CN105764335B - 产生免疫细胞缺陷型小型猪的方法及等位基因突变的细胞 - Google Patents
产生免疫细胞缺陷型小型猪的方法及等位基因突变的细胞 Download PDFInfo
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Abstract
本发明涉及白介素2受体γ(IL2RG)基因打靶载体,一种用于产生具有引入其中的载体的免疫细胞缺陷型转基因克隆的小型猪的方法,及其应用。
Description
技术领域
本发明涉及白介素2受体γ(IL2RG)基因打靶载体,用于产生具有引入所述载体的免疫细胞缺陷型转基因克隆的小型猪的方法,及其应用。
背景技术
人类中出现了重症综合性免疫缺陷(下文称为“SCID”)(Buckley, R.H. Annualreview of immunology 22, 625-655 (2004))。但是,由于反映人类SCID类型的动物模型有限,因此不易开发出治疗SCID的药剂。猪具有与人类类似的生理学特征,并且以比啮齿动物模型高的相似性模拟了很多人类疾病(Whyte, J.J. & Prather, R.S. Molecularreproduction and development 78, 879-891(2011))。因此,SCID猪能代表模拟人类疾病的模型。此外,在癌症研究、细胞移植和药物开发研究中,都能使用SCID猪模型。白介素2受体γ(下文中称为“IL2RG”)与X连锁SCID类型相关,并且IL2RG突变导致小鼠中的T细胞和NK细胞缺乏而且导致B细胞的功能受损(Cao, X. et al. Immunity 2, 223-238 (1995))。最近报道了通过中断IL2RG生成的X连锁SCID猪,该X连锁SCID猪显示出人类X连锁SCID的表型(Suzuki, S. et al. Cell stem cell 10, 753-758 (2012))。
在先文件
韩国公开专利公开号:10-2004-0074108。
发明内容
【技术问题】
基于以上需要进行了本发明,本发明的目标是提供一种用于产生显示出SCID表型的基因工程猪的有效方法。
【技术方案】
为了实现上述目标,本发明提供了一种用于产生免疫细胞缺陷型转基因克隆的小型猪的方法,所述免疫细胞缺陷型转基因克隆的小型猪在白介素2受体γ(IL2RG)中具有等位基因突变,所述方法包括以下步骤:使用TALEN(转录激活样效应因子核酸酶)处理猪(Susscrofa)的染色体X上的由SEQ ID NO: 1所示的TALEN识别序列位点,以诱导等位基因突变;以及,使用具有诱导的等位基因突变的细胞,通过体细胞核移植(SCNT)产生突变体胚胎。在本发明的实施方式中,优选使用编码TAL效应因子核酸酶的载体转染感兴趣的细胞,来进行TALEN对TALEN识别序列位点的处理,但并不限于此。本发明还提供了一种通过本发明的生产方法产生的,在白介素2受体γ(IL2RG)中具有等位基因突变的免疫细胞缺陷型转基因克隆的小型猪。本发明还提供了一种分选细胞的方法,该方法包括:将以下载体一起引入到细胞中:编码TAL效应因子核酸酶的载体,所述编码TAL效应因子核酸酶的载体能识别猪(Susscrofa)的染色体X上的由SEQ ID NO: 1所示的TALEN识别序列位点,以在感兴趣的基因序列中或在所述基因序列两侧的区域中诱导突变;和报告载体,所述报告载体包括单体红色荧光蛋白(RFP)基因、如SEQ ID NO: 1所示的可编程(programmable)的核酸酶靶向序列、增强型绿色荧光蛋白(GFP)和H-2KK基因;以及,分选出RFP、GFP和H-2KK阳性的细胞,作为白介素2受体γ(IL2RG)中靶细胞。在本发明的实施方式中,通过抗体进行H-2KK的检测。在本发明的另一实施方式中,优选通过流式细胞术检测RFP和GFP的表达,但并不限于此。本发明还提供了具有等位基因突变的细胞,所述等位基因突变是通过使用TALEN(转录激活样效应因子核酸酶)处理猪(Sus scrofa)的染色体X上的由SEQ ID NO: 1所示的TALEN识别序列位点来诱导的。本发明的细胞于2013年10月2日保藏在韩国典型菌种保藏中心,韩国生物科学和生物技术研究所(韩国大田市儒城区)中,保藏号为KCTC 12497BP。下面将对本发明进行描述。