WO2018045727A1 - 粘多糖贮积症ii型动物模型的构建方法及应用 - Google Patents

粘多糖贮积症ii型动物模型的构建方法及应用 Download PDF

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WO2018045727A1
WO2018045727A1 PCT/CN2017/075127 CN2017075127W WO2018045727A1 WO 2018045727 A1 WO2018045727 A1 WO 2018045727A1 CN 2017075127 W CN2017075127 W CN 2017075127W WO 2018045727 A1 WO2018045727 A1 WO 2018045727A1
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mouse
mice
ids
animal model
sgrna
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高恩
侯增淼
李晓颖
李敏
杨小琳
赵金礼
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陕西慧康生物科技有限责任公司
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • C—CHEMISTRY; METALLURGY
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • the invention belongs to the technical field of medical biology, and in particular, the invention relates to a method for constructing an animal model.
  • the mouse disease model plays a key role in studying the pathogenesis and drug screening of human diseases, especially in evaluating the therapeutic effects of drugs and the choice of treatment methods.
  • Gene knockout is a new molecular biology technique developed since the late 1980s. It is a technique for inactivating or deleting organism-specific genes through certain pathways. In general, gene knockout is mainly based on the principle of DNA homologous recombination, and the target gene fragment is replaced by a designed homologous fragment to achieve the purpose of gene knockout. This is a complex molecular biology technique, also known as "gene targeting" technology. So far, thousands of genetically modified mouse models have been constructed using this technology. However, this traditional gene knockout method is complicated, time consuming, and expensive. In recent years, everyone is looking for a new method that is faster, more economical, and more adaptable.
  • Mucopolysaccharidosis type II (MPS II, MIM309900), also known as Hunter syndrome, is an X-linked recessive genetic disease caused by gene mutations that cause the lysosomal enzyme, idose-2-sulfate.
  • the enzyme iduronate-2-sulfatase, IDS
  • IDS iduronate-2-sulfatase
  • MPS mucop01ysaccharides
  • DS dermatin sulfate B
  • HS heparitin sulfate
  • the patient's performance at birth is generally normal, but as more and more mucopolysaccharides are stored in the body, the course of MPS II is progressively aggravated, with typical symptoms and poor prognosis.
  • the IDS gene is located at Xq27.3-Xq28, and MPS II is highly correlated with mutations in the IDS gene. Because of the extremely low incidence, it is also called "orphan disease.” At present, the research and treatment of the disease in the world is still difficult.
  • the present invention provides a problem in the complexity of the current animal model construction, the harsh conditions, the high cost, and the lack of a researchable animal model in the current mucopolysaccharidosis type II study.
  • the present invention provides a method for constructing a mucopolysaccharidosis type II animal model, which comprises the following steps:
  • mice were mated with wild type male mice to obtain F1 mice.
  • Ms Ids E5-1-sgRNA1 TAGGACAAAGCAGATTGGCG and AAACCGCCAATCTGCTTTGT;
  • Ms Ids E5-2-sgRNA2 TAGGATGTGGCAGATGTGCCTGA and AAACTCAGGCACATCTGCCACAT.
  • step (2) female mice of 4-5 weeks old are used as donors, and PMSG and hCG are intraperitoneally injected for superovulation, and then mated with male mice, and the mouse fertilized eggs are obtained the next day. .
  • step (4) the primer comprising the target of sgRNA action is:
  • Ms Ids E5 C9 reverse primer AGGCATCCTGGTAGGTAGGTTAT.
  • the PCR reaction system is 50 ⁇ L, and the conditions of the PCR reaction are: pre-denaturation at 95 ° C for 5 min, followed by denaturation at 95 ° C for 30 s, annealing at 55 ° C for 30 s, extension at 72 ° C for 30 s, 30 cycles, It was then extended at 72 ° C for 10 min.
  • step (4) digestion with a T7 endonuclease is carried out.
  • the mutant mouse is selected, the mutation is further detected by TA cloning and sequencing, and then the Founder mouse is determined.
  • the foregoing construction method further includes genotype identification of a mucopolysaccharidosis type II animal model, comprising the steps of: extracting genomic DNA of F1 generation mice; designing a primer containing a target of sgRNA, and extracting the F1 generation mouse
  • the genomic DNA is used as a template, and a PCR reaction is carried out to sequence the products of the PCR reaction.
