CN105813456B - Scid样小型猪的生产方法及双等位基因突变的细胞 - Google Patents
Scid样小型猪的生产方法及双等位基因突变的细胞 Download PDFInfo
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Abstract
本发明提供了一种Rag‑2(重组激活基因2)基因打靶载体,用于产生引入有该载体的SCID样小型猪的方法,及其应用。
Description
技术领域
本发明涉及Rag-2(重组激活基因Recombination activating gene 2)基因打靶载体,用于产生引入该载体的SCID样小型猪的方法,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,还未开发出治疗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,SCID猪模型可用于癌症研究、细胞移植和药物开发研究。重组激活基因2(下文称为“RAG2”)参与了与人类常染色体相关的SCID疾病(Villa, A., Santagata, S.,Bozzi, F., Imberti, L. & Notarangelo, L.D. Journal of clinical immunology 19,87-97 (1999))。RAG2中的突变导致B细胞和T细胞受损,而且因为RAG2对于V(D)J重排很重要,因此RAG2中的突变破坏适应性免疫系统(Shinkai, Y. et al. Cell 68, 855-867(1992))。[专利文献] 专利文献1:韩国公开专利公开号1020040074108
发明内容
【技术问题】为了解决传统问题,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,本发明提供了一种具有重组激活基因(RAG)2的等位基因突变的SCID样小型猪的生产方法,其中所述生产方法包括:使用转录激活样效应因子核酸酶(TALEN)处理猪(Sus scrofa)的染色体2上的由SEQ ID NO:1所示的TALEN识别序列位点,来诱导等位基因突变;以及,使用具有诱导的等位基因突变的细胞,通过体细胞核移植产生突变体胚胎。根据本发明的实施方式,优选通过使用在编码TAL效应因子DNA核酸酶的载体类型中的TALEN转染相关细胞,来诱导等位基因突变,但本发明并不限于此。此外,本发明提供了具有重组激活基因2的等位基因突变的SCID样小型猪,该SCID样小型猪是通过本发明的生产方法产生的。此外,本发明提供了一种分选RFP、GFP和H-2KK阳性细胞作为重组激活基因(RAG)2中靶细胞的方法,其中该方法包括:将编码TAL效应因子DNA核酸酶的载体与报告载体一起引入到细胞中,其中所述编码TAL效应因子DNA核酸酶的载体能通过识别猪(Sus scrofa)的染色体2上的由SEQ ID NO: 1所示的TALEN识别序列,诱导相关基因区域或周围位点的突变;所述报告载体包括单体红色荧光蛋白(RFP)基因、SEQ ID NO: 1所示的可编程的核酸酶靶向序列、增强型绿色荧光蛋白(GFP)基因和H-2KK基因。根据本发明的实施方式,优选通过抗体进行H-2KK的检测,但本发明并不限于此。根据本发明的另一实施方式,优选通过流式细胞术检测RFP和GFP的表达,但本发明并不限于此。此外,本发明提供了一种具有诱导的等位基因突变的细胞,该等位基因突变是通过使用转录激活样效应因子核酸酶(TALEN)处理猪(Sus scrofa)的染色体2上的由SEQ ID NO:1所示的TALEN识别序列区域来诱导的。上述细胞于2013年10月2日保藏在位于韩国大田市儒城区的韩国生物科学和生物技术研究所的基因库(韩国典型菌种保藏中心)中,保藏号为KCTC 12496BP。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)介导的基因打靶,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RAG2,设计出特定的TALEN并且由ToolGen(韩国首尔)合成。设计TALEN,以在RAG2的外显子2上产生突变(图1a)。使用含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组和报告基因一起引入到HEK 293T细胞中(图1c),验证所设计的TALEN活性。验证之后,通过电穿孔,将编码TALEN和报告基因的构建体引入到猪成纤维细胞中。48小时之后,分选出GFP阳性的细胞(图4),并且铺在96孔板中;一个孔中有一个细胞。