CN106916849A - 一种新型Fndc5基因敲除小鼠模型的创建 - Google Patents

一种新型Fndc5基因敲除小鼠模型的创建 Download PDF

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CN106916849A
CN106916849A CN201510990840.1A CN201510990840A CN106916849A CN 106916849 A CN106916849 A CN 106916849A CN 201510990840 A CN201510990840 A CN 201510990840A CN 106916849 A CN106916849 A CN 106916849A
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匡世焕
金文�
吴子环
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Abstract

本发明公开了一种新型Fndc5基因敲除小鼠模型的创建方法，属于病理动物模型的药理用途领域。本发明通过TALEN技术，使Fndc5基因DNA序列发生移码突变，从而蛋白质发生相应的移码突变，导致提前产生终止密码子，结果建立了Fndc5基因敲除小鼠模型，由于Fndc5与糖尿病、肥胖疾病密切相关，表明其可用于建立二型糖尿病的小鼠模型。

Description

一种新型Fndc5基因敲除小鼠模型的创建
技术领域
本发明涉及一种新型Fndc5基因敲除小鼠模型的创建，并涉及其在制作糖尿病、肥胖等病理模型的用途，属于病理模型的药理用途领域。
背景技术
随着饮食结构和生活方式的改变，系统能量稳态失衡，糖尿病发生率逐年增加，已成为倍受关注的公共卫生问题。骨骼肌在调控系统能量稳态上扮演着重要的角色。一方面骨骼肌自身发生物质代谢如糖代谢等调控系统能量稳态；另一方面骨骼肌能分泌肌因子作用于脂肪、肝脏等器官调节系统能量稳态。
研究发现Fndc5在运动的刺激下作为PGC1-α依赖的产物，随后裂解产生Irisin进入血液作用于白色脂肪，刺激UCP1表达提高产热；促使白色脂肪棕色化，从而提高和改善了肥胖和糖尿病患者的代谢状态，为治愈肥胖和糖尿病提供了可能，表明Fndc5可以作为药效筛选的新的靶点。
本发明利用技术创建了Fndc5敲除小鼠动物模型，本发明解决了筛选出对该靶点发挥作用药物的问题。
发明内容
本发明的主要目的是提供一种新的糖尿病并发症-周围神经病变的大鼠模型。
本发明的主要目的是通过以下技术方案来实现的：
本发明将Fndc5-T1和Fndc5-T3的TALEN质粒对，体外转录成mRNA后，共显微注射mRNA到小鼠的受精卵中，一共注射120个受精卵，出生23只小鼠。两周龄后，剪取4周龄出生小鼠任一只耳朵大约三分之一，提取基因组DNA。以此基因组DNA为模板进行PCR反应，将产物进行测序。通过测序图可分析小鼠的突变情况：如果测序峰全是单峰说明该小鼠是WT(野生型)；如果测序峰在预定位置出现双峰且2种峰恰好对应不同的突变类型则该小鼠为Fndc5基因敲出首建鼠(Founder)。根据发生移码突变情况，选取雄性Founder小鼠分别与野生型C57小鼠(♀)交配，出生的小鼠两周龄后按上述方法进行基因型鉴定。通过测序图可分析F1代小鼠的突变情况：如果测序峰全是单峰说明该小鼠是WT(野生型)；如果测序图出现双峰则该小鼠为杂合子；如果测序图也是单峰但是在预期位置出现缺失突变则该小鼠为纯合基因敲除小鼠，建立稳定遗传F1代小鼠品系，从而可以应用于相关药物的药效评价。
图1是Fndc5的基因组及蛋白质一级结构图，注：A为Fndc5蛋白质的一级结构，B为Fndc5基因组结构(箭头为TALEN的靶点位置)；
图2是Fndc5TALEN的SSA活性检测统计结果图；
图3是Fndc5TALEN的SSA活性检测报告图；
图4是Fndc5基因敲除首建鼠的鉴定图，注：突变型1的DNA序列(向下箭头所指峰形)和突变型2的DNA序列(向上箭头所指峰形)。
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下，可以对本发明技术方案的细节和形式进行修改和替换，但这些修改和替换均落入本发明的保护范围内。
实验例
1.1材料
1.1.1实验动物
野生型C57BL/6小鼠品系，购自北京维通利华有限公司，合格证号：SCXK(京)2014-0001。饲养于中国医学科学院药用植物研究所SPF级动物房，实验动物使用许可证：SYXK(京)2013-0023，动物房温度20～26℃，相对湿度40～70％。
1.1.2 TALEN质粒
TALEN质粒pCS5-eTALEN-T由北京唯尚立德生物科技有限公司合成。靶位点位置T1：TTTCTAGAAGAAGGATGT gcggatgctccggtt CATTCAGGAGGTGAACA，其中左边TALE识别序列：TTCTAGAAGAAGGATGT 17bp，右边TALE识别序列：GTTCACCTCCTGAATG 16bp，间隔15bp。靶位点位置T3：TCCGGCACCTCAAGGCC aactctgccgtggtcagCTGGGATGTCCTGGAGGA，其中左边TALE识别序列：CCGGCACCTCAAGGCC 16bp，右边TALE识别序列：CCTCCAGGACATCCCAG 17bp，间隔17bp。
1.1.3引物