在本发明中,发明人报道了一种有效方法,其中,使用转录激活样效应因子核酸酶(TALEN)介导的打靶,利用体细胞核移植(SCNT),来产生显示出SCID表型的两种类型的基因工程猪。为了靶向猪的IL2RG,设计出特定的TALEN并且由ToolGen(韩国首尔)合成。设计每一种TALEN,以在染色体X的IL2RG上产生突变(图1a)。使用包括TALEN识别位点的报告基因,鉴定出适当表达TALEN组的细胞(图3和图1b)。通过将TALEN和报告基因的组合插入到HEK293T细胞中,来证实所设计的TALEN的活性(图1c)。在证实之后,通过电穿孔,将编码TALEN和报告基因的构建体插入到猪的成纤维细胞中。48小时之后,分选出GFP阳性的细胞(图3),并且将分选出的细胞以1个细胞/孔的密度铺在96孔板中。10天之后,将细胞进行传代培养,并且使用一半细胞进行基因分型。对预测的TALEN剪切位点两侧的小PCR片段(约400 bp)进行测序示出,在IL2RG组中存在TALEN诱导的插入/缺失(indel);筛选了总共30个克隆的IL2RG(参见下面表1中的引物信息)。对于IL2RG,基因打靶的效率是30%(9/30)。本发明中使用的TALEN对于它们的靶标是特异性的,因此发明人没有观察到脱靶剪切(图4和表2)。使用得到证实的中靶细胞进行体细胞核移植(SCNT)(图5)。通过SCNT产生突变体胚胎,并且将733个胚胎转移到四只代孕母猪中,在第25天发现这四只代孕母猪全部怀孕。出生了一只活的IL2RG突变体(图6和下面的表3)。基因分型表明猪具有IL2RG的中靶突变(图1d)。IL2RG中的突变是缺失四个核苷酸,产生了提前终止密码子(IL2RG+/Δ69-368)。图7示出了产生的IL2RG +/-猪崽,和产生的IL2RG+/-猪崽的解剖照片。图8示出了分析IL2R的表达的结果。可以看出,通过实时RT-PCR和IHC发现产生的IL2RG+/-示出了高靶标特异性,并且示出在IL2受体中,IL2Rα和IL2Rβ正常表达,仅IL2Rγ受到了影响。为了分析IL2RG (+/-)杂合子猪的特征,分析了脾脏中IL2RG的蛋白表达。还分析了IL2RG+/-后代的脾脏中IL2Rα蛋白和IL2Rγ蛋白的表达。分析了IL2RG+/-脾脏中的IL2受体α的表达,结果示出IL2Rα在野生型猪和IL2RG+/-敲除猪中都正常表达。为了再次证实上述结果,检验了这些基因的mRNA表达,检验结果如图8c所示。这些结果与蛋白表达的结果相同。即,可以看出IL-2Rα和IL-2Rβ的表达在野生型猪和IL2RG+/-敲除猪之间存在很小的差异或没有差异,而IL2RG+/-猪中IL-2Rγ的表达比野生型中IL-2Rγ的表达至少低3倍。图9示出了使用T细胞标记物(CD25和CD3)分析CD3+ T细胞子集的结果。此外,检验了猪胸腺中的T细胞发育过程。图10示出了使用IL2RG+/-组织和正常组织进行的微阵列芯片分析的结果。通过DNA芯片测定分析了脾脏和胸腺中的基因表达,并且相对于对照组进行了标准化,然后确定了被上调或下调的基因。如图10中所示,对照组示出了与IL2RG敲除组明显不同的表达模式。有趣的是,胸腺中的表达差异比脾脏中更明显,这似乎是因为IL2RG KO猪的胸腺的发育非常不明显(图10a)。在猪的DNA芯片分析中最大的困难是,仍然不可能进行不同物种之间的功能分析,因为即使报道了猪的全长基因组DNA序列,基因库储存的蛋白的功能基因序列也是不足的。因此,使用与小鼠基因的对比分析,来克服猪基因组分析的局限性。为此,在本发明中,仅分选出功能与小鼠基因一致的猪基因。结果分析出下调2倍或更多的基因是胸腺中的4487个基因和脾脏中的1050个基因,而下调4倍或更多的基因是胸腺中的1217个基因和脾脏中的231个基因(图10b)。IL-2RG敲除猪示出了免疫缺陷。为此,为了对比与B细胞、T细胞和NK细胞相关的基因表达,使用对小鼠所报道的现有信息,分析了Studio通路。基于分析的结果,筛选了参与T/B/NK细胞发育和分化的主要基因,并且检验了这些基因的表达。检验结果示于图10c中。图11示出了影响IL2受体的因子,和受到IL2受体影响的因子。在IL-2RG上游的一组基因的情况中,CEACAM1、GNRH1、IL-4、DNM2和IL21基因受到IL-2RG基因的影响,使得这些基因的表达被上调或下调。