CN103952424B - 生产mstn双侧基因敲除的双肌性状体细胞克隆猪的方法 - Google Patents
生产mstn双侧基因敲除的双肌性状体细胞克隆猪的方法 Download PDFInfo
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Abstract
本发明公开了生产MSTN双侧基因敲除的双肌性状体细胞克隆猪的方法,其利用高效简便的基因修饰技术TALEN技术,设计构建能高效敲除MSTN基因的质粒;通过电转染法转染猪胎儿成纤维细胞,磁珠分选阳性细胞作为供体细胞,化学辅助去核方法去核后进行体细胞核移植;经胚胎移植确定妊娠后26‑36天取出胎儿进行基因测序,以MSTN双侧基因敲除胎儿细胞作为供体细胞再次体细胞核移植,胚胎移植;足月分娩后免疫组化及形态学观察,确定为双肌性状克隆猪。获得MSTN‑/‑双侧敲除的双肌性状克隆公猪,取精液对发情母猪进行人工受精,获得大量双肌猪。本发明工艺简单,易于实施,且可产业化生产双肌猪,利于农业生产和医学研究。
Description
技术领域
本发明具体涉及一种克隆动物模型的构建方法,特别是一种生产MSTN双侧基因敲除的双肌性状体细胞克隆猪的方法。
背景技术
随着人们对营养与健康的日渐重视,市场的瘦肉需求量也越来越大,而这种现象却导致了“注水猪肉”、“瘦肉精”、“三聚氰胺”等事件的发生,对人们的健康和生活造成了严重的困扰。中国是全世界最大的猪肉生产和消费大国,在生猪存栏量减少的情况下,人均猪肉占有量呈现上升势头。肌肉是动物胴体的最重要的组成部分,肌肉的生产是动物生产学中三大生产方向(肉、蛋、奶)之一,肌肉的产量是肉用畜禽的重要的经济性状,也是畜牧生产中最重要的生产方向。为此,长期以来育种学家们都将肌肉生产列为主要的研究目标。同时在医学方面肌肉消耗和衰竭症状的患者正逐年增多,为患者生产能够使双肌性状的克隆猪模型项目的启动迫在眉睫。
肌生成抑制素(Myostatin,MSTN)是近几年来发现的骨骼肌生长的负调控因子,其活性的丧失,会引起动物肌肉的过度发育,肌纤维直径变大或肌纤维数增加,表现为动物的“双肌”性状。各国科学工作者分别将基因敲除、基因诊断、分子标记辅助育种、转基因等技术应用到畜牧生产上,力求培育出一些高产肉率、肉质好和饲料报酬高的肉用品种。
体细胞核移植技术是生产转基因猪的最有效方法。体细胞核移植技术与基因修饰技术结合代表了未来转基因技术的发展方向。2000年,Betthauser等系统地优化了猪体细胞核移植的各个步骤,包括卵母细胞的来源和体外成熟培养、供体细胞的培养、核移植后卵的激活、胚胎的体外培养和胚胎移植代孕,建立了一套稳定的猪体细胞核移植程序,为通过核移植技术生产基因修饰猪的技术路线奠定了基础。2001年,Park等报道以转有绿色荧光蛋白基因的猪成纤维细胞为供体细胞进行体细胞核移植获得转基因猪,第一次打通了通过体细胞核移植技术生产转基因猪的技术路线。在国内2002年,赖良学等采用这一技术路线,利用同源重组技术敲除小型猪成纤维细胞α-1,3半乳糖苷转移酶(GGTA1)基因,得到四头GGTA1基因敲除克隆猪,第一次实现了猪的基因定点修饰,但此类核移植克隆猪技术还存在效率低的问题。
参阅CN101892264A等文献,虽然研究人员通过不同方法获得MSTN基因敲除胎儿,但均为MSTN单侧基因敲除,未达到理想的MSTN双侧基因敲除双肌性状健康猪个体。
发明内容
本发明的目的提供一种生产MSTN双侧基因敲除的双肌性状体细胞克隆猪的方法,以解决现有技术中的技术问题。
为实现上述目的,本发明提供如下技术方案:
一种敲除MSTN双侧基因的TALENs基因表达盒,其特征在于包含SEQ.NO.1~2所示的核酸序列。
上述TALENs基因表达盒在生产MSTN双侧基因敲除的双肌性状体细胞克隆猪中的应用。
一种敲除MSTN双侧基因的TALENs质粒,包含用以切割MSTN基因特异位点而形成上述基因表达盒的TALENs质粒。
上述TALENs质粒在生产MSTN双侧基因敲除的双肌性状体细胞克隆猪中的应用。
利用上述TALENs基因表达盒或上述TALENs质粒构建的MSTN基因敲除阳性细胞系。
