CN107034221A - 一种能在小鼠体内表达的部分碱基缺失肌肉抑制素基因及应用 - Google Patents
一种能在小鼠体内表达的部分碱基缺失肌肉抑制素基因及应用 Download PDFInfo
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Abstract
本发明公开了一种能在小鼠体内表达的部分碱基缺失肌肉抑制素基因及应用,MSTN基因包含两个内含子和三个外显子序列,三号外显子175‑180位6个碱基缺失,构建了含有部分碱基缺失肌肉抑制素基因的切割载体,制备了含有部分碱基缺失肌肉抑制素基因的敲除小鼠,含有部分碱基缺失肌肉抑制素基因的切割载体应用于MSTN生物体性状研究,增加小鼠骨骼肌的肌肉量,产生双肌现象,并降低小鼠基础代谢率和小鼠体温。本发明通过基因工程的方法,对小鼠原有MSTN基因进行碱基敲除,可增加小鼠骨骼肌的肌肉量而不影响机体正常的生理功能,可快速提高小鼠的肌肉量,具有较大应用前景。
Description
技术领域
本发明属于生物技术领域中的基因工程技术,具体为一种能在小鼠体内表达的部分碱基缺失肌肉抑制素基因及应用。
背景技术
MSTN是一种肌肉抑制素基因,属于TGF-β超家族,它的合成主要是由骨骼肌完成,属于多肽中的分泌型,作为TGF-β超家族的成员,它具有与这个家族共同的生物结构:包括位于N-末端的分泌信号肽、蛋白酶水解位点(proteolytic processing site,RSRR)和位于C-末端的成熟肽区,含有半胱氨酸(cystine knot)结构,这些组件组成一个52kDa的没有活性的前体蛋白,通过中间的蛋白水解位点的加工后才可形成一个26kDa的活性肽,从而发挥该有的生物功能。MSTN基因cDNA的组成:1个可读框(ORF)、3个外显子、2个内含子的核苷酸序列。
1997年Mcpherron等人在筛选小鼠的肌肉cDNA文库首次发现myostatin(MSTN)基因,同年Grobet等人对自然双肌的比利时兰牛进行了测序,结果发现比利时蓝牛MSTN的p.D273RfsX13发生了纯合突变,从而引起双肌性状。1999年,Carlson等人也发现若大量的MSTN存在于骨骼肌中会使得肌肉生长受到抑制,形成肌肉萎缩,同时Lee等人则发现该基因由特定的肌肉组织产生,调节肌肉组织的生长,该基因缺失后,会使一些动物出现双肌现状。此后,人们开展了一系列与MSTN相关的研究,发现MSTN突变小鼠除了出现双肌性状,还可以以肌肉再生增强以及纤维化程度降低的方式提高肌肉愈伤能力;糖的消耗和糖摄入加强,对胰岛素的敏感性加强;心脏增大且压力应激增强;脂肪含量减少,脂肪的发生也会受到抑制,且白色脂肪向棕色脂肪转变加强从而促进机体的生热作用;骨密度、骨矿物质含量也会有所增强,同时还可以可增加骨痂、骨折的尺寸和强度而加快骨折后的愈合。通过调控胎盘的建立和葡萄糖的摄入,以此来调控子宫平滑肌细胞和内膜上皮细胞的增殖及乳腺的发育,参与雌性哺乳动物的生殖调控。
以上研究说明,肌肉抑制素突变基因在动物体内是可以发挥肌肉含量增加的功效,而且没有表现出影响机体正常生理功能的征兆。然而,在动物体上自然突变的MSTN很少,因此如何对小鼠原有MSTN基因进行碱基敲除,使得其能够在哺乳动物中发挥作用是本领域亟待解决的技术问题,在进行gRNA优化过程中面临诸多挑战:首先是如何获得能够表达的基因序列,其次,还要构建打靶载体,考虑到打靶载体的打靶效率,外源基因mRNA的稳定性和mRNA二级结构对翻译效率的影响,优化的同时一定要避免阻碍表达的特殊的mRNA的二级结构的形成。上述挑战阻碍了对基因序列的优化。
发明内容
本发明的目的在于提供一种能在小鼠体内表达的部分碱基缺失肌肉抑制素基因及应用。
为实现上述目的,本发明提供如下技术方案:
一种能在小鼠体内表达的部分碱基缺失肌肉抑制素基因,包含两个内含子和三个外显子序列,三号外显子175-180位6个碱基缺失。
作为本发明进一步的方案:构建了含有部分碱基缺失肌肉抑制素基因的切割载体。
作为本发明进一步的方案:制备了含有部分碱基缺失肌肉抑制素基因的敲除小鼠。
在小鼠体内表达的部分碱基缺失肌肉抑制素基因在MSTN生物体性状研究中的应用。
作为本发明进一步的方案:含有部分碱基缺失肌肉抑制素基因的切割载体用于增加小鼠骨骼肌的肌肉量,产生双肌现象,并降低小鼠基础代谢率和小鼠体温。
与现有技术相比,本发明的有益效果是:
通过基因工程的方法,对小鼠原有MSTN基因进行碱基敲除,获得能在小鼠体内表达的部分碱基缺失肌肉抑制素基因,构建了含有该基因的切割载体,制备了含有该基因的敲除小鼠,含有部分碱基缺失肌肉抑制素基因的切割载体可应用于增加小鼠骨骼肌的肌肉量而不影响机体正常的生理功能,可快速提高小鼠的肌肉量,具有较大应用前景。