尽管需要进一步的研究,但是目前与对照不同的这些基因的结果表明,这些基因在IL-2RG表达中起到了直接或间接的作用,同时交换了顺式信号或反式信号。在参与IL-2RG下游信号的基因中,大部分基因的结果与通过Pathway Studio软件分析的结果一致,但是CCL2、IFNG和STAT1基因的表达与通过Pathway Studio软件分析的结果不同。出现该现象是因为:(1)分析的IL-2RG敲除(KO)猪使用了杂合器官,并且(2)猪中的这些基因表达机制与小鼠中的不同。在本发明中,发明人发现通过TALEN介导的基因打靶,能有效产生SCNT来源的SCID猪。使用报告载体和TALEN构建体诱导了高效率的突变。与传统的基因打靶(其中将打靶载体插入到基因组中)不同,本发明能产生基因工程猪,同时不会在基因组中留下任何特征。在本发明中产生的基因工程猪可用作SCID研究的模型,该SCID研究的模型包括可显示出人类欧门(Omenn)综合征的第一猪模型(猪可获自国家猪资源和研究中心(National Swine Resource and Research Center),http://nsrrc.missouri.edu/)。
【有益效果】
具有重症综合性免疫缺陷(SCID)表型的猪将用于干细胞治疗、癌症研究和异种移植开发中。本发明人描述了通过TALEN介导的打靶产生的两种类型的SCID猪(IL2RG敲除)。
附图说明
图1示出了使用TALEN产生SCID猪。具体地,图1(a)示意性示出了IL2RG和RAG2的TALEN介导的敲除。图1(b)示出了包括TALEN识别位点的供体报告载体。供体报告载体中的TALEN识别位点的剪切诱导GFP的表达。图1(c)示出了体外证实设计的TALEN的结果。仅当TALEN与供体报道基因一起转染到HEK 293T细胞中时,才能检测到GFP的表达。图1(d)示出了通过基因工程方法产生的猪的基因分型。
图2示出了用于富集经TALEN打靶的细胞的代孕报告载体(surrogate report vector)的构建。(a)该报告载体包括单体RFP基因、可编程的核酸酶靶向序列(左半部位点和右半部位点)、增强型GFP和H-2KK基因。如果由于没有可编程的核酸酶活性而使GFP和H-2KK序列不在阅读框中,因此仅表达RFP基因。当通过可编程的核酸酶在靶向序列中插入双链切口时,通过非同源性末端接合(NHEJ)修复切口,这常常引起移码突变。这样的突变使得GFP与RFP处于同一阅读框中,并且诱导mRFP-eGFP-H-2KK融合蛋白的表达。(b)示意性示出通过两个系统(H-2KK抗体的磁性分离和通过RFP和GFP的表达流式细胞术),对分选的mRFP+eGFP+H-2KK+细胞中核酸酶诱导的突变进行富集。在细胞内,示出了报告质粒和染色体靶位点。突变由黑点表示。
图3示出了在引入TALEN之后,对GFP阳性细胞的FACS分选。(a)转染48小时之后,RFP和GFP的高度共表达。(b)箭头下面的方框表明GFP阳性细胞。分选出GFP表达最强的前10%的细胞,并且放置在96孔板中。GFP阳性细胞在23.0%至38.0%的范围内。比例尺= 20 μm。
图4示出了对IL2RG突变体猪的IL2RG基因的脱靶分析。顶部)使用Surveyor核酸酶对异源双链DNA的处理示出,在与IL2RG基因同源性最高的9个基因座中没有额外的脱靶突变。SM:大小标记物,泳道1:LRRIQ1;泳道2:BNC2; 泳道3:SLC17A5;泳道4:ZNF334;泳道5:TTN;泳道6:PGRMC2;泳道7:AVPR2;泳道8:CCDC18;和泳道9:ZSWIM2。底部)用于排除脱靶突变的具有IL2RG-相关序列的基因,和基因之间的序列同源性。
图5示出了用于鉴定SCNT候选细胞克隆的PCR测序结果。由于NHEJ,引入TALEN诱导了接近TALEN结合位点处的多态性。分析多态性的类型,并且用作SCNT的细胞克隆。
图6是在本发明中产生的SCID猪模型的图像。该猪的遗传背景是明尼苏达迷你猪(Minnesota mini-pig)。图7示出了所产生的IL2RG +/-猪崽。
图8示出了验证IL2R表达的结果。图9示出了使用T细胞标记物(CD25和CD3)分析CD3+ T细胞子集的结果。图10示出了使用IL2RG+/-组织和正常组织进行的微阵列芯片分析的结果。图11示出了影响IL2受体的因子,和受到IL2受体影响的因子。