上述MSTN基因敲除阳性细胞系在生产双肌性状体细胞克隆猪中的应用。
一种生产MSTN双侧基因敲除的双肌性状体细胞克隆猪的方法,包括如下步骤:
(1)提供上述TALENs质粒,其作用靶位点位于MSTN基因第一外显子的特定序列,其如SEQ.NO.3所示;
(2)提供猪胎儿成纤维细胞,并通过电转染法以所述质粒转染猪胎儿成纤维细胞,再分选出其中的阳性细胞作为供体细胞,且利用化学辅助去核方法去核后进行体细胞核移植;
(3)经胚胎移植确定妊娠后26-36天取出胎儿建立胎儿成纤维细胞后进行基因测序;
(4)以步骤(2)所获MSTN双侧基因敲除胎儿细胞作为供体细胞再次体细胞核移植,实现胚胎移植;
(4)足月分娩后免疫组化及形态学观察,确定为双肌性状克隆猪;
(5)利用步骤(4)所获MSTN双侧敲除的双肌性状克隆公猪,取精液对发情母猪进行人工受精,进而批量生产双肌猪。
与现有技术相比,本发明的优点包括:开发了一种生产MSTN双侧基因敲除的双肌性状体细胞克隆猪的方法,其工艺简单,易于实施,且可产业化生产双肌猪,当应用于农业方面时,将改进肌肉质量和重量,生产具有商业化性质的优良品种家畜,而在医学领域也会对人的肌肉萎缩、肌营养不良等疾病提供良好的治疗切入点。
附图说明
图1是本发明生产MSTN双侧基因敲除的双肌性状体细胞克隆猪的工艺流程图;
图2所示是本发明中TALEN介导的猪胎儿成纤维细胞及磁珠分选MACS后的富集细胞;
图3所示是本发明中公猪胎儿成纤维细胞的MSTN基因测序结果,其中,黑色加粗部分为碱基插入,“---”为碱基缺失。
图4所示是本发明所获的MSTN-/-双侧敲除体细胞克隆公猪的照片;
图5所示是本发明中免疫印迹分析图谱,MSTN-/-双侧敲除公猪Myostatin基因没表达;
图6所示是本发明中肌肉形态测定分析图谱,经HE染色MSTN-/-双侧敲除公猪比对照组肌纤维明显肥大;
图7所示是本发明中以Scion Image软件测量肌纤维直径分析400个肌纤维,并计算平均值所获图谱,结果显示MSTN-/-双侧敲除公猪肌纤维直径比对照组肌纤维明显大;
图8所示是本发明中大量生产MSTN-/-猪的方法的示意图。
具体实施方式
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明中记载的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
本发明的主旨在于提供一种生产MSTN双侧基因敲除的双肌性状体细胞克隆猪的方法,其利用目前最高效简便的基因修饰技术TALEN技术,结合体细胞克隆技术获得MSTN双侧基因敲除的双肌性状克隆猪,并可应用双肌猪的规模化生产。
更为具体的讲,参阅图1,本发明的工艺包括:利用TALEN技术,设计构建能高效敲除肌生成抑制素基因的质粒和编码TALENs的信使核糖核酸;通过电转染法转染猪胎儿成纤维细胞,磁珠分选阳性细胞作为供体细胞,化学辅助去核方法去核后进行体细胞核移植;经胚胎移植确定妊娠后26-36天取出胎儿建立胎儿成纤维细胞后进行基因测序,以MSTN双侧基因敲除胎儿细胞作为供体细胞再次体细胞核移植,胚胎移植;足月分娩后免疫组化及形态学观察,确定为双肌性状克隆猪;获得MSTN双侧敲除的双肌性状克隆公猪,取精液对发情母猪进行人工受精,获得大量双肌猪。
体细胞克隆获得的胎儿建立成纤维细胞后进行基因测序,10种体细胞体株的MSTN基因发生突变,其中3株细胞为MSTN基因双侧突变具体为F1、F2、19-3,7株细胞为MSTN基因单侧基因突变具体为F3、F5、F9、18-2、18-3、19-1、19-4,突变部位的碱基序列如图2所示。对获得的10个阳性突变胎儿细胞系永久冷冻保存并提供未来大量克隆基因突变猪。
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行详细的描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。
需要说明的是,在本文中,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