  • the primer comprising the target of sgRNA action is:
  • Ms Ids E5 C9 reverse primer AGGCATCCTGGTAGGTAGGTTAT.
  • the reaction system of the PCR reaction is 50 ⁇ L, and the reaction conditions are: pre-denaturation at 95 ° C for 5 min, followed by denaturation at 95 ° C for 30 s, annealing at 55 ° C for 30 s, extension at 72 ° C for 30 s, 30 cycles, followed by extension at 72 ° C for 10 min. .
  • the foregoing construction method further includes phenotypic analysis of a mucopolysaccharidosis type II animal model, comprising the steps of separately extracting four tissues of heart, liver, spleen and kidney of F1 mice and male wild type mice, and preparing the same. Tissue sections, tissue sections were stained with Alcian blue, and tissue sections of F1 mice were compared with tissue sections of male wild type mice.
  • the present invention provides a mucopolysaccharidosis type II animal model constructed by the construction method of the first aspect.
  • the nucleotide of the X-chromate Idose-2-sulfatase gene is 18956-18975 bp, and the sequence is AACTCCACGCCAATCTGCTT.
  • the present invention provides a mucopolysaccharidosis type II animal model constructed by the construction method of the first aspect or a mucopolysaccharidosis type II animal model of the second aspect in the study of mucopolysaccharidosis type II use.
  • the mucosal storage disease type II mouse animal model established by the CRISPR/Cas9 gene knockout technique of the present invention provides an extremely important study for further research on the pathogenic mechanism and therapeutic effect evaluation of mucopolysaccharidosis type II. Animal model.
  • the method for obtaining a mucopolysaccharidosis type II mouse animal model by the method of the present invention has a short cycle of only 2 months.
  • the IDS knockout mouse model of the present invention is obtained, genotype labeled X -ids X ids, for the phenotype of normal mice, normally grown in the laboratory, by wild type male C57BL / 6
  • the mouse (genotype is X ids Y) mating, its gene can be stably inherited, and the inventors succeeded in establishing a gene stable genetic model of the IDS knockout model. It was mated with wild-type C57BL/6 male mice (genotype X ids Y) to obtain F1 mice. In F1 mice, 25% of the mice had a genotype of X- ids Y, ie, mucopolysaccharide.
  • mice X -ids Y storage disease type II mouse model
  • IDS knockout mouse model X genotype -ids X ids
  • the method of the present invention obtains an IDS knockout model mouse capable of simultaneously producing an IDS knockout model mouse and a mucopolysaccharidosis type II mouse animal model, which is the most ingenious in the technical solution of the present invention.
  • Figure 1 is a schematic diagram showing the mechanism of RNA-mediated directed genomic modification of the Cas9 system
  • Figure 2 is a CRISPR/Cas9-mediated analysis of IDS gene modification
  • Figure 3 is a schematic diagram showing the structure of a DNA sequence at a target
  • Figure 4 (A) and Figure 4 (B) are genotype identification sequencing results of a mucopolysaccharidosis type II mouse animal model
  • Fig. 5 is a result of Alcian blue staining of tissue sections of a mucopolysaccharidosis type II mouse animal model.
  • the CRISPR/Cas9 gene knockout technology involved in the present invention is the latest generation method for constructing gene knockout mice by a technique such as zinc finger nuclease and TALEN, and exhibits great potential in directional modification of genes.
  • the invention provides a mucopolysaccharidosis type II mouse animal model based on CRISPR/Cas9 gene knockout technology and a construction method thereof, and further studies on mucopolysaccharide storage The pathogenic mechanism and evaluation of therapeutic effects of Type II provides an extremely important animal model.
  • the synthesized oligonucleotide was annealed (naturally reduced to room temperature after 5 min at 95 ° C), and ligated into the pUC57-T7-sgRNA expression vector (Addgene, NO. 51132) recovered by BsaI digestion to construct an sgRNA expression vector. It is verified by sequencing whether the ligated fragment is correct, the correct clone is selected, and the plasmid is extracted after expansion to prepare an in vitro transcription template.
  • Cas9 (D10A) expression plasmid (Pst1374-NLS-Cas9-ZF) was linearized by AgeI, purified by phenol chloroform extraction, and dissolved in nuclease-free water as a template for in vitro transcription.
  • the synthesis of Cas9 mRNA was performed in vitro by the T7 Ultra Kit (Ambion, AM1345) using T7 RNA polymerase.