具体实施方式
下面,将通过非限制性实施例进一步详细描述本发明。但是应理解,这些实施例仅是用于例示的目的,并不限制本发明的范围。本发明中使用的所有动物和实验都获得了密苏里大学(University of Missouri)动物保护和利用委员(Institutional Animal Care andUse Committee)的批准。
实施例1:细胞转染和基因打靶 对于基因打靶,使用报告载体和TALEN构建体转染2~3百万个细胞(2 μg各构建体/百万个细胞)。使用BTX Electro Cell Manipulator (HarvardApparatus, Holliston, MA),以490 V、1毫秒和3次脉冲,用上述构建体对细胞进行电穿孔。接着,将细胞铺在T75培养瓶中保持48小时,然后使用Beckman Coulter MoFlo XDP分选GTP阳性细胞。将经分选的细胞铺在96孔板中。10天之后,使用一半细胞进行基因分型。在插入TALEN之后,为了检验插入缺失(indel)的存在,通过PCR扩增TALEN剪切位点两侧的基因组DNA的片段。使用细胞裂解缓冲液,从培养的细胞中分离基因组DNA,然后用于PCR。在以下条件下进行PCR扩增:在95℃进行初始变性2 min,然后进行32次由在94℃变性30 sec、55℃退火30 sec和72℃延伸30 min组成的循环(参见下面表1中的PCR引物对)。IL2RG的PCR产物的预测大小是417 bp,RAG2的PCR产物的预测大小是426 bp。对PCR产物进行测序,以鉴定indel的存在。
实施例2:体细胞核移植 为了产生SCNT胚胎,从ART(Madison, WI)购买了母猪的卵母细胞。在成熟培养基(具有2.9 mM Hepes、5 μg/ml胰岛素、10 ng/ml EGF、0.5 μg/ml p-FSH、0.91 mM丙酮酸盐、0.5 mM半胱氨酸、10%猪卵泡液和25 ng/ml庆大霉素的TCM199)中连夜运送卵母细胞。24小时后,将卵母细胞转移到新鲜的培养基中。成熟40~42小时之后,在0.1%透明质酸酶的存在下,通过涡旋从卵母细胞中去除卵丘细胞。在操作过程中,将卵母细胞放置在补充有7.0 μg/ml细胞松弛素B的操作培养基中。与一部分邻近的细胞质(可能含有中期II板)一起去除极体,并且使用细玻璃毛细管将供体细胞放置在卵周隙中。接着,使用BTX Electro Cell Manipulator(Harvard Apparatus),通过1.2 kV/cm的两次DC脉冲(1-sec间隔)30 μsec,在融合介质(0.3 M 甘露醇、0.1 mM CaCl2、0.1 mM MgCl2、0.5 mMHepes)中融合重构的胚胎。融合之后,在黑暗条件下,使用200 μM硫柳汞持续10 min并且使用8 mM二硫代苏糖醇持续30 min,使融合的胚胎完全活化。然后,将胚胎与含0.5 μMscriptaid(即组蛋白脱乙酰酶抑制剂)的Porcine Zygote Media 3(PZM3)3孵育14~16小时。第二天,将SCNT胚胎移植到代孕猪中。对于囊胚期胚胎移植,从scriptaid中冲洗胚胎,并且在存在10 ng/ml CSF2的PZM3中再培养五天。将SCNT胚胎用外科手术方法移植到代孕猪的壶腹峡部连接处。
实施例3:免疫组织化学(IHC) 对于IHC,使用在具有10%福尔马林的中性缓冲液中固定的组织。将组织放置在用于IHC的载玻片上。首先,在3%过氧化氢酶中阻断内源性过氧化物酶活性。样品经Borg Decloaker预处理,然后在背景Sniper溶液中进行封闭。冲洗之后,样品与对B细胞(CD79A; Diagnostic Biosystems- # Mob118,1:100)或T细胞(CD3; DAKO- #A0452,1:400)具有特异性的初级抗体孵育。孵育之后,冲洗样品,并且与HRP缀合的二级抗体孵育。然后,使用Romulin Red Chromogens(Romulin红色原)对样品进行染色,以使信号可视化。此外,还使用IP FLX苏木精对样品进行染色以提供背景。Borg、Sniper、Romulin红和IP FLX苏木精都购买自Biocare(Concord, CA)。使用配备有Olympus DP70高分辨率数字显微照相机(Olympus, Center Valley, PA)的Zeiss Axiophot显微镜(Carl Zeiss, 上科亨(Oberkochen), 德国),获取所有的显微照片。