实施例1:TALEN的构建
针对MSTN基因特异位点的TALENs设计和构建由韩国ToolGen公司完成(NCBI基因编号:399534;基因组序列号:NC_010457.3)。经生物信息学方法分析和在酵母中的DNA切割能力比较,最终确定了1对TALENs,作用靶位点位于MSTN基因第一外显子特定序列(表1)。
TALEN是由一个TAL复制域和一个核酸内切酶的切割域(FokⅠ)构成。FokⅠ的切割域形成RR或DAS的异质二聚体,可以打靶MSTN基因突变exon 1位点。TALENs由ToolGen公司构建。424TAL效应子阵列质粒构建由一共84个TAL效应子质粒构成,其中包括64个三单元模块质粒,16个二单元模块质粒和4个多单元模块质粒排列成NN、HD、NI和NG的所有可能组合。RVD模块的建立采用GenScript技术。为了避免不良反应,本发明排除了人类罕见的密码子和有限的最大序列鉴别不同的RVDs 81%。每84个质粒由适当的引物进行PCR扩增,根据6个TAL效应子阵列的位置由BsaI消化。PCR扩增子在以卡那霉素抗药性选择标记的主链载体扩增亚克隆。8个FokI表达质粒含有氨苄西林抗药性基因、CMV启动子、一个HA附加表位、核定位信号、AvrBs3的n端135个氨基酸,其中一个中心四个RVD半复制和sharkey FokI切割域(DAS或RR)32。
表1 TALEN-MSTN对应的DNA结合位点
实施例2:猪胎儿成纤维细胞系的建立
供体细胞为来自成年母猪怀孕40天后取出胎儿,建立初代公猪胎儿成纤维细胞并传代,采用常规组织块接种培养,建立细胞系。用眼科剪剪掉猪头部及脏器,用75%的酒精充分清洗消毒,无菌条件下剪取胎膜,置于含有双抗的PBS中,反复漂洗3-5次,直到漂洗液清亮透明为止,移至培养皿中,用眼科剪将其剪成0.5-1mm3大小的组织块,放于DMEM培养基(Gibco)包含0.25%胰蛋白酶中,在38.5℃的CO2培养箱中消化4分钟,取出后加入含血清的DMEM培养基终止消化,用吸管将组织块转移至15ml离心管内,1500rmp离心10分钟,离心后去掉上清液,加入新鲜DMEM培养基(Gibco)添加20%胎牛血清(FBS,Gibco)重悬,移入38.5℃预温的新鲜DMEM培养基添加20%胎牛血清约3ml,用无菌吸管将组织块转移至处理过的专用培养皿中,并均匀铺开在培养皿中,每小块间距5mm左右,将培养皿置于在38.5℃,CO2培养箱进行培养,每日在观察细胞形态和生长情况,当细胞长出后,将其转入培养瓶中继续培养。细胞在体外经3-5次传代用于细胞转染实验。
实施例3:电转染法转染猪胎儿成纤维细胞,磁珠筛选阳性细胞
母猪怀孕40天后取出胎儿,建立初代猪胎儿成纤维细胞并传代,置于含有15%胎牛血清、1%非必须氨基酸、100mg/mL青霉素和100mg/mL链霉素的DMEM中培养。细胞电转染(Amaxa4D,Lonza)的比率为45:45:10(TALEN单体编码的质粒:其他TALEN单体编码的质粒:报告基因)。具体方法:胰蛋白酶消化总个数约为1×106个细胞并用HBSS(WelGen,首尔,韩国)洗涤,离心。最终将转染的细胞悬浮在含有TALEN和报告基因质粒的缓冲液中,于95%的空气湿度,5%CO2,37℃的培养箱中培养2天。突变体细胞通过磁分离富集(图2)。通过体细胞核移植技术获得携带等位基因突变的胎儿,建立细胞并培养在含有15%胎牛血清的DMEM中,4-8代的细胞作为体细胞核移植的供体细胞。
实施例4:卵母细胞体外成熟培养
从屠宰场取初情期前青年母猪卵巢,用吸卵泵抽吸卵巢上直径为3~8mm卵泡。挑选卵丘包裹3层以上、致密且胞质均匀卵丘细胞-卵母细胞复合体(CumuLus-oocyte-complexes,COCs),用TALP液洗涤3遍,放在卵母细胞体外成熟培养液(IVM液)中。IVM培养液成分为:TCM-199添加10%猪卵泡液(pFF)、0.6mmol·L-1L-半胱氨酸、10IU·mL-1人绒毛膜促性腺激素(hCG)、10IU·mL-1孕马血清促性腺激素(PMSG)和10ng·mL-1表皮生长因子(EGF)。22h后更换无激素IVM液。COCs在38.5℃、5%CO2饱和湿度环境培养38h后,移入含0.1%透明质酸酶中,用直径为170~180μm剥卵针,反复吹吸卵母细胞脱去卵丘细胞,挑选卵周隙明显,卵细胞完整,且排出第一机体卵母细胞备用。