对于靶点位置1，设计基因型检测PCR引物：CATGTTTCCTTAGCTCTACTGTGG(正向)，GGAGAAAGCATGCATGGCAGTCTC(反向)PCR片段长度为518bp；对于靶点位置3，设计基因型检测PCR引物：GGACCCTTGGTTTGGCCAGTCTA(正向)，CTCTACCATCATCCTCCATGCCTG(反向)PCR片段长度为479bp。Real-Time引物：FNDC5-F：ATGAAGGATGGGGAGGAA，FNDC5-R：GCGGCAGAAGAGAGCTATAACA，18s-F：CATGCA GAACCCACGACAGTA，18s-R：CCTCACGCAGCTTGTTGTCTA。
1.1.4试剂
本实验的引物由北京睿博兴科生物技术有限公司合成。Amp PCRmix(TAKARA，Ro74A)试剂盒、TransZol Up Plus RNA Kit(Tran，ER501-01)、EasyScript First-Strand cDNA Synthesis SuperMxi(Tran，AE301-03)、TransStrt Top Green qPCR SupreMix(Tran，AQ131-02)、Base(碱液)、Neutrlization(中和液)等。
1.2方法
1.2.1四品系Fndc5基因敲除鼠的构建
将Fndc5-T1和Fndc5-T3的TALEN质粒对，体外转录成mRNA后，共显微注射mRNA 到小鼠的受精卵中，一共注射120个受精卵，出生23只小鼠。两周龄后，剪取4周龄出生小鼠任一只耳朵大约三分之一，放置在做好标记的1.5ml的离心管中，加入75ul碱液(Base)95℃以上40分钟；然后加入75ul中和液(Neutrlization)，常温静置几分钟，溶液即为提取的基因组DNA。以此基因组DNA为模板进行PCR反应，将产物进行测序。通过测序图可分析小鼠的突变情况：如果测序峰全是单峰说明该小鼠是WT(野生型)；如果测序峰在预定位置出现双峰且2种峰恰好对应不同的突变类型则该小鼠为Fndc5基因敲出首建鼠(Founder)。共有15只小鼠带有突变，其中3只移码：7(T1一条缺失2bp，一条缺失4bp)、Founder13(T1缺失2bp)、Founder 18(T1一条缺9bp，一条缺2bp；T3缺2bp)。根据发生移码突变情况，选取雄性Founder7(m-fn-7，♂)小鼠、Founder13(m-fn-13，♂)小鼠、和Founder 18(m-fn-18，♂)小鼠分别与野生型C57小鼠(♀)交配，出生的小鼠两周龄后按上述方法进行基因型鉴定。通过测序图可分析F1代小鼠的突变情况：如果测序峰全是单峰说明该小鼠是WT(野生型)；如果测序图出现双峰则该小鼠为杂合子；如果测序图也是单峰但是在预期位置出现缺失突变则该小鼠为纯合基因敲除小鼠。我们从中选出4中突变类型#7-T1-1(T1缺失2个碱基CT)、#7-T1-2(T1缺失4个碱基GCTC)、#13-T1-1(T1缺失2个碱基TG)、#18-T1-1(T1缺失2个碱基TC)建立稳定遗传F1代小鼠品系。
2.结果
2.1四品系Fndc5基因敲出小鼠的建立
2.1.1 Fndc5基因敲除TALEN靶点位置的设计
根据Fndc5的基因组结构，Fndc5 protein Domain的分析，决定破坏the FNIII domain，因此TALEN的靶点位置将设计在外显子3上或者外显子3前，如箭头所示结果见附图1。
2.1.2 Fndc5 TALEN的SSA活性检测
根据SSA活性结果，我们选择活性比较高的靶点Fndc5-T1和Fndc5-T3进行后续的Fndc5基因敲除小鼠的构建结果见附图2。
2.1.3 Fndc5基因敲除首建鼠的鉴定
将受精卵注射后出生23只小鼠，两周龄后，按照上述的方法对出生的小鼠进行基因型分析和鉴定。结果如图3所示，我们选取Founder7(T1一条缺失2bp，一条缺失4bp)进行测序图分析。测序峰形图在突变位点开始出现双峰，表明为杂合子。通过峰形分析，可以将突变型1的DNA序列(黑色箭头所指峰形)和突变型2的DNA序列(红色箭头所指峰形)分析出来。结果为一条在CCGGTTCATTCAGGA前缺失2个碱基CT；一条在CGGTTCATTCAGG前缺失4个碱基GCTC，均发生移码突变。
2.1.2 F1四品系Fndc5基因敲除鼠的鉴定
根据发生移码突变情况，选取的Founder7小鼠、Founder13小鼠、和Founder 18小鼠分别与野生型C57小鼠交配，出生的小鼠两周龄后按上述方法进行基因型鉴定。通过测序图可分析F1代小鼠的突变情况：如果测序峰全是单峰说明该小鼠是WT(野生型)；如果测序图出现双峰则该小鼠为杂合子；如果测序图也是单峰但是在预期位置出现缺失突变则该小鼠为纯合基因敲除小鼠，这个图3中已经进行了类似分析。我们从中选出4种突变类型#7-T1-1(T1缺失2个碱基CT)、#7-T1-2(T1缺失4个碱基GCTC)、#13-T1-1(T1缺失2个碱基TG)、#18-T1-1(T1缺失2个碱基TC)建立稳定遗传F1小鼠品系。四品系F1小鼠的突变位点及突变后导致提前产生终止密码子的位置见表1。其中红色标记部分是TALEN设计敲除的靶位点序列，这些缺失突变都会导致提前产生终止密码子。
表1四品系F1小鼠的突变情况

Claims (3)

1.一种新型Fndc5基因敲除小鼠模型的创建在药效学评价方面的用途。
2.Fndc5基因敲除小鼠模型在制作糖尿病、肥胖等病理模型的用途。
3.按照权利要求2所述的用途，其特征在于：所述的糖尿病是二型糖尿病。
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Application publication date: 20170704