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剪切的52个位点两侧的小PCR片段(约400 bp)进行的测序示出,在RAG2组中存在TALEN诱导的插入/缺失(indel);筛选了总共57个克隆的RAG2(参见下面表1中的引物信息)。对于RAG2,基因打靶的效率是42.1%(24/57)(图1d)。在使用RAG2 TALEN诱导的细胞中,我们观察到14%(8/57)的双等位基因改变。对于RAG2,基因打靶的效率是42.1%(24/57)。在本发明中使用的TALEN对打靶区域是特异性的,因此我们没有观察到任何脱靶剪切(图6和表2)。使用得到证实的中靶细胞进行体细胞核移植(SCNT)(图7)。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。除了3只死的RAG2突变体之外,生出13只RAG2突变体(图1d)。有趣的是,发明人从一个细胞克隆中产生了携带RAG2的单等位基因或双等位基因改变的RAG2突变体。具体地,所有RAG2基因工程猪都在一个等位基因上具有相同的改变,而一些猪在另一等位基因上具有另一改变。单等位基因突变体由于丢失了三个核苷酸而具有一个氨基酸缺失和一个氨基酸替换(RAG2+/△140, S141H);而双等位基因突变体具有相同的改变,另外,因为缺少了14个核苷酸而在另一等位基因上形成了提前终止密码子(RAG2△140,S141H/△140-527)。对RAG2双等位基因突变体(RAG2Δ140,S141H/Δ140-527)的基因分型表明突变体可能缺乏功能性RAG2,因为丢失和被替换的氨基酸在各物种的RAG2的β螺旋区域中是高度保守的(图9)。在第17天,处死野生型、RAG2单等位基因突变体和双等位基因突变体,并且进行尸检。虽然在RAG2Δ140,S141H/Δ140-527猪中没有检测到胸腺,但是在RAG2+/Δ140, S141H猪中检测到大小与野生型类似的胸腺(图2a)。此外,双等位基因突变体的脾脏的大小小于对照猪的脾脏(图2b和图2c)。该结果与小鼠中观察到的结果一致,表明在RAG2+/Δ140,S141H猪中检测到免疫缺陷。对RAG2Δ140,S141H/Δ140-527猪脾脏的免疫组织化学(IHC)和苏木精和伊红(H & E)染色,显示出白髓明显发育不全,缺乏生发中心和外周淋巴鞘(PALS)的形成,并罕有B细胞和T细胞(图2d和图10)。另一方面,来自RAG2+/Δ140,S141H猪的B细胞和T细胞的水平是正常的。类似地,根据使用脾白细胞进行的流式细胞术示出,成熟的B细胞和T细胞在RAG2Δ140,S141H/Δ140-527猪中是不足的(图2e)。在4周的监控过程中,发明人观察到体重增加在野生型、RAG2单等位基因突变体和RAG2双等位基因突变体之间存在差异。所有猪在两周中都显示出类似的体重增加,但是RAG2Δ140,S141H/Δ140-527猪的体重达到稳定期(图2f)。四周之后,RAG2双等位基因突变体的体重小于单等位基因突变体的体重。这些动物生长在非特定灭菌(SPF)的房屋中,RAG2Δ140,S141H/Δ140-527猪最后在第29天死亡。剩余的RAG2单等位基因突变体是健康的且生长正常。在本发明中,发明人发现通过TALEN介导的基因打靶,能有效生产由SCNT制备的SCID猪。通过使用TALEN构建体与报告载体,能高效率地诱导突变。与传统的基因打靶(其中打靶载体整合到基因组中)不同,上述途径使我们能产生基因工程猪,同时不会在基因组中留下任何特征。根据本发明产生的基因工程猪可用作SCID研究的模型,该SCID研究的模型包括能显示出人类欧门(Omenn)综合征的第一猪模型(猪可获自美国国家猪资源和研究中心(National Swine Resource and Research Center),http://nsrrc.missouri.edu/)。为了测试RAG2Δ140,S141H/Δ140-527猪是否支持人类iPSC的增殖和分化,我们将来自多潜能细胞系的细胞s.c.注射到猪中,以确定细胞是否会引起畸胎瘤,其中该多潜能细胞系已从人类脐带成纤维细胞通过使用非整合质粒载体进行重编程而生成。在注射之前,细胞已经是碱性磷酸酶阳性的,并且表达多能标记物[POU类5同源框1(POU class 5 homeobox 1,POU5F1)、Nanog同源框(NANOG)、阶段特异性胚胎抗原3(SSEA-3)](图12中C~E)。因为RAG2Δ140,S141H/Δ140-527猪是免疫功能不全的,因此用于本实验的第二组新生猪崽独立饲养,与动物接触的所有人员都穿戴个人防护装备(PPE)以使病原体暴露最小化。在实验的第1天,将5百万或1千万个细胞s.c.注射到接近右耳底部处,和动物的左侧腹(双等位基因猪,n=3;单等位基因猪,n=2)。一只双等位基因猪崽第7天因为未知原因死亡,并且没有示出形成肿瘤的任何迹象,但是另外两只在两个注射位点的每一个位点都形成了肿瘤。