  • the sgRNA expression vector was linearized by DraI, purified by phenol chloroform extraction, and dissolved in nuclease-free water as a template for in vitro transcription.
  • In vitro synthesis of sgRNA was performed in vitro using the kit MEGAshortscript Kit (Ambion, AM1354) using T7 RNA polymerase.
  • the donor female mice were treated with PMSG (serum gonadotropin), hCG (human chorionic gonadotropin) was injected 46 hours later, and the male mice were mated with the cage, and the fertilized eggs were taken for microinjection the next day.
  • PMSG serum gonadotropin
  • hCG human chorionic gonadotropin
  • the well-transcribed Cas9 mRNA and sgRNA were mixed and adjusted to concentrations of 20 ng/ ⁇ l and 12.5 ng/ Ll each sgRNA, microinjection method to inject the RNA mixture into the cytoplasm of the mouse fertilized egg, transplant the surviving 143 fertilized eggs to 5 pseudopregnant C57BL/6 female mice, and transplant each pseudopregnant female mouse 28 fertilized eggs. After about 3 weeks, the mice were born, and a total of 19 F0 mice were obtained, and 17 survived.
  • the embryo-transplanted mice will be born about 19 days after the operation. After the mice are born for 20 days, the DNA is extracted from the tail and identified by PCR.
  • a pair of primers was designed to include a sgRNA target (ms Ids E5C9 For: AGTTCTGGTCTGGAGACACAATT and ms Ids E5 C9 Rev: AGGCATCCTGGTAGGTAGGTTAT), and the primers were synthesized by Bioengineering (Shanghai) Co., Ltd.
  • the PCR reaction system was 50 ⁇ L (the PCR reaction related reagent was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.).
  • Reaction conditions 95 ° C for 5 min; (95 ° C 30 s, 55 ° C 30 s, 72 ° C 30 s), 30 cycles; 72 ° C 10 min; storage 4 ° C.
  • the amplified fragment was 466 bp in size.
  • the PCR product was purified using a PCR purification kit (Beijing Tiangen). 100 ng of the purified PCR product was denatured and renatured in NEB Buffer 2, and then incubated with T7 endonuclease (NEB, M0302L) at 37 ° C for 40 min, and then separated by 1.5% agarose gel electrophoresis.
  • T7 endonuclease recognizes incompletely paired double-stranded DNA and cleaves it, and if CRISPR/Cas9 causes a mutation in the target, it will be recognized by the enzyme and cause double-stranded DNA breaks. Therefore, a band other than the PCR product band was present in the agarose gel electrophoresis, indicating that the annealing product could be recognized and cleaved by the T7 endonuclease, indicating that there may be a mutation in the target DNA sequence (Fig. 2). To further verify the presence of mutations, as well as the specifics of the mutations, mutations were further detected by TA cloning and sequencing (Fig. 3).
  • mice are mated with wild type mice to obtain F1
  • mice Female Founder mice were mated to wild-type male mice by 4 weeks of age, and the mice were identified by PCR 20 days after birth. If a positive mouse is born, it means that the transgene has been integrated into the germ cell and the marker line is successfully established. IDS obtain knockout mouse model, the genotype labeled X -ids X ids. Selection No. 23-1 F0 generation female mice (genotype X -ids X ids) to wild type male C57BL / 6 mice were obtained in F1 mice (genotype X ids Y) mate.
  • PCR reaction system was 50 ⁇ L (the PCR reaction related reagent was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.).
  • the amplified fragment was 466 bp in size.
  • the PCR product was purified by PCR purification kit (Beijing Tiangen), and the purified PCR product was directly sent to the sequencing. The sequencing results are shown in Fig. 4 (A) and Fig. 4 (B), and the mucopolysaccharidosis type II was obtained.
  • the mouse model has a genotype of X- ids Y.
  • the sequencing result shows that it lacks 20 bases of the nucleotide 18956-18975bp of Mouse BAC-146N21 Chromosome X contains iduronate-2-sulfatase gene (Accession: AC002315), and the sequence is AACTCCACGCCAATCTGCTT. This result is consistent with the design, that is, the genotype identification of the correct mucopolysaccharidosis type II mouse animal model (genotype is X- ids Y).
  • mice were sacrificed by cervical dislocation, and the animal models of the mucopolysaccharidosis type II mouse (genotype X- ids Y) and the heart, liver, spleen and kidney of male wild mice were extracted.
