRISPR/Cas9 system was microinjected into mouse zygotes for gene cleavage to induce homologous recombination repair.

CRISPR/Cas9基因编辑系统与基因组的靶位点进行结合，Cas9发挥切割活性，产生DNA双链断裂，从而诱发DNA损伤修复，细胞通过同源重组修复DNA，将修复模板定点敲入到小鼠Rho基因中。The CRISPR/Cas9 gene editing system is combined with the target site of the genome, and Cas9 exerts cutting activity to generate DNA double-strand breaks, thereby inducing DNA damage repair. Cells repair DNA through homologous recombination, and the repair template is knocked into mouse Rho. in genes.

(4)基因编辑后的受精卵发育为胚胎，转入假孕母鼠子宫中，待生产后得到基因敲入小鼠。(4) The gene-edited fertilized egg develops into an embryo, which is transferred into the uterus of a pseudo-pregnant female mouse, and a knock-in mouse is obtained after delivery.

T17M基因敲入小鼠Rho基因部分序列如SEQ ID NO.3所示：The partial sequence of the Rho gene of the T17M knock-in mouse is shown in SEQ ID NO.3:

gcgttagtatgatatctcgcggatgctgaatcagcctctggcttagggagagaaggtcactttataagggtctggggggggtcagtgcctggagttgcgctgtgggagccgtcagtggctgagctcgccaagcagccttggtctctgtctacgaagagcccgtGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCGATGggcgtggtgcggagccccttcgagcagccgcagtactacctggcggaaccatggcagttctccatgctggcagcgtacatgttcctgctcatcgtgctgggcttccccatcaacttcctcacgctctacgtcaccgtacagcacaagaagctgcgcacacccctcaactacatcctgctcaacttggccgtggctgacctcttcatggtcttcggaggattc，文中DNA序列(大写)为人类的75ntDNA片段，突变位点(RHO,p.T17M)标有下划线。gcgttagtatgatatctcgcggatgctgaatcagcctctggcttagggagagaaggtcactttataagggtctggggggggtcagtgcctggagttgcgctgtgggagccgtcagtggctgagctcgccaagcagccttggtctctgtctacgaagagcccgtGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCGAT Gggcgtggtgcggagccccttcgagcagccgcagtactacctggcggaaccatggcagttctccatgctggcagcgtacatgttcctgctcatcgtgctgggcttccccatcaacttcctcacgctctacgtcaccgtacagcacaagaagctgcgcacacccctcaactacatcctgctcaacttggccgtggctgacctcttcatggtcttcggaggattc，文中DNA序列(大写)为人类的75ntDNA片段，突变位点(RHO,p.T17M)标有下划线。

在本发明中，小鼠繁殖并进行后代鉴定的步骤包括：In the present invention, the steps of mouse breeding and progeny identification include:

假孕母鼠产仔后，即为F0代小鼠；取小鼠的尾巴和脚趾，PCR扩增后利用BstXI酶切验证或者进行基因测序鉴定。F0代阳性小鼠与野生型小鼠进行杂交，获得F1代小鼠，取F1代小鼠的尾巴和脚趾，提取全基因组，进行PCR扩增，BstXI酶切验证或者进行基因测序鉴定。F1代小鼠继续配种以获得更多的疾病小鼠。Pseudo-pregnant female mice are F0 generation mice after giving birth; the tails and toes of the mice are taken, and PCR amplification is used for verification by BstXI digestion or for identification by gene sequencing. F0-generation positive mice were crossed with wild-type mice to obtain F1-generation mice. The tails and toes of F1-generation mice were taken, and the whole genome was extracted for PCR amplification, BstXI digestion verification or gene sequencing identification. The F1 generation mice continued to be bred to obtain more diseased mice.