肿瘤的形成与SCID小鼠中通常很多周的时间跨度相比,迅速发生。在一只接受了1千万个细胞注射的双等位基因突变体猪的耳位点下,在第12天观察到可触摸的肿瘤(图14中A和图13)。在第28天,该肿瘤是囊包裹的、实体且部分囊性的(大小3.4 x 2.5 cm;重量,3.6 g)(图14中A和B),而且似乎对宿主还没有带来不良影响。尸检时,将它切成5块,并且通过H&E染色检验组织切片。每个肿瘤切片都含有组织的紊乱混合物(图14中C)。横纹肌(中胚层)(图14中C和E)特别明显,并且在整个肿瘤中形成了随机分布的岛。其他多个区域包括组成腺状结构的上皮样细胞。它们包括分泌性上皮:即,具有杆状细胞的上皮(内胚层)和神经上皮(外胚层)。通过使用分别抗CTNNB1和VWF[-连环蛋白和血管性假血友病因子(VWF):内胚层],DES和ACTG2(肌间线蛋白和平滑肌肌动蛋白:中胚层),以及GFAP和ENO2(胶质原纤维酸性蛋白和神经元特异性烯醇化酶:外胚层)的抗体进行免疫组织化学分析,证实一般的组织学评价。还注意到对造血谱系的标记物呈阳性的细胞和多潜能细胞(图17)。在7.5周,第二只双等位基因突变体猪在耳部位(其中注射了5百万个细胞)和侧腹(其中注射了1千万个细胞)处形成生长较慢且较小的肿瘤(0.75 g和0.35 g)。在第二只猪接近左侧腹注射位点的腹膜腔中,发现了额外的肿瘤。在第二只猪的肿瘤中观察到成比例减少的肌肉组织和增多的囊性结构(图15中A),但是仍然存在所有三种胚层的衍生物(图15中B~D)。畸胎瘤特征上的这些差异不能归因于iPSC来源,因为每只猪都使用了相同的细胞。肿瘤还清楚地是人类来源,而不是猪来源的,因为从人类iPSC品系及其衍生的畸胎瘤的DNA中成功扩增出人类特异性MFN1(线粒体融合蛋白-1)的扩增子,但从突变体猪的DNA中没有扩增出人类特异性MFN1(线粒体融合蛋白-1)的扩增子(图16)。为了探究RAG2Δ140,S141H/Δ140-527猪是否能接受同种异体的猪细胞,我们注射了之前推测为猪的滋养层干细胞的细胞,所述细胞在裸鼠中已产生主要由包裹的上皮层和接近外周的横纹肌组织岛组成的实体瘤。在第17天,这些相同的细胞在猪中产生了在某种程度上类似的包在囊中的肿瘤。与来自小鼠的肿瘤类似,该猪中的肿瘤主要由上皮组织层组成。【有益效果】显示出重症综合性免疫缺陷(SCID)表型的猪有助于干细胞治疗、癌症研究和异种移植开发。发明人描述了通过TALEN介导的打靶产生两种类型的SCID猪和RAG2敲除。RAG2单等位基因突变导致得到白髓发育不全证实的免疫缺陷,显示出未检测到的胸腺以及缺乏B细胞和T细胞。
附图说明
图1示出使用TALEN产生SCID猪的示意图。其中,(a)示出TALEN介导的RAG2敲除;(b)示出了包括TALEN识别结构域的供体报告载体,其中供体报告载体中的TALEN识别结构域的剪切诱导GFP表达;(c)示出对体外设计的TALEN的验证,其中仅当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;以及(d)示出基因工程猪的基因分型。图2示出显示出欧门(Omenn)综合征的SCID猪的特征。其中,(a)示出在RAG2双等位基因突变体中没有胸腺;(b)示出野生型猪、单等位基因突变体猪和双等位基因突变体猪的脾脏大小,其中与野生型和单等位基因突变体相比,RAG2双等位基因突变体具有较小的脾脏;(c)示出野生型猪、单等位基因突变体猪和双等位基因突变体猪的脾脏重量,其中与其它类型的猪相比,RAG2双等位基因突变体具有较轻的脾脏;(d)示出双等位基因突变体猪中的SCID表型,其中根据对脾脏中的CD79A(B细胞)和CD3(T细胞)进行IHC染色,可证实RAG2双等位基因突变体罕有B细胞和T细胞;在野生型和单等位基因突变体中的生发中心周围观察到B细胞;并且在PALS中主要观察到T细胞;但是双等位基因突变体中B细胞或T细胞的数量大大减少,而且没有观察到成簇的这些细胞,而仅以单个细胞稀少地分布在脾脏基质中;(e)流式细胞术的结果示出,与野生型猪相比,RAG2双等位基因突变体的脾脏中的B细胞(CD21)和T细胞(CD3)显著降低。仅来自双等位基因突变体的脾细胞的2%~3%对B细胞或T细胞标记物呈阳性,大约相当于使用同种型对照抗体获得的荧光;以及(f)对照、单等位基因突变体猪和双等位基因突变体猪的体重。RAG2双等位基因突变体的体重在两周后停止增加。图3示出用于富集经TALEN打靶的细胞的代孕报告载体(surrogate report vector)的构建。(a)该报告载体由单体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基因组成。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,则仅表达RFP基因。