CN106172238B - miR-124基因敲除小鼠动物模型的构建方法和应用 - Google Patents
miR-124基因敲除小鼠动物模型的构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种miR‑124基因敲除小鼠动物模型,所述小鼠动物模型是被敲除了miR‑124‑1、miR‑124‑2、miR‑124‑3基因的小鼠。本发明使用Crispr‑cas9基因敲除技术,敲除了小鼠大脑中表达量最高的microRNA基因miR‑124。所获得的小鼠均表现出明显的自主活动降低、学习记忆力下降、可溶性β淀粉样蛋白增多等神经系统疾病状态。可以为神经系统疾病病理的研究,神经系统疾病药物的筛选提供简单、可靠、经济的动物模型。
Description
技术领域
本发明属于生物技术领域,具体涉及miR-124基因敲除小鼠动物模型及其构建方法和应用。
背景技术
现代人生活一方面节奏快、压力大,另一方生面生活质量普遍提高,人均寿命增加。因此各类精神疾病,尤其是老年性退行疾病的发病率迅速上升。目前神经系统类疾病的诊断麻烦,分期困难,缺乏有效治疗手段。究其原因,是因为这类疾病的病因复杂,发病机理不明,目前的研究缺乏合适的动物模型。因此建立有效的疾病动物模型是目前研究的迫切任务。本专利所构建的基因敲除小鼠均表现出明显的自主活动降低、学习记忆力下降、可溶性β淀粉样蛋白增多等神经系统疾病状态。可以为神经系统疾病病理的研究,神经系统疾病药物的筛选提供简单、可靠、经济的动物模型。
神经系统重大疾病(如脑血管病、阿尔茨海默病、帕金森病、亨廷顿病等)严重危害人类的生命健康,其发病率,死亡率,致残率高,每年给国家带来巨大的经济损失。目前神经系统类疾病的诊断麻烦,分期困难,缺乏有效治疗手段。究其原因,是因为这类疾病的病因复杂,发病机理不明,目前的研究缺乏合适的动物模型。
传统的神经系统重大疾病动物建模方式以自然衰老、物理损伤(电、热等手段损伤小鼠Meynert基底核)、化学诱导(乙酰胆碱M受体阻断剂、6-羟多巴胺、D-半乳糖、鱼藤酮模型等)或者手术处理。这些建模方式耗时长,价格不菲,需要专业的技术技巧,所建的模型一致性不高。
miR-124是一种在神经系统中特异性表达的miRNA。其在进化中十分保守,在46类种属中均能检测到miR-124的表达,其成熟序列在人类及小鼠中均为UAAGGCACGCGGUGAAUGCC(SEQ ID NO.4)。miR-124在人类及小鼠中均有3个拷贝的编码基因,分别叫做miR-124-1,miR-124-2,miR-124-3;它们的前体序列,所在染色体位置均不相同(见表1)。也就是说miR-124在人体内(还有鼠内)的染色体上只有3个地方有,其他地方没有。
表1.miR-124的前体序列及所在位置
miR-124是哺乳动物神经系统内表达最多的miRNA,占哺乳动物大脑皮质总miRNA的5%~48%,但在其它组织中表达量极低。其在分化和成熟的神经元中,特别是视网膜的感光细胞(视杆细胞、视锥细胞)中高度表达,但在神经干细胞、神经前体细胞和胶质细胞内表达很低。神经发生、分化,学习记忆,神经免疫,视觉感光等多种生理功能都有 miR-124的参与;多种神经系统疾病与miR-124的异常表达有关。
Laterza课题组及Weng课题组都发现血浆miR-124在短暂性(缺血60~90min)和永久性大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)小鼠脑缺血模型中均有不同程度的增加;表明miR-124与缺血性脑血管病相关。Smith等发现阿尔茨海默病(Alzheimer’s disease,AD)患者脑内miR-124表达减少,其导致AD发生的可能机制为:miR-124靶向作用于PTBPl,从而调节淀粉样前体蛋白mRNA的选择性剪切,而异常的选择性剪切导致β淀粉样蛋白沉积。Johnson等发现亨廷顿病患者和亨廷顿病模型鼠R6/2脑内的miR-124表达下降,其导致亨廷顿病发生可能与miR-124靶向基因Atp6voe、Vamp3、 Plod3、Ctdspl和Itgbl的异常表达有关。Baudet发现miR-124可通过调控CoREST基因诱导视锥细胞的生长,从而影响明视觉(photopic vision)。
我们前期研究也发现miR-124在小鼠神经系统,特别是视网膜中高度表达(图2A);测序结果也表明小鼠大脑中表达量最高的miRNA分别为miR-124及miR-9。(图2B)。