由本发明的参照图2所示，设计了sgRNA1和sgRNA2分别靶向目的序列以及195nt的修复模板，其中sgRNA1和sgRNA2分别靶向目的序列如图2A所示。所述修复模板包含75nt的人类DNA序列和致病突变(RHO,p.T17M)。对小鼠受精卵进行编辑并发育为胚胎。然后将基因编辑后的胚胎移植到假孕的母鼠子宫内，生产后得到基因敲入小鼠(如图1)。在设计修复模板时，由于该模板包含了75nt的人类DNA序列，因此与野生型小鼠Rho基因序列做对比，发现该修复模板包含BstXI酶切位点(5’-CCANNNNNNTGG-3’)，因此基因敲入小鼠Rho基因序列中包含该酶切位点，而野生型(WT)小鼠Rho基因序列中无。通过PCR扩增并进行BstXI酶切后，琼脂糖凝胶电泳出现两个条带的说明是基因敲入小鼠，只有一个条带的代表是野生型小鼠，从而进行鉴定(图2B)。通过Sanger测序也可进行基因型鉴定(图2C)。Referring to Figure 2 of the present invention, sgRNA1 and sgRNA2 were designed to target the target sequence and a repair template of 195nt, respectively, wherein sgRNA1 and sgRNA2 target the target sequence respectively as shown in Figure 2A. The repair template contains 75 nt of human DNA sequence and a pathogenic mutation (RHO, p.T17M). A mouse fertilized egg is edited and developed into an embryo. The gene-edited embryos were then transplanted into the uterus of pseudopregnant female mice, and knock-in mice were obtained after delivery (Figure 1). When designing the repair template, because the template contains a 75nt human DNA sequence, it is compared with the wild-type mouse Rho gene sequence, and it is found that the repair template contains the BstXI restriction site (5'-CCANNNNNNTGG-3'), so The knock-in mouse Rho gene sequence contains this restriction site, but the wild-type (WT) mouse Rho gene sequence does not. After PCR amplification and BstXI digestion, two bands appeared on agarose gel electrophoresis, indicating that they were knock-in mice, and only one band represented wild-type mice for identification (Fig. 2B). Genotyping was also performed by Sanger sequencing (Figure 2C).

由本发明的参照图3所示，进一步观察基因敲入小鼠视网膜的结构变化。OCT(Optical Coherence Tomography，即光学相干断层扫描技术)显示野生型(WT)小鼠视网膜结构清晰，而基因敲入小鼠视网膜感光细胞层明显变薄，尤其是外核层(图3A、B)。进一步做苏木素-伊红染色如图3C，与WT小鼠相比，基因敲入小鼠视网膜结构异常，最明显的是外核层变薄至几乎消失。As shown in FIG. 3 with reference to the present invention, the structural changes of the retina of gene knock-in mice were further observed. OCT (Optical Coherence Tomography, optical coherence tomography) showed that the retinal structure of wild-type (WT) mice was clear, while the retinal photoreceptor cell layer of gene knock-in mice was significantly thinner, especially the outer nuclear layer (Fig. 3A, B). . Further hematoxylin-eosin staining was performed as shown in Figure 3C. Compared with the WT mice, the retinal structure of the knock-in mice was abnormal, the most obvious being the thinning of the outer nuclear layer to almost disappear.

由上可知，由本发明的参照图4所示，由上可知，T17M基因敲入小鼠视网膜结构变化明显，对小鼠视网膜的电生理功能进行了评估。视网膜电图是光刺激视网膜时从相应部位记录到的视网膜总电生理反应。在暗适应视网膜电图检测视杆细胞反应时(A、E)，与WT相比，b波波幅明显减小(n＝10,P<0.0001)；在暗适应混合反应时，正常的野生型小鼠依次有一个负相较小的a波和一个较大的正相b波，T17M基因敲入小鼠的a波和b波波幅(B、D、E)明显减小，差异显著(n＝10，P<0.0001)；在检查视锥细胞功能时(C、D、E)，与WT相比，a波(n＝10,P>0.05)无明显差异，b波波幅明显减小(n＝10,P<0.01)，这提示T17M基因敲入小鼠视网膜中有大量视杆细胞凋亡，但是视锥细胞还保留了部分功能。T17M基因敲入小鼠视网膜的功能明显降低与OCT检测到的视网膜外核层变薄结果是一致的。As can be seen from the above, as shown in FIG. 4 with reference to the present invention, it can be seen from the above that the retinal structure of the T17M gene knock-in mice changed significantly, and the electrophysiological function of the mouse retina was evaluated. The electroretinogram is the total electrophysiological response of the retina recorded from the corresponding site when the retina is stimulated by light. In the dark-adapted electroretinogram to detect the rod photoreceptor response (A, E), the b-wave amplitude was significantly reduced compared with WT (n=10, P<0.0001); in the dark-adapted mixed response, the normal wild-type The mice had a negative smaller a wave and a larger positive b wave in turn. The amplitudes of the a and b waves (B, D, E) of the T17M knock-in mice were significantly reduced, and the difference was significant (n =10, P<0.0001); when examining cone cell function (C, D, E), compared with WT, there was no significant difference in a wave (n=10, P>0.05), and the amplitude of b wave was significantly reduced ( n=10, P<0.01), which suggested that a large number of rod cells were apoptotic in the retina of T17M gene knock-in mice, but cone cells still retained some functions. The significantly reduced retinal function of T17M knock-in mice was consistent with the thinning of the retinal outer nuclear layer detected by OCT.