实施例5:化学辅助去核方法去核后转基因体细胞核移植
参照Yin等去核方法,选取做受体卵母细胞在含有0.4ug·mL-1秋水酰胺(Demecolcine)和0.05mol·L-1蔗糖培养液处理1h后,移入含5mg·mL-1细胞松弛素b(CB)和0.4mg·mL-1Demecolcine操作液中。在显微操作系统(IX71,Olympus,Japan)下,利用去核针先去除M II卵母细胞第一极体及突起处细胞核,然后在注核液中用注入针将体细胞从去核切口放入卵黄间隙。
实施例6:重构卵融合与激活
将重构卵在融合液(0.28mmol·L-1甘露醇+0.1m mol·L-1MgCl2)中平衡后,放入铺满融合液融合槽内,用玻璃针使供体细胞-受体卵细胞膜接触面与电极平行,再用融合仪(LF 301),施加1次2kV·cm-1、20μs直流电脉冲进行融合。融合后重构卵在含有0.4mg·mL- 1demecolcine培养液中培养1h后,在激活液(0.28mmol·L-1甘露醇+0.1mmol·L-1MgCl2)+0.1mmol·L-1Ca2+中给予一次1.5kV·cm-1、100μs直流电脉冲进行激活,然后在含有2mmol·L-1 6-DMAP培养液中培养4h。
实施例7:胚胎移植及代孕受体的妊娠诊断
融合的重构胚于NCSU-37培养液中,培养条件为38℃5%CO2饱和湿度环境,以胚胎构建当天为第0天,对重构胚胎培养7-8天后,在第38小时和第7天时分别观察卵裂率和囊胚胎发育情况并记录。
胚胎移植以发情周期正常,生殖器官无疾病大白猪作为受体,注射1000IU孕马血清促性腺激素(PMSG)72h后,对肌肉注射1000IU人绒毛膜促性腺激素(hCG)进行诱导发情在注射hCG后48h,用手术法将培养1-3d卵裂正常的1-8细胞期的胚胎移植到代孕母猪输卵管内,胚胎移植后24-26天进行B超监测妊娠情况。
表2 MSTN阳性细胞作为供体细胞进行体细胞核移植得到的胎儿
实施例8:克隆胎儿基因型鉴定
对B超监测妊娠确定的36天受体猪,将子宫从两个子宫角位置摘除,冰上放置带回实验室剥离胎儿。用G-DEX IIc DNA提取试剂盒(iNtRON)提取胎儿的基因组DNA。提取的DNA被用作嵌套式PCR的模板,引物设计为扩增整个靶位点。PCR操作设置一个正向(5'-ctggtcccgtggatctgaatg-3')和反向(5'-gatcgtttccgtcgtagcgtg-3')引物,得到一个302bp的扩增产物。然后将这些PCR产物用于第二次PCR反应。第二个PCR设置一个正向(5'-gaatgagaacagcgagcaaaagg-3')和反向(5'-catcttccaaggagccatcac-3')引物,得到一个257bp的扩增产物。扩增子经过加热变性和退火形成异源双链DNA,继续在37℃下用5单位T7核酸内切酶Ι(New England Biolabs,Massachusetts,US)处理20分钟,然后进行2.5%琼脂糖凝胶电泳分析。在第二次PCR扩增子中,经T7E1测定为阳性基因组的DNA样品用T-BluntPCR Cloning Kit(SolGent)克隆入T-Blunt载体中。克隆质粒在测序公司用M13引物测序。参阅图2,经过测序分析,10个胎儿的MSTN基因发生突变,其中3个是MSTN双侧基因突变(F1、F2、19-3),7个是MSTN单侧基因突变(F3、F5、F9、18-2、18-3、19-1、19-4),突变部位的碱基序列如图3所示。对获得的10个阳性突变胎儿成功建立了细胞系并冻存保管于液氮内。
表3 公猪胎儿成纤维细胞MSTN基因突变类型
实施例9:再次体细胞核移植,胚胎移植
经基因测序,确定MSTN-/-双侧敲除胎儿F2,以此胎儿细胞系作为供体细胞再次进行核移植,重复第7步。妊娠个体分离饲养管理,诱导分娩。最终产下MSTN双侧基因敲除的克隆猪。通过该方法生产的MSTN-/-双侧敲除的体细胞克隆猪,与对照组同月龄猪(2个月龄)比较表现为明显的双倍肌肉性状(参阅图4)。