当通过可编程的核酸酶在靶向序列中引入双链切口时,则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在同一阅读框中,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)分选的mRFP+eGFP+H-2KK+细胞中,富集了核酸酶诱导的突变。在细胞内,示出了报告质粒和染色体靶位点。突变由图中的黑点表示。图4示出在引入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5示出使用TALEN对RAG2进行的单等位基因改变或双等位基因改变。示出在TALEN结合位点两侧的PCR产物。在几种情况中,在野生型大小处没有PCR条带,清楚地表明RAG2发生了双等位基因改变(泳道14)。将PCR产物加载到2.0%的琼脂糖凝胶上。图6示出对RAG2突变猪的RAG2基因的脱靶分析。(a)顶部)对异源双链DNA进行Surveyor核酸酶消化示出,在与RAG2基因同源性最高的7个基因座中没有额外的脱靶突变;SM:分子量标记物,泳道1:MAN2A1;泳道2:FAM82A; 泳道3:MTDH;泳道4:TRIM31;泳道5:PCSK2;泳道6:PRKACB;泳道7:GCFC2。底部)以排除脱靶突变的RAG2-相关序列的基因和序列同源性。大写字母:TALEN结合位点;小写字母:TALEN剪切位点;同源碱基对为红色。图7示出用于鉴定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,然后使用细胞克隆进行SCNT。图8示出本发明产生的SCID猪模型的图像,其中猪的遗传背景是明尼苏达小型猪(Minnesota miniature pig)。图9示出在RAG2保守结构域上的突变。(a)来自RAG2突变的预测的蛋白序列。14 bp的缺失产生提前终止密码子。(b)来自RAG2突变的预测的蛋白序列。丢失3个碱基对产生一个氨基酸的缺失和错配。(c)被改变的氨基酸在不同物种中高度保守。这表明突变体可能具有无功能的RAG2。图10示出在RAG2双突变体中脾脏的发育缺陷。来自双等位基因突变体的H&E染色切片显示出显著的白髓发育不全,缺乏生发中心和外周淋巴鞘(PALS)的形成。图11示出四周之后的RAG2双等位基因突变体和RAG2单等位基因突变体的图像。RAG2双等位基因突变体的体重在两周之后停止增加。四周之后,RAG2双等位基因突变体猪和RAG2单等位基因突变体猪在表型上存在明显差异。图12示出使用附加型质粒生成的人类iPSC。(A)在质粒转染至脐带外生长物(umbilical cord outgrowth)后14天,iPSC克隆的相差图像。(B)分离的iPSC克隆在无饲养层培养条件下的图像。(C~E)在细胞中表达多能核蛋白POU5F1(C)、NANOG(D)和细胞表面分子SSEA-4(E)。下图示出使用4',6-二脒基-2-苯基吲哚(DAPI)进行的核染色。A & B中,比例尺= 200 μm;E中,比例尺= 100 μm. 图13示出RAG2突变体猪中的畸胎瘤生长的进展。(a)猪接受了1千万个细胞。在注射后约12~14天,检测注射位点上的生长。(b)猪接受了5百万个细胞。在注射后5周之后,检测生长。箭头指示潜在的畸胎瘤生长的位点。在RAG2单等位基因突变体中没有观察到生长。图14示出RAG2Δ140,S141H/Δ140-527猪中的畸胎瘤形成。(A)在耳下注射1千万个细胞之后,第16天(上插图,红色箭头)和4周(下插图)肿瘤的外视图。(B)从注射位点收集的囊包裹的肿瘤的总体形态学。(C~L)肿瘤的组织学检验。切片经H&E(C~F从耳部的肿瘤收集)染色,并且对一系列诊断抗原进行免疫组织化学(G~L)。C是组织的放大倍数较低的视图。(比例尺:200 μm)图15示出对形成在第二只RAG2Δ140,S141H/Δ140-527猪的侧腹区域上的畸胎瘤的组织学分析,其中肿瘤发展较慢。肿瘤切片包括组织的紊乱混合物,与在第一只突变体猪的畸胎瘤中所观察到,该组织具有成比例较少的肌肉组织和较多的囊性结构。(a)畸胎瘤切片的放大倍数较小的视图;(b~d)例示了三种胚层存在的代表性切片(B内胚层;C中胚层;D外胚层)。比例尺= 100 μm。图16示出畸胎瘤的人类来源。使用PCR扩增人类特异的线粒体融合蛋白1(MFN1)的片段。人类扩增子的预期大小是236 bp。1,来自人类iPSC #1品系的基因组DNA;2,来自人类iPSC #2品系的基因组DNA;3,来自用作受体的RAG2Δ140,S141H/Δ140-527猪的基因组DNA;4,来自畸胎瘤的基因组DNA;(-),-阴性对照(没有DNA)。图17示出使用造血标记物进行的染色。(a、b)畸胎瘤内存在造血标记物阳性细胞。(c)畸胎瘤中存在未分化的POU5F1+细胞(绿色荧光,POU5F1;蓝色,DNA的DAPI染色)。
具体实施方式