由本发明的参照图5所示，通过眼底视网膜检测和血管造影(如图5，箭头指示)可以观察到T17M基因敲入小鼠视网膜眼底出现明显视网膜色素变性，视网膜动脉逐渐变细，并且出现串珠样改变。As shown in FIG. 5 in reference to the present invention, it can be observed that T17M gene knock-in mice have obvious retinitis pigmentosa in the retinal fundus of T17M gene knock-in mice by fundus retinal detection and angiography (indicated by arrows in FIG. 5 , retinal arteries are gradually thinned, and beading appears. kind of change.

综上可知，T17M基因敲入小鼠视网膜的结构和功能出现明显异常，这与临床病人表征相同，说明成功构建了T17M基因敲入小鼠疾病模型。In summary, the structure and function of the retina of T17M knock-in mice were obviously abnormal, which was the same as that of clinical patients, indicating that the disease model of T17M knock-in mice was successfully constructed.

以上所述，仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭示如上，然而并非用以限定本发明，任何本领域技术人员，在不脱离本发明技术方案范围内，当可利用上述揭示的技术内容做出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简介修改、等同变化与修饰，均仍属于本发明技术方案的范围内。The above descriptions are only preferred embodiments of the present invention, and do not limit the present invention in any form. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art , without departing from the scope of the technical solution of the present invention, when the technical content disclosed above can be used to make some changes or modifications to equivalent embodiments of equivalent changes, as long as it does not depart from the technical solution content of the present invention, according to the technical solution of the present invention Substantially any brief modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the technical solutions of the present invention.

Claims (9)