表4 MSTN-/-公猪胎儿成纤维细胞F2核移植胚胎发育结果
实施例10:免疫印迹分析
在蛋白质水平检测猪Mstn-/-基因敲除猪和野生型上的表达差异,用GAPDH(SantaCruz,CA,USA)作为内参,Myostatin基因的一抗为Myostatin(3E7)(Young In Frontierco.LTD,South Korea)。用RIPA裂解液(含有50mM Tris-HCl(pH值8.0),150mM NaCl,1%NP-40,0.5%钠脱氧胆酸盐和0.1%SDS)提取细胞总蛋白质。Quick StartTM BIO-RAD试剂盒测定蛋白质浓度后,经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,转移到PVDF膜(GE water&Process Technologies.,Trevose,PA.USA)上,在5%脱脂奶粉溶液中室温孵育1h以封闭非特异结合,封闭过的膜加入一抗(anti-GDF8/Myostatin)4℃过夜。加入HRP标记的二抗体(抗鼠IgG/抗羊IgG,Santa Cruz,CA,USA)1:5000稀释,以结合一抗和GAPDH,室温孵育膜1小时。增强型化学发光底物检测(Animal Genetics Inc.),经X胶片曝光显影(AgfaHealthCare)。结果显示(图5)Mstn-/-基因敲除猪不表达Myostatin。
实施例11:免疫组化及形态学观察,确定为双肌性状克隆猪
出生后25天死亡的Mstn-/-小猪用于组织化学分析。分别从对照组同日龄小猪和mstn-/-小猪的身上提取了肱二头肌。之后肌肉组织在甲醛中固定10个小时,梯度酒精脱水、氯仿透明、石蜡包埋,用切片机切片厚度一般为3-5μm。切片时先将组织片平摊于一块玻璃上,迅速滴加30%的酒精水溶液使组织片完全展开,再移入40℃恒温热水器中。也可直接将组织切片移入40℃的恒温热水器中,待组织片完全展开后将其贴附于载玻片上,经56-60℃烤片30-60min后,做HE染色。肌纤维直径和数量按照之前的方法进行测量。实验结果:细胞核呈蓝色;肌肉呈红色。显示MSTN双侧敲除的仔猪比对照组肌肉切片肌纤维肥大紧密(图6)。用Scion Image软件测量肌纤维直径和数量,分析了400个肌纤维并且计算了400个猪肌纤维的平均值(图7)。
获得MSTN-/-双侧敲除的双肌性状克隆公猪,取精液对发情母猪进行人工授精,获得大量双肌猪,产业化生产。根据研究文献,MSTN+/-(杂合子)、MSTN-/-(突变纯合子)肌肉发达程度与野生型小鼠相比均有增加,且突变纯合子更明显。MSTN-/-双肌性状克隆公猪性成熟后,采取精液,精子经活力检测,稀释后,对普通成年母猪进行人工受精。分娩后可获得大量F1MSTN+/-仔猪(参阅图8),有助于在畜牧业生产上改良育种,提高胴体瘦肉率。
应当理解,以上所述仅是本发明的具体实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种敲除MSTN双侧基因的TALENs基因表达盒,其特征在于,包含SEQ.NO.1~2所示的核酸序列。
2.一种敲除MSTN双侧基因的TALENs质粒,其特征在于,包含用以切割MSTN基因特异位点而形成权利要求1所述基因表达盒的TALENs质粒。
3.根据权利要求2所述的TALENs质粒,其特征在于,所述MSTN的NCBI基因编号为399534;基因组序列号为NC_010457.4。
4.根据权利要求2或3所述的TALENs质粒,其特征在于,所述质粒的作用靶位点位于MSTN基因第一外显子特定序列,该特定序列如SEQ.NO.3所示。
5.利用权利要求1所述TALENs基因表达盒或权利要求2至4中任一项所述的TALENs质粒构建的MSTN双侧基因敲除阳性细胞系。
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