附图说明
图1为本发明中CRISPR/Cas9介导的MSTN基因敲除图,其中A:sgRNA在小鼠MSTN基因上的靶位点,两个位于二号外显子,两个位于三号外显子,其序列如表格中所示,B:四个sgRNA序列,sg1-4在原序列中用红色标注,PAM用绿色标注,*代表碱基置换,后面的数字代表置换了几个碱基。
图2为本发明中41只子代小鼠MSTN琼脂糖凝胶电泳结果图。
图3为本发明中小鼠敲除位点图,其中A为F1代小鼠待敲除位点图,B为F2代小鼠待敲除位点图。
图4为本发明中MSTN-/-小鼠体重增加图,其中A为F1代雌性小鼠的体重对比图,B为F1代雄性小鼠的体重对比图,C为F2代雌性小鼠的体重对比图,D为F2代雄性小鼠的体重对比图。
图5为本发明中MSTN-/-小鼠与野生小鼠性状对比图。
图6为本发明中MSTN-/-小鼠与对照小数单个肌纤维图,其中A为野生型(上)和突变型(下)腓肠肌部分单肌纤维图,B为野生型(上)和突变型(下)骨刺头部分单肌纤维图。
图7为本发明中MSTN表达量western blotting图,其中Con为野生小鼠,MT为MSTN-/-小鼠。
图8为本发明中MSTN-/-小鼠与对照小鼠基础能量代谢图。
图9为本发明中MSTN-/-小鼠与对照小鼠体温对比图。
具体实施方式
下面结合具体实施方式对本专利的技术方案作进一步详细地说明。
一、MSTN靶位点设计与合成
1、登陆NCBI网站,搜索网站上公布的小鼠MSTN基因序列并下载(本试验所用的基因ID:17700)。
2、打开靶点预测网站(http://www.genome-engineering.org/crispr/),打开网站中靶位点在线设计工具“CRISPR design tool”,在文本框中输入基因序列,选择物种提交后,即可给出该序列所有的靶位点,并给出各位点的潜在脱靶位点和综合评分等。
3、根据网站给出的靶位点序列,综合考虑各项参数后选取其中预测脱靶效应较低的4组靶位点。位点如图1所示。
二、构建切割载体
设计好的gRNA两端添加酶切位点并送TaKaRa公司合成,得到带有粘性末端的双链gRNA序列(gA、gB、gC、gD),连入pCas-Guide载体,构建好pCas-Guide-gRNA(A/B/C/D)载体并测序得到正确载体,Cas9载体购自origene公司pCas-Guide cloning kit(SKU GE100001)。
三、原核注射获得MSTN基因敲除小鼠
取合笼0.5d见栓的雌鼠,输卵管中收集原核期的受精胚胎,借助显微操作系统将浓度为3ng/μl的质粒DNA注射到雄原核中,注射成功的受精胚胎移植到见栓1.5d的受体雌鼠输卵管中,19d后获得子代小鼠。待小鼠断乳后,提取鼠尾DNA,PCR测序鉴定MSTN基因敲除小鼠。
四、MSTN基因敲除小鼠鉴定
1.鼠尾DNA提取
(1)无菌条件下剪取少许鼠尾于1.5mlEP管中,置于冰上。
(2)在鼠尾中加入冷冻的500μl Nuclei lysis solution+120μl EDTA,再加入17.5μl 20ng/μl的蛋白酶K,混匀后,置于55℃固浴上孵育一夜。
(3)加入200μl protein precipitation solution,涡旋振荡20s后置于冰上5min。
(4)13000-16000r离心5min,将上清转移至一个新管中,加入600μl异丙醇,颠倒混匀。
(5)13000-16000r离心1min,弃上清,加入600μl 70%乙醇,颠倒混匀。
(6)13000-16000r离心1min,弃酒精,倒置风干酒精。
(7)加水溶解,65℃孵育1h或4℃过夜。
(8)风光光度计测定OD值。
2.PCR及测序
将提好的鼠尾DNA进行MSTN第二外显子和第三外显子靶位点PCR,电泳确认条带大小正确后,交由华大公司进行基因测序。二号外显子和三号外显子PCR引物序列如序列表中SEQ ID NO:1至SEQ ID NO:4所示。La-Taq PCR程序如表1所示。PCR结果如图2所示,测序鉴定如图3所示。
表1 La Taq 50μl PCR体系
La Taq Pcr程序如下:
五、MSTN基因敲除小鼠性状检测
1.生长性状
因0-1月小鼠处于哺乳期且太小,无法分笼和打耳标,因此从小鼠出生三周开始称重,每隔一周称重一次。结果如图4-A所示,MSTN-/+雌性小鼠体重始终大于野生小鼠,并从检测的第三周后增长速显著上升(P<0.05)。MSTN-/+雄性小鼠体重变化如图4-B所示,检测的前三周体重增长缓慢,三周后体重逐渐超越野生小鼠。MSTN-/-雌性小鼠和雄性小鼠体重变化如图4-C及4-D所示,MSTN-/-小鼠体重始终高于对照小鼠,且在检测的第六周后趋于平稳。