实施例4:流式细胞术 将来自安乐死的野生型猪崽和双等位基因猪崽的脾脏收集在补充有10%胎牛血清的RPMI-1640培养基(Mediatech, Inc., Manassas, VA)中,使用解剖刀片切碎,经20号针头抽吸几次,然后通过70 μm尼龙网细胞筛(BD Biosciences, 圣何塞(San Jose), CA)。然后,脾细胞悬浮液与药物溶解溶液(Pharm Lyse Solution,BDBiosciences)孵育15分钟以裂解红细胞,然后在200 × g离心5分钟形成沉淀块。丢弃上清液之后,将沉淀块重悬在冷的染色缓冲液(BD Pharmingen)中,并且在血细胞计数器上对细胞进行计数。然后,将细胞分成在200 μL染色缓冲液中的5×106个细胞的等份。以0.5 μg/1× 106个细胞的量,将FITC标记的小鼠抗猪CD21、小鼠抗猪CD 3ε和小鼠抗T-2真菌毒素IgG1k(同型对照组)(SouthernBiotech, 伯明翰(Birmingham), AL)添加到细胞中,在黑暗条件下,在4℃孵育30分钟。然后,将细胞冲洗两次,并且重悬在新鲜的染色缓冲液中。使用密苏里大学的细胞和免疫生物学核心(Cell and Immunobiology Core)设施中的CyAn ADP流式细胞仪(Beckman Coulter, 布雷亚(Brea),CA),分析细胞。使用Summit v4.3软件(Beckman Coulter)分析数据。
实施例5:脱靶分析 为了鉴定从本发明使用的TALEN中脱靶的序列,使用生物信息学工具从最新的猪基因组集合(Sscrofa10.2)中,鉴定出与每一TALEN结合位点类似的序列。基于核苷酸差异的数目,设计最可能的脱靶位点两侧的PCR引物。在种源动物中扩增这些区域,并且使用Surveyor核酸酶测定法(下面的表2)测试脱靶事件。PCR扩增之后,将300~500ng PCR产物(10~15 μl)转移到新管中,然后按照热循环仪的程序(95℃ 2 min,以-2℃/秒的速度从95℃降低至85℃,以-0.1℃/秒的速度从85℃降低至25℃,无限期地处于4℃)变性并且再退火。向其中添加1μl Surveyor核酸酶和1μl Surveyor强化剂,然后在42℃孵育30分钟。之后,将反应立即放在冰上,并且向反应中添加6X Surveyor核酸酶终止缓冲液和6X染料。样品在2.0%琼脂糖凝胶上进行电泳。
表1
上面的表1示出了用于对TALEN诱导的IL2RG突变体进行基因分型的引物。
表2
上面的表2示出了用于鉴定猪IL2RG的脱靶的引物对。
表3
上面的表3示出了在本发明中获得的核移植效率。
保藏号 保藏单位的名称:韩国生物科学和生物技术研究所;保藏号:KCTC12497;保藏日期:2013年10月2日。
Claims (4)
1.一种用于产生免疫细胞缺陷型转基因克隆的小型猪的方法,所述免疫细胞缺陷型转基因克隆的小型猪在白介素2受体γ(IL2RG)中具有等位基因突变,所述方法包括以下步骤:
使用TALEN(转录激活样效应因子核酸酶)处理猪的染色体X上的由SEQ ID NO:1所示的TALEN识别序列位点,以诱导等位基因突变;以及
使用具有诱导的等位基因突变的细胞,通过体细胞核移植(SCNT)产生突变体胚胎,
其中,所述TALEN识别序列位点是SEQ ID NO:1的第1~19位核酸和SEQ ID NO:1的第32~50位核酸。
2.根据权利要求1所述的方法,其中,使用编码TAL效应因子核酸酶的载体转染感兴趣的细胞,来进行TALEN对所述TALEN识别序列位点的处理。
3.一种具有等位基因突变的细胞,所述等位基因突变是通过使用TALEN(转录激活样效应因子核酸酶)处理猪的染色体X上的由SEQ ID NO:1所示的TALEN识别序列位点来诱导的,
其中,所述TALEN识别序列位点是SEQ ID NO:1的第1~19位核酸和SEQ ID NO:1的第32~50位核酸。
4.根据权利要求3所述的细胞,所述细胞是IL2RG敲除的小型猪的成纤维细胞系KCTC12497。
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