下面,将参考非限制性实施例,更详细地描述本发明。但是下面的实施例仅是用于例示本发明,并不应理解为限制本发明的范围。本发明中使用的所有动物和实验都获得了位于密苏里大学(University of Missouri)的动物伦理委员(Animal Ethics 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TALEN构建体与报告载体转染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,用所述构建体对细胞进行电穿孔。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GFP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。在引入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,然后使用基因组DNA进行PCR。进行扩增的PCR条件为:在95℃进行初始变性2 min,然后进行32个由在94℃变性30sec、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)购买了来自母猪的卵母细胞。在成熟培养基(具有2.9 mM Hepes、5 μg/ml胰岛素、10 ng/mlEGF、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,并且在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。然后,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 sec,在融合介质(0.3 M 甘露醇、0.1 mM CaCl2、0.1 mM MgCl2、0.5 mM Hepes)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 μM scriptaid(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冲洗胚胎,并且在存在10 ng/ml CSF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,使用固定在具有10%福尔马林的中性缓冲液中的组织。将组织包埋在用于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红色原(Romulin RedChromogens)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ZeissAxiophot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,BD Biosciences)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,将连接FITC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℃进行培养。然后,将细胞冲洗两次,并且重悬在新鲜的染色缓冲液中。在密苏里大学的细胞和免疫生物学核心设施(Cell and Immunobiology Core Facility),使用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(BeckmanCoulter)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(表2和表3)测试脱靶事件。PCR扩增之后,将300~500 ng 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。实施例6:TUNEL测定组织在含4%(w/v)多聚甲醛的0.01 M PBS(pH 7.4)中进行固定,在PBS中冲洗,在乙醇(70%、90%和100%)中进行脱水,并且包埋在石蜡中。在进行TUNEL染色之前,切片(6μm)进行再水化(二甲苯 5 min;乙醇100%、95%、70%,每次2 min)并在蒸馏水中冲洗。在室温,将切片与蛋白酶K(10mM Tris/HCl中的20 g/ml,pH 7.5)孵育30 min。在室温,将切片在保湿室中与TUNEL混合物(原位细胞死亡检测试剂盒(In situ Cell Death Detection kit), Roche, 瑞士(Swiss))孵育10min。经过PBS冲洗三次之后,将载玻片安置在含DAPI的VECTASHIELD封片剂(VECTASHIELDMounting Media)(VECTOR, USA)中。