CN107460196A - 一种免疫缺陷小鼠动物模型的构建方法及应用 - Google Patents

一种免疫缺陷小鼠动物模型的构建方法及应用 Download PDF

Info

Publication number
CN107460196A
CN107460196A CN201710741236.4A CN201710741236A CN107460196A CN 107460196 A CN107460196 A CN 107460196A CN 201710741236 A CN201710741236 A CN 201710741236A CN 107460196 A CN107460196 A CN 107460196A
Authority
CN
China
Prior art keywords
mouse
sgrna
il2rg
seq
exon
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710741236.4A
Other languages
English (en)
Inventor
祝献民
范国平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tongji University
Original Assignee
Tongji University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tongji University filed Critical Tongji University
Priority to CN201710741236.4A priority Critical patent/CN107460196A/zh
Publication of CN107460196A publication Critical patent/CN107460196A/zh
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knockout animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0387—Animal model for diseases of the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Abstract

本发明涉及一种免疫缺陷小鼠动物模型的构建方法及应用，属于动物基因工程和遗传修饰领域，主要通过CRISPR/Cas9技术获得Il2rg基因敲除的小鼠，Il2rg基因敲除的小鼠具有免疫缺陷的表型，可以广泛应用于人源化动物模型、干细胞和肿瘤研究等多种领域。