  • the paraffin tissue sections of the tissue were sliced and the sections were subjected to Alcian blue staining.
  • Alcian blue is a cationic dye and is the most specific dye for acidic mucus.
  • the heart, liver, spleen, and kidney tissues of the mucopolysaccharidosis type II mouse model (genotype X- ids Y) were found by comparison with the Alcian blue staining of wild-type mouse tissue sections (Fig. 5). There is obvious aggregation of mucopolysaccharide, which proves that the animal model of mucopolysaccharidosis type II mouse is successfully constructed.

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Abstract

提供了粘多糖贮积症II型动物模型的构建方法，包括针对小鼠IDS基因设计靶点，合成寡核苷酸链，合成的寡核苷酸链经退火与pUC57-T7-sgRNA质粒进行连接，获得sgRNA表达载体；获得小鼠受精卵；将转录好的Cas9 mRNA和sgRNA混合均匀得到RNA混合物，将RNA混合物显微注射至小鼠受精卵的细胞质，随后将存活的受精卵植入雌性小鼠体内，繁殖获得F0代小鼠；提取F0代小鼠的基因组DNA，作为模板，进行PCR反应，产物经酶切，选择突变小鼠，从而确定Founder小鼠；Founder小鼠与野生型雄性小鼠交配获得F1代小鼠。还提供了由该方法构建的动物模型以及该模型在研究粘多糖贮积症II型中的用途。

Description

粘多糖贮积症II型动物模型的构建方法及应用 技术领域
本发明属于医学生物学技术领域，具体地，本发明涉及一种动物模型的构建方法。
背景技术
小鼠疾病模型在研究人类疾病致病机理和药物筛选中起到了关键作用，特别是在评价药物治疗效果及治疗方式的选择上具有重要价值。
基因敲除是自80年代末以来发展起来的一种新型分子生物学技术，是通过一定的途径使机体特定的基因失活或缺失的技术。通常意义上的基因敲除主要是应用DNA同源重组原理，用设计的同源片段替代靶基因片段，从而达到基因敲除的目的。这是一项复杂的分子生物学技术，又称为“基因打靶”技术，到目前为止，利用该技术已构建了数千种基因突变小鼠模型。然而这项传统的基因敲除方法因流程复杂、耗时长、费用大，近些年来，大家都在寻找一种更快速、更经济且适用性强的新方法。
粘多糖贮积症II型(Mucopolysaccharidosis type II，MPS II，MIM309900)，又称Hunter综合征，是一种X连锁隐性遗传病，由于基因突变导致溶酶体酶艾杜糖-2-硫酸酯酶(iduronate-2-sulfatase，IDS)缺乏，以致粘多糖(mucop01ysaccharides，MPS)在体内大量沉积，尿中大量排出硫酸皮肤素(dermatin sulfate B，DS)、硫酸已酰肝素(heparitin sulfate，HS)。患者出生时表现一般正常，但随着越来越多的粘多糖贮积在体内，MPS II病程进行性加重，症状典型，预后甚差。IDS基因定位于Xq27.3-Xq28，MPS II与IDS基因发生突变高度相关。由于发病率极低，又被称为“孤儿病”。目前，世界上该疾病的研究及治疗仍比较困难。
发明内容
针对当前动物模型构建时存在的过程复杂、条件苛刻、费用巨大，以及当前粘多糖贮积症II型研究中缺少可研究的动物模型的问题，本发明提供了 一种粘多糖贮积症II型动物模型的构建方法，由该方法获得的动物模型，以及该动物模型在研究粘多糖贮积症II型中的用途。