2.双肌性状
取三月龄MSTN-/-小鼠和同窝野生小鼠进行解剖拍照,结果如图5所示,MSTN-/-小鼠全身的肌肉量明显大于野生小鼠,且更加致密。解剖前对两只小鼠称重,MSTN-/-小鼠重量39.80g,野生小鼠19.7g,在体长无明显差异的情况下,MSTN-/-小鼠体重约为对照小鼠的两倍,MSTN-/-小鼠的前后肢有明显的双肌现象,身体厚度大于野生小鼠。
3.骨骼肌中MSTN含量检测以及单肌纤维对比
分离小鼠骨骼肌,浸泡于生理盐水中,并在显微镜下剥离单个肌纤维,观察其结构,发现MSTN-/-小鼠的肌纤维显著粗于对照小鼠,且在MSTN-/-小鼠的肌纤维上我们看到了一些均一排布的纹路,而对照组小鼠的肌纤维则纤细光滑,结果如图6所示。
利用western blotting的方法对MSTN-/-小鼠及野生小鼠肌肉组织中MSTN蛋白表达情况进行半定量检测,如图7所示骨骼肌组织中MSTN-/-小鼠的MSTN表达量显著低于野生小鼠。
4.小鼠基础代谢及温度测定
基础代谢是指人体维持生命活动所需的最低能量,具体是指生物体在清醒、空腹、无肌肉紧张和思维活动,处于18~25℃环境中,且12小时前已停止进食情况下产生的维持生命活动所需的能量。基础代谢或称基础代谢率(basal metabolic rate,BMR),是以每小时、每平方米体表面积的产热量(kJ·m-2·h-1)来表示。
呼吸商(respiratoryquotient,RQ),又称气体交换率,指生物体在同一时间内,释放二氧化碳与吸收氧气的体积之比或摩尔数之比,即指呼吸作用所释放的CO2和吸收的O2的分子比,我们对测得的CO2、O2数据进行处理得到小鼠呼吸商,进而查出该小鼠的热氧价,之后根据雷伯纳(Rupner)公式查出小鼠体表面积,能量代谢=4.825×O 2耗量千卡/小时/体表面积(m2),计算结果图8所示。结果显示MSTN-/-小鼠的基础能量代谢值小于对照组的能量代谢值。
将DST nano温度检测探针放入7月龄的MSTN-/-小鼠和对照小鼠背部皮下进行温度跟踪,放置7d,每隔15min测量小鼠体温一次,取出后检测芯片记忆数据。发现7天的体温变化趋势基本相同,因而随机截取一天的数据处理如图9所示,显示MSTN-/-小鼠的体温较同窝对照小鼠略低,但都处于正常体温之内。
六、结论
利用Crispr/Cas9技术获得MSTN敲除小鼠后,通过扩繁获得稳定遗传的MSTN-/-三号外显子176-180位敲除的纯合小鼠,会产生肌肉量明显增大,体重显著增加,且基础能量代谢下降,体温下降的性状。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
SEQUENCE LISTING
<110> 内蒙古大学
<120> 一种能在小鼠体内表达的部分碱基缺失肌肉抑制素基因及应用
<130> 2017
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> MSTN2号外显子
<400> 1
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<210> 2
<211> 22
<212> DNA
<213> MSTN二号外显子
<400> 2
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<210> 3
<211> 18
<212> DNA
<213> MSTN三号外显子
<400> 3
agtcaaggtg acacaccc 18
<210> 4
<211> 22
<212> DNA
<213> MSTN三号外显子
<400> 4
gtgcttgaat tcacagtttc ga 22
Claims (5)
1.一种能在小鼠体内表达的部分碱基缺失肌肉抑制素基因,其特征在于,包含两个内含子和三个外显子序列,三号外显子175-180位6个碱基缺失。
2.根据权利要求1所述的能在小鼠体内表达的部分碱基缺失肌肉抑制素基因,其特征在于,构建了含有部分碱基缺失肌肉抑制素基因的切割载体。
3.根据权利要求1所述的能在小鼠体内表达的部分碱基缺失肌肉抑制素基因,其特征在于,制备了含有部分碱基缺失肌肉抑制素基因的敲除小鼠。
4.根据权利要求1所述的能在小鼠体内表达的部分碱基缺失肌肉抑制素基因在MSTN生物体性状研究中的应用。
5.根据权利要求4所述的能在小鼠体内表达的部分碱基缺失肌肉抑制素基因的应用,其特征在于,含有部分碱基缺失肌肉抑制素基因的切割载体用于增加小鼠骨骼肌的肌肉量,产生双肌现象,并降低小鼠基础代谢率和小鼠体温。
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