Description

一种免疫缺陷小鼠动物模型的构建方法及应用
技术领域
本发明属于动物基因工程和遗传修饰领域，尤其是涉及一种基于基因敲除技术的免疫缺陷小鼠动物模型的构建方法及应用。
背景技术
人源化动物模型体内重建了人源免疫系统，能够更好的模拟人体肿瘤微环境，在肿瘤的在体研究中越来越受到重视。目前，人源化小鼠模型已经应用在新型抗癌药物的筛选以及治疗方法(如细胞治疗和抗体治疗)的探索等各方面。Il2rg基因编码一种跨膜蛋白，是几种白介素受体复合物的共同亚基，在免疫细胞分化和功能中起重要作用。敲除Il2rg基因能够清除小鼠体内的NK细胞，因此在建立免疫缺陷动物模型方面有着重要的作用。人源化小鼠模型SCID-hu小鼠，通过肾包膜下移植人胎儿胸腺、淋巴结、脾脏的同时从尾静脉注射胎儿肝脏细胞，在该小鼠外周血中检测到人源的CD4+、CD8+的T细胞以及B细胞所产生的免疫球蛋白(Ig)。Hu-SRC-SCID小鼠，通过将成年人骨髓中分离的或从胎儿肝脏或脐带血中分离的造血干细胞(hematopoietic stem cell，HSC)注射到小鼠体内，从而增加了包括初始免疫细胞在内的白细胞类型。而Rag和Il2rg基因敲除品系小鼠进一步提高了人源组织和细胞的移植效率，成功在小鼠体内分化出了不同亚型的造血细胞，包括T、B淋巴细胞、髓系的巨噬细胞、树突状细胞和血小板、红细胞等。目前常用的NOG(NOD/Shi-scid Il2rg–/–)、NSG(NOD/LtSz-scid Il2rg–/–)和BRG(BALB/c-Rag2–/–Il2rg–/–)小鼠更加适合移植人源造血干细胞和组织。
传统的转基因动物制作方法需要首先得到相应的干细胞，然后将干细胞植入囊胚，并生产出嵌合型动物，并通过杂交最终得到纯合型动物。整个过程耗时严重。
发明内容
本发明的目的就是为了提供一种免疫缺陷小鼠动物模型的构建方法及应用。
本发明的目的可以通过以下技术方案来实现：
本发明第一方面：提供了小鼠Il2rg基因第1外显子在制备免疫缺陷小鼠动物模型中的用途，所述小鼠Il2rg基因第1外显子的核苷酸序列如SEQ ID NO.2所示，即：5’-ATGTTGAAACTAT TATTGTCACC TAGATCCTTC TTAGTCCTTC AGCTGCTCCT GCTGAGGGCA GGGTGGAGCTCCAAGGTCCT CATGTCCAGT GCGAATGAAG ACATCAAAGC TG-3’。
本发明第二方面：提供了所述Il2rg基因第1外显子的sgRNA在敲除小鼠Il2rg基因中的应用。
本发明第三方面：提供了含有针对所述Il2rg基因第1外显子的sgRNA的DNA序列的载体。在本发明的一个优选实施方式中，提供的上述载体为pX330。
特异性靶向小鼠Il2rg基因第1外显子的sgRNA的表达载体，由载体pX330和与载体pX330连接的靶向DNA构成，靶向DNA序列为Seq ID No.3和Seq ID No.4序列。
所述Seq ID No.3和Seq ID No.4针对Il2rg基因第1外显子的sgRNA靶点。
本发明提供了用于敲除小鼠Il2rg基因的CRISPR-Cas9表达系统，含有特异性靶向小鼠Il2rg基因第1外显子的sgRNA的表达载体，pX330载体能表达Cas9 mRNA。
本发明第四方面：还提供免疫缺陷小鼠动物模型的构建方法，包括以下步骤：
(1)构建特异性靶向小鼠Il2rg基因第1外显子的sgRNA的表达载体；
(2)通过体外转录分别表达得到小鼠Il2rg基因第1外显子的sgRNA，以及Cas9mRNA；
(3)将sgRNA和Cas9 mRNA纯化后，混合，注射入小鼠受精卵细胞中，移植入小鼠输卵管中，以生产敲除小鼠Il2rg基因的动物。
步骤(2)转录产生的Cas9 mRNA和sgRNA分别进行吸附柱纯化，将纯化后的Cas9mRNA和sgRNA分别利用分光光度计测定浓度。
步骤(3)中，sgRNA与Cas9 mRNA混合时，二者的质量比为1:2。
本发明第五方面：提供了一种免疫缺陷小鼠动物模型，通过以下检测指标验证：
(1)Il2rg基因敲除小鼠的DNA具有相应的突变序列；
(2)外周血NK细胞显著减少。
本发明第六方面：提供一种非人类哺乳动物细胞，细胞内含有构建方法步骤(2)得到的sgRNA和Cas9 mRNA。
本发明涉及免疫缺陷小鼠动物模型和构建方法，主要通过CRISPR/Cas9技术获得Il2rg基因敲除的小鼠，Il2rg基因敲除小鼠缺少NK细胞，为免疫和肿瘤相关领域研究提供一种可靠的动物模型。本发明不仅可以促进免疫系统相关领域的基础研究，而且可以为肿瘤等疾病的新药研发提供高水平的动物模型。
附图说明
图1为利用CRISPR/Cas9技术建立Il2rg基因敲除小鼠的示意图；
图2图示了Il2rg基因敲除导致外周血NK细胞显著减少。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中描述的具体方法进行，或者按照试剂盒和产品说明书进行。
实施例1
针对Il2rg基因的CRISPR-Cas9表达系统的构建
利用CRISPR/Cas9技术建立Il2rg基因敲除小鼠，图1中，A.针对Il2rg基因的第1外显子设计靶向序列。B.产生的碱基缺失和插入的突变。
1、根据小鼠Il2rg基因序列，进行sgRNA的设计并获得了sgRNA的序列信息。其中特异性靶向Il2rg基因第1外显子的sgRNA，其DNA序列如SEQ ID NO.1所示，即GAGGGCAGGGTGGAGCTCCA。其中SEQ ID NO.1靶向SEQ ID NO.2(SEQ ID NO.2为Il2rg基因的一部分)。分别合成正反单链Seq ID No.3和Seq ID No.4，然后退火形成SEQ ID NO.1。