第一方面，本发明提供了一种粘多糖贮积症II型动物模型的构建方法，其特征在于，包括如下步骤：
(1)针对小鼠IDS基因设计靶点，合成寡核苷酸链，合成的寡核苷酸链经退火与pUC57-T7-sgRNA质粒进行连接，获得sgRNA表达载体；
(2)获得小鼠受精卵；
(3)将转录好的Cas9 mRNA和sgRNA混合均匀得到RNA混合物，将RNA混合物显微注射至小鼠受精卵的细胞质，随后将存活的受精卵植入雌性小鼠体内，繁殖获得F0代小鼠；
(4)提取F0代小鼠的基因组DNA，作为模板，采用包含sgRNA作用靶点的引物，进行PCR反应，产物经酶切，选择突变小鼠，从而确定Founder小鼠；
(5)Founder小鼠与野生型雄性小鼠交配获得F1代小鼠。
前述的构建方法，步骤(1)中合成如下两对寡核苷酸链：
ms Ids E5-1-sgRNA1：TAGGACAAAGCAGATTGGCG和AAACCGCCAATCTGCTTTGT；
ms Ids E5-2-sgRNA2：TAGGATGTGGCAGATGTGCCTGA和AAACTCAGGCACATCTGCCACAT。
前述的构建方法，在步骤(2)中，以4-5周龄的雌性小鼠为供体，腹腔注射PMSG和hCG进行超数排卵，随后与雄性小鼠交配，次日获得小鼠受精卵。
前述的构建方法，步骤(4)中，包含sgRNA作用靶点的引物是：
ms Ids E5 C9正向引物：AGTTCTGGTCTGGAGACACAATT；
ms Ids E5 C9反向引物：AGGCATCCTGGTAGGTAGGTTAT。
前述的构建方法，步骤(4)中，PCR反应的体系是50μL，PCR反应的条件是：95℃预变性5min，随后95℃变性30s，55℃退火30s，72℃延伸30s，30个循环，随后72℃延伸10min。
前述的构建方法，步骤(4)中，采用T7核酸内切酶进行酶切。
前述的构建方法，步骤(4)中，选择突变小鼠之后，先通过TA克隆、测序进一步检测确定突变，之后再确定Founder小鼠。
前述的构建方法，还包括粘多糖贮积症II型动物模型的基因型鉴定，包括步骤：提取F1代小鼠的基因组DNA；设计包含sgRNA作用靶点的引物，并以提取的F1代小鼠的基因组DNA为模板，进行PCR反应，对PCR反应的产物进行测序。
前述的构建方法，所述包含sgRNA作用靶点的引物是：
ms Ids E5 C9正向引物：AGTTCTGGTCTGGAGACACAATT；
ms Ids E5 C9反向引物：AGGCATCCTGGTAGGTAGGTTAT。
前述的构建方法，所述PCR反应的反应体系是50μL，反应条件是：95℃预变性5min，随后95℃变性30s，55℃退火30s，72℃延伸30s，30个循环，随后72℃延伸10min。
前述的构建方法，还包括粘多糖贮积症II型动物模型的表型分析，包括步骤：分别提取F1代小鼠和雄性野生型小鼠的心脏、肝脏、脾脏和肾脏四种组织，制作成组织切片，对组织切片阿尔新蓝染色，并将F1代小鼠的组织切片与雄性野生型小鼠的组织切片进行比较。
第二方面，本发明提供了第一方面的构建方法构建的粘多糖贮积症II型动物模型。
前述的粘多糖贮积症II型动物模型，X染色体的艾杜糖-2-硫酸酯酶基因的核苷酸18956-18975bp缺失，其序列是AACTCCACGCCAATCTGCTT。
第三方面，本发明提供了第一方面的构建方法构建的粘多糖贮积症II型动物模型或者第二方面的粘多糖贮积症II型动物模型在研究粘多糖贮积症II型中的用途。
本发明的技术方案至少具有如下有益效果：
(1)本发明基于CRISPR/Cas9基因敲除技术建立的粘多糖贮积症II型小鼠动物模型为进一步深入研究粘多糖贮积症II型的致病机理及治疗效果评价提供了极其重要的动物模型。
(2)采用本发明的方法获得粘多糖贮积症II型小鼠动物模型的周期短，仅需2个月。