Translated fromChinese

1.一种视网膜色素变性小鼠疾病模型，其特征是：以视网膜色素变性病人RHO基因为基础，利用CRISPR/Cas9将病人的75ntDNA片段敲入到小鼠基因组中对应位点，该片段含有致病突变，从而构建T17M基因敲入小鼠疾病模型，所述致病突变由核酸水平命名为c.C50T，所述致病突变由蛋白质水平命名为p.T17M。1. A mouse disease model of retinitis pigmentosa, characterized in that: based on the RHO gene of a patient with retinitis pigmentosa, a 75nt DNA fragment of the patient is knocked into the corresponding site in the mouse genome by using CRISPR/Cas9, and the fragment contains a causative agent. To construct a T17M knock-in mouse disease model, the pathogenic mutation was named c.C50T from the nucleic acid level, and the pathogenic mutation was named p.T17M from the protein level.2.根据权利要求1所述的一种视网膜色素变性小鼠模型，其特征是：突变型Rho基因对应的蛋白质序列如SEQ ID NO.1所示。2 . The retinitis pigmentosa mouse model according to claim 1 , wherein the protein sequence corresponding to the mutant Rho gene is shown in SEQ ID NO.1. 3 .3.根据权利要求1或2所述的一种视网膜色素变性小鼠疾病模型，其特征是：T17M基因敲入小鼠Rho部分基因序列如SEQ ID NO.3所示。3 . The mouse disease model of retinitis pigmentosa according to claim 1 or 2 , wherein the Rho partial gene sequence of the T17M gene knock-in mouse is shown in SEQ ID NO.3. 4 .4.一种视网膜色素变性小鼠模型的构建方法，其特征是：用CRISPR/Cas9构建基因敲入小鼠包括如下步骤，4. a method for constructing a retinitis pigmentosa mouse model, characterized in that: constructing a knock-in mouse with CRISPR/Cas9 comprises the following steps,(1)设计两条特定的sgRNA序列；(1) Design two specific sgRNA sequences;(2)在小鼠Rho基因外显子1上引入错义突变RHO,p.T17M，设计基因敲入的修复模板，修复模板序列如SEQ ID NO.2；(2) Introduce the missense mutation RHO, p.T17M into the exon 1 of the mouse Rho gene, and design a repair template for gene knock-in. The repair template sequence is as shown in SEQ ID NO.2;(3)采用显微注射的方式将体外转录的Cas9 mRNA，sgRNA和修复模板注射入C57BL/6J小鼠受精卵中，CRISPR/Cas9基因编辑系统在小鼠受精卵中进行基因切割，诱发同源重组修复，受精卵发育为胚胎；(3) The in vitro transcribed Cas9 mRNA, sgRNA and repair template were injected into C57BL/6J mouse zygotes by microinjection, and the CRISPR/Cas9 gene editing system performed gene cleavage in the mouse zygotes to induce homology Recombination repair, the fertilized egg develops into an embryo;(4)基因编辑后的胚胎立即转入假孕母鼠子宫中，生产后得到基因敲入小鼠。(4) The gene-edited embryos were immediately transferred into the uterus of pseudopregnant female mice, and gene knock-in mice were obtained after delivery.5.根据权利要求4所述的一种视网膜色素变性小鼠模型的构建方法，其特征是：两条特定的sgRNA序列包括sgRNA1和sgRNA2，分别靶向基因组特定位点的正向和反向序列，所述sgRNA1和sgRNA2的序列如下:5. the construction method of a kind of retinitis pigmentosa mouse model according to claim 4 is characterized in that: two specific sgRNA sequences comprise sgRNA1 and sgRNA2, the forward and reverse sequences of targeting genome specific sites respectively , the sequences of the sgRNA1 and sgRNA2 are as follows:sgRNA1:CGGCTCTCGAGGCTGCCCCACGG；sgRNA1:CGGCTCTCGAGGCTGCCCCCACGG;sgRNA2:CTTCTCCAACGTCACAGGCGTGG。sgRNA2: CTTCTCCAACGTCAACAGGCGTGG.6.根据权利要求4所述的一种视网膜色素变性小鼠模型的构建方法，其特征是：6. the construction method of a kind of retinitis pigmentosa mouse model according to claim 4 is characterized in that:CRISPR/Cas9基因编辑系统与基因组的靶位点进行结合，Cas9发挥切割活性，产生DNA双链断裂，从而诱发DNA损伤修复，细胞通过同源重组修复DNA，将修复模板定点敲入到小鼠Rho基因中。The CRISPR/Cas9 gene editing system is combined with the target site of the genome, and Cas9 exerts cutting activity to generate DNA double-strand breaks, thereby inducing DNA damage repair. Cells repair DNA through homologous recombination, and the repair template is knocked into mouse Rho. in genes.7.根据权利要求4或5或6所述的一种视网膜色素变性小鼠模型的构建方法，其特征是：设计195nt的修复模板，所述修复模板包含75nt的患者DNA序列和致病突变RHO,p.T17M，通过与野生型小鼠Rho基因序列进行对比，所述修复模板包含BstXI酶切位点5’-CCANNNNNNTGG-3’，而野生型小鼠Rho基因序列中无，因此该酶切位点可用作基因型鉴定。7. the construction method of a kind of retinitis pigmentosa mouse model according to claim 4 or 5 or 6, is characterized in that: the repair template of design 195nt, described repair template comprises the patient DNA sequence of 75nt and pathogenic mutation RHO ,p.T17M, by comparing with the wild-type mouse Rho gene sequence, the repair template contains the BstXI restriction site 5'-CCANNNNNNTGG-3', but there is no wild-type mouse Rho gene sequence, so this restriction enzyme cut The loci can be used for genotyping.8.根据权利要求7所述的一种视网膜色素变性小鼠模型的构建方法，其特征是：8. the construction method of a kind of retinitis pigmentosa mouse model according to claim 7 is characterized in that:所述修复模板序列如SEQ ID NO.2所示；The repair template sequence is shown in SEQ ID NO.2;T17M基因敲入小鼠Rho部分基因序列如SEQ ID NO.3所示。The partial Rho gene sequence of T17M knock-in mouse is shown in SEQ ID NO.3.9.根据权利要求4所述的一种视网膜色素变性小鼠模型的构建方法，其特征是:其中小鼠鉴定的步骤包括，9. the construction method of a kind of retinitis pigmentosa mouse model according to claim 4, is characterized in that: wherein the step of mouse identification comprises,假孕母鼠产仔后，即为F0代小鼠；After the pseudo-pregnant female mice give birth, they are the F0 generation mice;取F0代小鼠的尾巴和脚趾，提取全基因组，进行PCR扩增和测序，F0代阳性小鼠与野生型小鼠进行杂交，获得F1代小鼠，取小鼠的尾巴和脚趾，PCR扩增后利用BstXI酶切验证或者进行基因测序鉴定。The tail and toes of the F0 generation mice were taken, the whole genome was extracted, PCR amplification and sequencing were performed, the F0 generation positive mice were crossed with wild-type mice to obtain the F1 generation mice, and the tails and toes of the mice were taken, PCR amplification was performed. After the increase, use BstXI enzyme digestion to verify or perform gene sequencing identification.

Images