发明内容
本发明旨在克服现有技术的不足,提供了一种miR-124基因敲除小鼠动物模型及其构建方法和应用。
所述miR-124基因敲除小鼠动物模型是被敲除了miR-124-1、miR-124-2、miR-124-3 基因的小鼠。
上述小鼠动物模型的构建方法包括如下步骤:
(1)构建针对miR-124-1、miR-124-2、miR-124-3基因的sgRNA;所述miR-124-1 的sgRNA序列如SEQ ID NO.1所示;所述miR-124-2的sgRNA序列如SEQ ID NO.2 所示;所述miR-124-3的sgRNA序列如SEQ ID NO.3所示;
(2)PMSG处理C57/BL6雌性小鼠,46小时后注射hCG,与雄性小鼠合笼交配,次日取受精卵进行显微注射,将步骤(1)所述的sgRNA与Cas9核酸酶mRNA 体外转录后,注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,产出小鼠,即为F0代小鼠;
(3)提取F0代小鼠尾部DNA,PCR扩增并将产物送测序,鉴定是否为嵌合体;
(4)待雄性Founder小鼠到7周龄,雌性小鼠到4周龄,可分别与野生型异性小鼠交配获得F1代杂合子小鼠,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;
(5)将F1代杂合子小鼠杂交获得F2代纯合子小鼠,即为小鼠动物模型。
其中,SEQ ID NO.1:
GATCACTAATACGACTCACTATAGGCAAGGTCCGCTGTGAACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT;
SEQ ID NO.2:
GATCACTAATACGACTCACTATAGGCAAGGTCCGCTGTGAACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT;
SEQ ID NO.3:
GATCACTAATACGACTCACTATAGGCCCTCTGCGTGTTCACAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT。
本发明系统研究了miR-124-3基因敲除小鼠行为学,发现小鼠自发活动能力降低,但运动、平衡能力没有改变,也无焦虑或抑郁现象。进一步研究发现敲除小鼠的认知能力、空间学习和记忆能力以及长期学习记忆能力都受到损伤(图3,4)。
本发明使用Crispr-cas9基因敲除技术,敲除了小鼠大脑中表达量最高的microRNA基因miR-124。所获得的小鼠均表现出明显的自主活动降低、学习记忆力下降、可溶性β淀粉样蛋白增多等神经系统疾病状态。可以为神经系统疾病病理的研究,神经系统疾病药物的筛选提供简单、可靠、经济的动物模型。
所述基因敲除小鼠生下来不久(3-4个月内)即自然表现出神经系统不正常现象,造模时间短,不需要特殊的试剂、手术和物理方法,方法简单易行。小鼠可以自由交配,能产下可存活的纯合子后代,价格低廉。
附图说明
图1为CRISPR基因敲除小鼠模型建立示意图;
图2为miR-124的组织分布:(A)Northern Blot显示miR-124在大脑和视网膜中高度表达;miR-96为视网膜特异表达标记分子;Let-7为所有组织广泛表达的标记分子;TotalRNA作为定量标记;(B)小鼠大脑中高度表达的miRNA;
图3为miR-124-3基因敲除小鼠行为学研究:(A)旷场实验表明自发活动能力降低,但无焦虑现象;(B)强迫游泳实验表明无抑郁现象;(C)旋转实验表明平衡能力没有改变;(D)行走痕迹实验表明运动能力没有改变;
图4为miR-124-3基因敲除小鼠记忆力测试:(A)新物体识别实验表明敲除小鼠记忆力下降;(B)Morris水迷宫实验表明敲除小鼠空间记忆力下降;(C)恐惧记忆与消退实验表明敲除小鼠学习能力降低,长期记忆力下降。
具体实施方式
小鼠动物模型的构建方法:
(1)构建针对miR-124-1、miR-124-2、miR-124-3基因的sgRNA,分步测序,所需时间约45-60天;然后线性化及纯化DNA并在体外转录为sgRNA;纯化sgRNA 至适合转基因注射的纯度,所需时间为15天;所述miR-124-1的sgRNA序列如SEQ ID NO.1所示;所述miR-124-2的sgRNA序列如SEQ ID NO.2所示;所述 miR-124-3的sgRNA序列如SEQ ID NO.3所示;