(3)采用本发明获得的IDS基因敲除模型鼠，其基因型标记为X-idsXids，为表现型正常的小鼠，可在实验室中正常培养，通过与野生型C57BL/6雄性 小鼠(基因型为XidsY)交配，其基因可稳定遗传，发明人成功建立了一种基因可稳定遗传的IDS基因敲除模型鼠品系。其与野生型C57BL/6雄性小鼠(基因型为XidsY)交配获得F1代小鼠，F1代小鼠中理论上有25％的小鼠其基因型为X-idsY，即粘多糖贮积症II型小鼠动物模型(基因型为X-idsY)，同时理论上有25％的小鼠的基因型为X-idsXids，即IDS基因敲除模型鼠(基因型为X-idsXids)。这种情况下，一旦获得IDS基因敲除模型鼠(基因型为X-idsXids)，就只需在实验室中进行简单的自然交配过程就会获得粘多糖贮积症II型小鼠动物模型(基因型为X-idsY)，而不需要再重新进行复杂的基因敲除实验。因此，本发明的方法获得了一种可同时生产IDS基因敲除模型鼠和粘多糖贮积症II型小鼠动物模型的IDS基因敲除模型鼠，这是本发明技术方案中的最巧妙之所在。
附图说明
图1为RNA介导的Cas9系统定向基因组修饰作用机制示意图；
图2为CRISPR/Cas9介导的IDS基因修饰情况分析；
图3为靶点处DNA序列结构示意图；
图4(A)和图4(B)为粘多糖贮积症II型小鼠动物模型的基因型鉴定测序结果；
图5为粘多糖贮积症II型小鼠动物模型组织切片阿尔新蓝染色结果。
具体实施方式
为充分了解本发明之目的、特征及功效，借由下述具体的实施方式，对本发明做详细说明，但本发明并不仅仅限于此。下述具体实施方式中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。下述具体实施方式中所涉及的物质均为常规市购得到。
本发明所涉及的CRISPR/Cas9基因敲除技术，是继锌指核酸酶、TALEN等技术后可用于定点构建基因敲除鼠的最新一代方法，在定向对基因进行修饰上展现出巨大的潜力。本发明基于CRISPR/Cas9基因敲除技术提供一种粘多糖贮积症II型小鼠动物模型及其构建方法，为进一步深入研究粘多糖贮积 症II型的致病机理及治疗效果评价提供了极其重要的动物模型。
I.粘多糖贮积症II型小鼠模型的构建
1.CRISPR靶向修饰基因载体的构建
针对IDS基因发明人设计了两个靶点(针对Mouse BAC-146N21Chromosome X contains iduronate-2-sulfatase gene(小鼠BAC-146N21 X染色体含有的艾杜糖-2-硫酸酯酶基因)(Accession：AC002315)的5号外显子(核苷酸18934-19134bp)的18963-18981bp核苷酸区域和18985-19005bp核苷酸区域，其中ms Ids E5-1-sgRNA1针对18963-18981bp核苷酸区域，ms Ids E5-2-sgRNA2针对18985-19005bp核苷酸区域)，合成两对寡聚核苷酸链(ms Ids E5-1-sgRNA1：TAGGACAAAGCAGATTGGCG和AAACCGCCAATCTGCTTTGT；ms Ids E5-2-sgRNA2：TAGGATGTGGCAGATGTGCCTGA和AAACTCAGGCACATCTGCCACAT)用于制备sgRNA(small guide RNA，小向导RNA)，其作用机理如图1所示。合成的寡聚核苷酸经退火(95℃5min后自然降至室温)，连入经BsaI酶切回收的pUC57-T7-sgRNA表达载体(Addgene，NO.51132)，构建sgRNA表达载体。通过测序验证连入片段是否正确，选择正确的克隆，扩大培养后提取质粒用于准备体外转录模板。
Cas9(D10A)表达质粒(Pst1374-NLS-Cas9-ZF)，经AgeI酶切线性化，经酚氯仿抽提纯化后，溶于无核酸酶的水中作为模板，用于体外转录。Cas9 mRNA的合成由试剂盒T7 Ultra Kit(Ambion，AM1345)在体外利用T7 RNA聚合酶完成。sgRNA表达载体DraI酶切线性化后，经酚氯仿抽提纯化，溶于无核酸酶的水中作为模板，用于体外转录。sgRNA的体外合成由试剂盒MEGAshortscript Kit(Ambion，AM1354)在体外利用T7 RNA聚合酶完成。
2.胚胎供体小鼠超排卵
PMSG(血清促性腺激素)处理供体雌性小鼠，46小时后注射hCG(人绒毛膜促性腺激素)，与雄性小鼠合笼交配，次日取受精卵进行显微注射。
3.显微注射及胚胎移植
转录好的Cas9 mRNA和sgRNA混合并调整浓度至20ng/μl和12.5ng/ μl每种sgRNA，显微注射法将RNA混合物注射到小鼠受精卵的细胞质，将存活的143枚受精卵移植至5只假孕C57BL/6雌性小鼠，每个假孕雌性小鼠体内移植28枚受精卵。约3周后小鼠出生，共获得19只F0小鼠，存活17只。