2、sgRNA载体质粒的构建：
(1)设计并合成识别Il2rg基因第1外显子的sgRNA识别区DNA序列，即Seq ID No.3和Seq ID No.4，具体如表2；
(2)每个DNA先合成单链，合成后的单链sgRNA序列进行磷酸化后梯度降温退火，形成双链DNA；具体步骤为将合成的oligoDNA(SEQ ID NO.3作为正链oligoDNA，Seq ID No.4作为反链序列，将正、反链oligoDNA与10×T4 Ligation Buffer和T4 PNK以2:2:1的体积比例混合后再加入3倍体积的水补齐体系，然后37℃孵育30min，再95℃，5min变性，之后在以5℃每分钟的速率降温至25℃完成反应以产生磷酸化粘性末端；同时BbsI酶切载体pX330产生粘性末端；退火反应体系如表1所示。
表1退火反应体系
表2sgRNA识别区DNA序列
表2中，ss是正链，as是负链。
(3)以T4连接酶将SEQ ID NO.1的双链DNA与pX330进行连接，获得真核CRISPR-Cas9表达系统载体。连接反应体系如表3。连接反应的温度为16摄氏度，连接反应时间为1h-3h。
表3连接反应体系
实施例2体外转录
利用T7-Cas9 PCR和T7-sgRNA PCR产物进行以T7启动子介导的体外转录，即以T7启动子作为体外转录的启动子，利用RNA聚合酶在体外实现从DNA到mRNA的转录过程，具体方法为：用mMESSAGE mMACHINE T7 ULTRA kit(Life Technologies)体外转录T7-Cas9 PCR产物，用MEGAshortscript T7 kit(Life Technologies)体外转录T7-sgRNA PCR产物。将转录产生的mRNA进行纯化，具体方法为：Cas9 mRNA用MEGAclear kit(Life Technologies)纯化，sgRNAs用乙醇沉淀方法纯化，用纯水溶解mRNA，将纯化后的mRNA利用分光光度计测定浓度。
7-Cas9 PCR引物见表4，T7-sgRNA PCR引物见表5，gRNA-Il2rg-F为针对SEQ IDNO.1的正向引物，反向引物为gRNA-R。PCR反应体系见表6，PCR反应条件见表7。
表4 T7-Cas9 PCR引物
表5 T7-sgRNA PCR引物
表6 PCR反应体系
表7 PCR反应条件
实施例3利用针对Il2rg基因的CRISPR-Cas9系统mRNA生产基因打靶小鼠
1、原核注射及胚胎移植
取小鼠的原核期受精卵，利用显微注射仪将预混好的Cas9mRNA/sgRNA混合物(Cas9mRNA终浓度为100ng/μl，sgRNA终浓度为50ng/μl)，注射至小鼠受精卵细胞质，然后移植至受体母鼠的输卵管中，生产基因打靶小鼠，注射量为0.5-1ul。
2、基因打靶小鼠的鉴定
代孕母鼠生产后，待仔鼠长至2周龄剪取1cm左右鼠尾，蛋白酶K，55℃消化后酚仿抽提提取鼠尾基因组DNA。以小鼠基因组DNA为模板，设计针对Il2rg基因第1外显子的引物，进行扩增，对获得的PCR产物进行测序，引物序列如下表8。其中，Il2rg F和Il2rg R对应于SEQ ID NO.5。
表8基因打靶小鼠的鉴定引物
如测序结果打靶位点附近出现双峰的情况，则可能为打靶成功。选择双峰的样品再次PCR，产物胶回收后TA克隆至T载体中，转化后挑取阳性克隆再次进行测序，如测序结果Il2rg基因靶位点附近发生碱基插入或碱基缺失，导致阅读框移码突变，则可判断为Il2rg基因敲除。
图1B中，针对SEQ ID NO.1的基因打靶大鼠均发生了碱基缺失和/或插入。
实施例4利用流式细胞验证一种免疫缺陷小鼠动物模型
取小鼠的外周血，裂红后用T、B、NK细胞相应的抗体孵育(分别是抗CD3、CD19和CD49的抗体)，通过流式细胞仪分析。图2中，A.野生型小鼠具有完备的T、B和NK细胞。B.免疫缺陷小鼠缺失相应的T、B和NK细胞。从图2看出，免疫缺陷小鼠外周血中的T、B、NK细胞都显著比野生型对照减少，说明其具有免疫缺陷的表型。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 同济大学
<120> 一种免疫缺陷小鼠动物模型的构建方法及应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gagggcaggg tggagctcca 20
<210> 2
<211> 115
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgttgaaac tattattgtc acctagatcc ttcttagtcc ttcagctgct cctgctgagg 60
gcagggtgga gctccaaggt cctcatgtcc agtgcgaatg aagacatcaa agctg 115
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
caccgagggc agggtggagc tcca 24
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aaactggagc tccaccctgc cctc 24