(2)PMSG处理C57/BL6雌性小鼠,46小时后注射hCG,与雄性小鼠合笼交配,次日取受精卵进行显微注射,将步骤(1)所述的sgRNA与Cas9核酸酶mRNA 体外转录后,注射到受精卵中,所需时间为10天,取注射后存活的受精卵移植到假孕母鼠体内,所需时间为30天,胚胎移植的小鼠将会在手术后19天左右出生,即为F0代小鼠,待小鼠出生20天后剪尾提取DNA并进行PCR鉴定。DNA抽提及PCR检测时间为2-3天。因此这个周期所需时间约为45天;
(3)待雄性Founder小鼠到7周龄,雌性小鼠到4周龄,可分别与野生型异性小鼠交配获得F1代杂合子小鼠,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,这个过程需要120天左右;
(4)将F1代杂合子小鼠杂交获得F2代纯合子小鼠,即为小鼠动物模型。
所获得的三个miR-124基因敲除小鼠的测序结果及检测引物如表2所示,测序结果中画删除线的文字为敲除掉的基因序列。
表2、所获得的三个miR-124基因敲除小鼠的测序结果及检测引物
Claims (3)
1.一种miR-124基因敲除小鼠动物模型的构建方法,其特征在于,所述方法包括如下步骤:
(1)构建针对miR-124-1、miR-124-2、miR-124-3基因的sgRNA;所述miR-124-1的sgRNA序列如SEQ ID NO.1所示;所述miR-124-2的sgRNA序列如SEQ ID NO.2所示;所述miR-124-3的sgRNA序列如SEQ ID NO.3所示;
SEQ ID NO.1所示序列中:GATCACTAATACGACTCACTATAGG为T7启动子区域;CAAGGTCCGCTGTGAACA为靶点特异性序列;GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT为引导RNA骨架序列;
SEQ ID NO.2所示序列中:GATCACTAATACGACTCACTATAGG为T7启动子区域;CAAGGTCCGCTGTGAACA为靶点特异性序列;GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT为引导RNA骨架序列;
SEQ ID NO.3所示序列中:GATCACTAATACGACTCACTATAGG为T7启动子区域;CCCTCTGCGTGTTCACAG为靶点特异性序列;GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT为引导RNA骨架序列;
(2)PMSG处理C57/BL6雌性小鼠,46小时后注射hCG,与雄性小鼠合笼交配,次日取受精卵进行显微注射,将步骤(1)所述的sgRNA与Cas9核酸酶mRNA体外转录后,注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,产出小鼠,即为F0代小鼠;
(3)提取F0代小鼠尾部DNA,PCR扩增并将产物送测序,鉴定是否为嵌合体;
(4)待雄性Founder小鼠到7周龄,雌性小鼠到4周龄,可分别与野生型异性小鼠交配获得F1代杂合子小鼠,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;
(5)将F1代杂合子小鼠杂交获得F2代纯合子小鼠,即为小鼠动物模型。
2.构建权利要求1所述小鼠动物模型的试剂盒,其特征在于,所述试剂盒中含有针对miR-124-1、miR-124-2、miR-124-3基因的sgRNA;所述miR-124-1的sgRNA序列如SEQ IDNO.1所示;所述miR-124-2的sgRNA序列如SEQ ID NO.2所示;所述miR-124-3的sgRNA序列如SEQ ID NO.3所示。
3.针对miR-124-1、miR-124-2、miR-124-3基因的sgRNA在制备神经系统及眼科疾病表征的模式动物中的应用,所述miR-124-1的sgRNA序列如SEQ ID NO.1所示;所述miR-124-2的sgRNA序列如SEQ ID NO.2所示;所述miR-124-3的sgRNA序列如SEQ ID NO.3所示。
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