4.Founder小鼠鉴定
胚胎移植的小鼠将在手术后19天左右出生，待小鼠出生20天后剪尾提取DNA并进行PCR鉴定。设计一对引物包含sgRNA作用靶点(ms Ids E5C9 For：AGTTCTGGTCTGGAGACACAATT和ms Ids E5 C9 Rev：AGGCATCCTGGTAGGTAGGTTAT)，引物由生工生物工程(上海)股份有限公司合成。PCR反应体系50μL(PCR反应相关试剂购自天根生化科技(北京)有限公司)。反应条件：95℃5min；(95℃30s，55℃30s，72℃30s)，30个循环；72℃10min；保存4℃。扩增片段大小466bp。用PCR纯化试剂盒(北京天根)纯化PCR产物。取100ng纯化后的PCR产物在NEB Buffer 2中变性、复性后，用T7核酸内切酶(NEB，M0302L)37℃孵育40min，然后用1.5％的琼脂糖凝胶电泳分离。T7核酸内切酶能够识别不完全配对的双链DNA，并进行切割，如果CRISPR/Cas9对靶点造成了突变，将能够被该酶识别，并造成双链DNA断裂。因此，在琼脂糖凝胶电泳中存在除PCR产物带之外的条带，说明退火产物能够被T7核酸内切酶识别并切割，表明靶点DNA序列可能存在突变(图2)。为进一步验证突变存在，以及突变的具体情况，通过TA克隆、测序进一步检测确定突变(图3)。
5.Founder小鼠与野生型小鼠交配得到F1
待雌性Founder小鼠到4周龄，可与野生型雄性小鼠交配，小鼠出生20天后PCR鉴定。若有阳性小鼠出生，则表示转基因已经整合到生殖细胞，标志品系建立成功。获得IDS基因敲除模型鼠，其基因型标记为X-idsXids。选用F0代23-1号雌性小鼠(基因型为X-idsXids)与野生型C57BL/6雄性小鼠(基因型为XidsY)交配获得F1代小鼠。
II.粘多糖贮积症II型小鼠动物模型的基因型鉴定
待F1代小鼠出生20天后，剪取F1代雄性小鼠和野生型C57BL/6雄性小鼠尾尖，提取小鼠基因组DNA，扩增目的基因。设计一对引物包含sgRNA 作用靶点(ms Ids E5 C9 For：AGTTCTGGTCTGGAGACACAATT和ms Ids E5C9 Rev：AGGCATCCTGGTAGGTAGGTTAT)，引物由生工生物工程(上海)股份有限公司合成。PCR反应体系50μL(PCR反应相关试剂购自天根生化科技(北京)有限公司)。反应条件：95℃5min；(95℃30s，55℃30s，72℃30s)，30个循环；72℃10min；保存4℃。扩增片段大小466bp。用PCR纯化试剂盒(北京天根)纯化PCR产物，纯化后的PCR产物直接送测序，测序结果如图4(A)和图4(B)所示，获得了粘多糖贮积症II型小鼠动物模型，其基因型为X-idsY，测序结果显示其缺失Mouse BAC-146N21Chromosome X contains iduronate-2-sulfatase gene(Accession：AC002315)核苷酸18956-18975bp的20个碱基，序列为AACTCCACGCCAATCTGCTT，此结果与设计一致，即获得基因型鉴定正确的粘多糖贮积症II型小鼠动物模型(基因型为X-idsY)。
III.粘多糖贮积症II型小鼠动物模型的表型分析
颈椎脱臼法牺牲小鼠，提取粘多糖贮积症II型小鼠动物模型(基因型为X-idsY)和雄性野生小鼠的心脏、肝脏、脾脏、肾脏四种组织，并制作以上四种组织的石蜡组织切片，切片制作好后，可对切片进行阿尔新蓝染色。阿尔新蓝属于阳离子染料，是显示酸性黏液物质最特异的染料。通过与野生型鼠组织切片的阿尔新蓝染色比对(图5)可发现，粘多糖贮积症II型小鼠动物模型(基因型为X-idsY)的心脏、肝脏、脾脏、肾脏组织中有明显的粘多糖聚集现象，由此证明，粘多糖贮积症II型小鼠动物模型构建成功。
最后，需要注意的是：以上列举的仅是本发明的具体实施例子，当然本领域的技术人员可以对本发明进行改动和变型，倘若这些修改和变型属于本发明权利要求及其等同技术的范围之内，均应认为是本发明的保护范围。