Claims (10)

1.小鼠Il2rg基因第1外显子在制备免疫缺陷小鼠动物模型中的用途，其特征在于，所述小鼠Il2rg基因第1外显子的核苷酸序列如SEQ ID NO.2所示。
2.小鼠Il2rg基因第1外显子的sgRNA在敲除小鼠Il2rg基因中的应用，其特征在于，所述小鼠Il2rg基因第1外显子的核苷酸序列如SEQ ID NO.2所示。
3.特异性靶向小鼠Il2rg基因第1外显子的sgRNA的表达载体，其特征在于，由载体pX330和与载体pX330连接的靶向DNA构成，靶向DNA序列为Seq ID No.3和Seq ID No.4序列。
4.根据权利要求3所述表达载体，其特征在于，所述Seq ID No.3和Seq ID No.4针对Il2rg基因第1外显子的sgRNA靶点。
5.特异性靶向Il2rg基因第1外显子的靶向DNA序列，其特征在于，靶向DNA序列为SeqID No.3和Seq ID No.4一对序列。
6.一种非人类哺乳动物细胞，细胞内含有sgRNA和Cas9mRNA，所述sgRNA为小鼠Il2rg基因第1外显子的sgRNA，小鼠Il2rg基因第1外显子的核苷酸序列如SEQ ID NO.2所示。
7.免疫缺陷小鼠动物模型的构建方法，包括以下步骤：
(1)构建特异性靶向小鼠Il2rg基因第1外显子的sgRNA的表达载体；
(2)通过体外转录分别表达得到小鼠Il2rg基因第1外显子的sgRNA，以及Cas9mRNA；
(3)将sgRNA和Cas9mRNA纯化后，混合，注射入小鼠受精卵细胞中，移植入小鼠输卵管中，以生产敲除小鼠Il2rg基因的动物，即免疫缺陷小鼠。
8.根据权利要求7所述免疫缺陷小鼠动物模型的构建方法，其特征在于，步骤(2)转录产生的Cas9mRNA和sgRNA分别进行吸附柱纯化，将纯化后的Cas9mRNA和sgRNA分别利用分光光度计测定浓度。
9.根据权利要求7所述免疫缺陷小鼠动物模型的构建方法，其特征在于，步骤(3)中，sgRNA与Cas9mRNA混合时，二者的质量比为1:2。
10.权利要求7所述构建方法得到的免疫缺陷小鼠动物模型在免疫调节药或抗肿瘤药物筛选中的应用。
CN201710741236.4A 2017-08-25 2017-08-25 一种免疫缺陷小鼠动物模型的构建方法及应用 Pending CN107460196A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710741236.4A CN107460196A (zh) 2017-08-25 2017-08-25 一种免疫缺陷小鼠动物模型的构建方法及应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710741236.4A CN107460196A (zh) 2017-08-25 2017-08-25 一种免疫缺陷小鼠动物模型的构建方法及应用

Publications (1)

Publication Number Publication Date
CN107460196A true CN107460196A (zh) 2017-12-12

Family

ID=60550537

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710741236.4A Pending CN107460196A (zh) 2017-08-25 2017-08-25 一种免疫缺陷小鼠动物模型的构建方法及应用

Country Status (1)

Country Link
CN (1) CN107460196A (zh)

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
CN108925500A (zh) * 2018-05-25 2018-12-04 广东药科大学 Lfa-1敲除的自发肠道肿瘤基因工程鼠的应用与研究方法
CN109868285A (zh) * 2018-05-29 2019-06-11 江苏艾尔康生物医药科技有限公司 一种免疫缺陷大鼠动物模型的构建方法及应用
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
CN110115248A (zh) * 2018-02-07 2019-08-13 南京普恩瑞生物科技有限公司 一种生长抑素基因缺陷的免疫缺陷小鼠、其制备方法及其用途
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
CN110495419A (zh) * 2018-05-16 2019-11-26 首都医科大学附属北京朝阳医院 一种多发性骨髓瘤髓外浆细胞瘤的小鼠模型
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
CN111485002A (zh) * 2020-05-06 2020-08-04 澎立生物医药技术（上海）有限公司 一种重度免疫缺陷的转基因小鼠的制备方法及其应用
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
CN113481238A (zh) * 2021-06-30 2021-10-08 创模生物科技(北京)有限公司 制备IL-2Rg敲除非人动物模型的方法及其应用
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106119284A (zh) * 2016-06-27 2016-11-16 北京维通达生物技术有限公司 一种用于构建免疫缺陷动物模型的产品及其应用
CN106148370A (zh) * 2016-06-21 2016-11-23 苏州瑞奇生物医药科技有限公司 肥胖症大鼠动物模型和构建方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106148370A (zh) * 2016-06-21 2016-11-23 苏州瑞奇生物医药科技有限公司 肥胖症大鼠动物模型和构建方法
CN106119284A (zh) * 2016-06-27 2016-11-16 北京维通达生物技术有限公司 一种用于构建免疫缺陷动物模型的产品及其应用

Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
CN110115248A (zh) * 2018-02-07 2019-08-13 南京普恩瑞生物科技有限公司 一种生长抑素基因缺陷的免疫缺陷小鼠、其制备方法及其用途
CN110495419A (zh) * 2018-05-16 2019-11-26 首都医科大学附属北京朝阳医院 一种多发性骨髓瘤髓外浆细胞瘤的小鼠模型
CN108925500A (zh) * 2018-05-25 2018-12-04 广东药科大学 Lfa-1敲除的自发肠道肿瘤基因工程鼠的应用与研究方法
CN109868285A (zh) * 2018-05-29 2019-06-11 江苏艾尔康生物医药科技有限公司 一种免疫缺陷大鼠动物模型的构建方法及应用
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
CN111485002A (zh) * 2020-05-06 2020-08-04 澎立生物医药技术（上海）有限公司 一种重度免疫缺陷的转基因小鼠的制备方法及其应用
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
CN113481238A (zh) * 2021-06-30 2021-10-08 创模生物科技(北京)有限公司 制备IL-2Rg敲除非人动物模型的方法及其应用

Similar Documents

Publication Publication Date Title
CN107460196A (zh) 一种免疫缺陷小鼠动物模型的构建方法及应用
CN109266656B (zh) 一种PD1人源化BALB/c小鼠模型的构建方法及其应用
CN107586835A (zh) 一种基于单链接头的下一代测序文库的构建方法及其应用
CN106148370A (zh) 肥胖症大鼠动物模型和构建方法
CN106661570A (zh) 通过与氨甲蝶呤选择偶联的睡美人转座子制备工程化t细胞
AU2017101108A4 (en) Construction method of animal model of mucopolysaccharidosis type II and use thereof
CN110055223A (zh) 一种B2m基因改造的免疫缺陷动物的制备方法及其应用
CN111718933B (zh) 一种rrbp1基因敲除热带爪蛙模型的制备方法与应用
CN109868285A (zh) 一种免疫缺陷大鼠动物模型的构建方法及应用
CN109280666A (zh) 一种基因敲除选育bai2基因缺失型斑马鱼的方法
CN106480099B (zh) 条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型及其构建方法
CN104334017B (zh) 尿激酶型纤溶酶原激活剂转基因小鼠
CN110894510A (zh) 一种基因敲除选育Lgr6基因缺失型斑马鱼的方法
CN112251463B (zh) 一种cd73人源化小鼠模型的构建方法
CN109652457A (zh) 一种基因敲除选育alpk2基因缺失型斑马鱼的方法
CN109929875A (zh) 一种lag3基因人源化动物模型的构建方法及其应用
CN112779258A (zh) 一种大黄鱼CRISPR/Cas9基因编辑方法
CN108300738B (zh) 一种nod遗传背景的中性粒细胞缺失的人源化小鼠模型的制备方法
CN111778278A (zh) 一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法及其应用
CN108251456B (zh) 一种nod遗传背景的动脉粥样硬化小鼠模型的制备方法
CN110066805A (zh) 基因敲除选育adgrf3b基因缺失型斑马鱼的方法
CN112626122B (zh) hKDR人源化小鼠模型及其建立方法和应用
CN111100877A (zh) 肥厚型心肌病小鼠模型的制备方法及其应用
CN114032240A (zh) 一种用于提高基因敲除效率的方法
CN110195057B (zh) Hr基因经遗传修饰的非人动物或其子代的制备方法及应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20171212

RJ01 Rejection of invention patent application after publication