Claims (14)

  1. 一种粘多糖贮积症II型动物模型的构建方法，其特征在于，包括如下步骤：
    (1)针对小鼠IDS基因设计靶点，合成寡核苷酸链，合成的寡核苷酸链经退火与pUC57-T7-sgRNA质粒进行连接，获得sgRNA表达载体；
    (2)获得小鼠受精卵；
    (3)将转录好的Cas9mRNA和sgRNA混合均匀得到RNA混合物，将RNA混合物显微注射至小鼠受精卵的细胞质，随后将存活的受精卵植入雌性小鼠体内，繁殖获得F0代小鼠；
    (4)提取F0代小鼠的基因组DNA，作为模板，采用包含sgRNA作用靶点的引物，进行PCR反应，产物经酶切，选择突变小鼠，从而确定Founder小鼠；
    (5)Founder小鼠与野生型雄性小鼠交配获得F1代小鼠。
  2. 根据权利要求1所述的构建方法，其特征在于，步骤(1)中合成如下两对寡核苷酸链：
    ms Ids E5-1-sgRNA1：TAGGACAAAGCAGATTGGCG和AAACCGCCAATCTGCTTTGT；
    ms Ids E5-2-sgRNA2：TAGGATGTGGCAGATGTGCCTGA和AAACTCAGGCACATCTGCCACAT。
  3. 根据权利要求1所述的构建方法，其特征在于，在步骤(2)中，以4-5周龄的雌性小鼠为供体，腹腔注射PMSG和hCG进行超数排卵，随后与雄性小鼠交配，次日获得小鼠受精卵。
  4. 根据权利要求1所述的构建方法，其特征在于，步骤(4)中，包含sgRNA作用靶点的引物是：
    ms Ids E5 C9正向引物：AGTTCTGGTCTGGAGACACAATT；
    ms Ids E5 C9反向引物：AGGCATCCTGGTAGGTAGGTTAT。
  5. 根据权利要求1所述的构建方法，其特征在于，步骤(4)中，PCR反应的体系是50μL，PCR反应的条件是：95℃预变性5min，随后95℃变性30s，55℃退火30s，72℃延伸30s，30个循环，随后72℃延伸10min。
  6. 根据权利要求1所述的构建方法，其特征在于，步骤(4)中，采用 T7核酸内切酶进行酶切。
  7. 根据权利要求1所述的构建方法，其特征在于，步骤(4)中，选择突变小鼠之后，先通过TA克隆、测序进一步检测确定突变，之后再确定Founder小鼠。
  8. 根据权利要求1-7任一项所述的构建方法，其特征在于，还包括粘多糖贮积症II型动物模型的基因型鉴定，包括步骤：提取F1代小鼠的基因组DNA；设计包含sgRNA作用靶点的引物，并以提取的F1代小鼠的基因组DNA为模板，进行PCR反应，对PCR反应的产物进行测序。
  9. 根据权利要求8所述的构建方法，其特征在于，所述包含sgRNA作用靶点的引物是：
    ms Ids E5 C9正向引物：AGTTCTGGTCTGGAGACACAATT；
    ms Ids E5 C9反向引物：AGGCATCCTGGTAGGTAGGTTAT。
  10. 根据权利要求8所述的构建方法，其特征在于，所述PCR反应的反应体系是50μL，反应条件是：95℃预变性5min，随后95℃变性30s，55℃退火30s，72℃延伸30s，30个循环，随后72℃延伸10min。
  11. 根据权利要求1-7任一项所述的构建方法，其特征在于，还包括粘多糖贮积症II型动物模型的表型分析，包括步骤：分别提取F1代小鼠和雄性野生型小鼠的心脏、肝脏、脾脏和肾脏四种组织，制作成组织切片，对组织切片阿尔新蓝染色，并将F1代小鼠的组织切片与雄性野生型小鼠的组织切片进行比较。
  12. 权利要求1-11任一项所述的构建方法构建的粘多糖贮积症II型动物模型。
  13. 根据权利要求12所述的粘多糖贮积症II型动物模型，其特征在于，X染色体的艾杜糖-2-硫酸酯酶基因的核苷酸18956-18975bp缺失，其序列是AACTCCACGCCAATCTGCTT。
  14. 权利要求1-11任一项所述的构建方法构建的粘多糖贮积症II型动物模型或者权利要求12或13所述的粘多糖贮积症II型动物模型在研究粘多糖贮积症II型中的用途。
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