实施例7:细胞增殖测定在24 h和48 h之后,通过对培养物中的细胞数目进行计数,测量来源于野生型猪、RAG2单等位基因猪和RAG2双等位基因猪的成纤维细胞的细胞增殖。将细胞以1x104个细胞/孔接种在包被有层粘连蛋白的12孔板上。对0、24 h和48 h时的每一孔中的细胞的数目进行计数。使用0.4%台盼蓝染料(Bio-Rad)对细胞进行染色,以验证它们的存活力。使用TC10自动细胞计数器(Bio-Rad),测量每一时间点的细胞数目;使用来自每只猪的三份独立样品。通过使用统计分析系统(StatisticalAnalysis System,SAS Institute, 卡瑞(Cary), NC),将细胞数目在24 h和48 h时的差异进行对比。实施例8:人类脐带成纤维细胞的衍生在大学医院(密苏里大学,哥伦比亚, MO(University of Missouri, Columbia, MO))中,无菌地收集新鲜的人类脐带组织。该组织收集(项目#1201132)得到了密苏里大学健康科学机构审查委员会(University ofMissouri Health Sciences Institutional Review Board)的批准。使用磷酸盐缓冲液(PBS)对组织冲洗两次或更多次,以去除血细胞,并且在DMEM培养基中用剪刀剪成12 mm3的碎块,以通过组织块贴壁法排出贴壁细胞。将成碎块的组织放置在包被有0.1%明胶的48孔板(1块/孔)中的含10% FBS、1%非必需氨基酸、2mM谷氨酰胺、0.1mM 2-巯基乙醇和4 ng/mlFGF2的DMEM培养基(Thermo)中,然后培养在37℃,含4% O2/5% CO2/91% N2的潮湿空气的培养箱中。在最初的5~7天,保持培养物不受干扰,并且补充Primocin(InvivoGen, 圣地亚哥(San Diego), CA),以降低在原代培养物中生长细菌和真菌的风险。之后,每两天更换新的没有Primocin或其它抗生素的培养基,直至成纤维贴壁细胞从组织碎片中出来,生长在孔中。在培养1周后,成纤维细胞的生长物开始出现在切碎的组织的边缘。经过10~11天,使用TrypLE(Invitrogen),将成纤维细胞从48孔板中传代到T25培养瓶中。经过约14天,培养瓶中的细胞达到融合,并且经扩充以用于进行转化成iPSC的重编程。实施例9:使用附加型载 体从脐带成纤维细胞生成iPSC利用Okita等开发的方案使成纤维细胞进行重编程,该方案中除了通用的重编程基因POU5F1、SOX2、KLF4和LIN-28之外,还使用携带用于p53抑制的shRNA和非转化L-MYC的附加型载体。按照制造商的说明书,使用Nucleofector II装置(Lonza, 巴塞尔(Basel), 瑞士(Switzerland))和Amaxa NHDF Nucleofector试剂盒(Lonza),将3 μg附加型质粒的Y4组合电穿孔到6×105个细胞中。使用装置中的电穿孔程序“U-020”。通过在上述条件下进行培养,使细胞能在2~4天中恢复。将细胞(2×105)放置在100 mm提前包被了基质胶(Matrigel)(BD Bioscience, 圣何塞(San Jose), CA)的培养皿中。第二天,将培养基替换成mTeSR1 (StemCell Technologies, 温哥华(Vancouver), 加拿大(Canada))。在转导后约14天,出现类似人类ESC的克隆;在约第20天,人工分离克隆,并且扩张到在Matrigel上的无饲养层的培养条件中。实施例10:iPSC的免疫组织化学检验使用配备有数字照像机Coolpix 5000(Nikon, 梅尔维尔(Melville), NY)的Olympus CKX41倒置显微镜,捕获iPSC的图像。对于免疫荧光分析,使细胞生长在包被有Matrigel的盖玻片上。在4%多聚甲醛/PBS中固定10 min之后,在1.0% Triton X-100/PBS中透化30 min,将盖玻片放置在PBS中的5%山羊血清/5% BSA中,保持1h。接着,将细胞与适当稀释的初级抗体POU5F1(1:200, sc-5279, Santa Cruz Biotechnology)、NANOG(1:100, ab109250,Abcam)和SSEA4 (1:100, #4755p, Cell Signaling Technology)在4℃孵育过夜,之后与Alexa Fluor 568或488标记的山羊抗小鼠或山羊抗兔抗体(1:500)孵育。使用配备有ORCA-AG CCD照像机(http://www.biotech.missouri.edu/mcc/Olympus.html)的Olympus IX70倒置显微镜,捕获图像。实施例11:畸胎瘤的形成在第一天,将来自两个个体的两个人类iPSC品系(传代数在4至9之间),以0.2ml的体积(25% Matrigel溶液)皮下注射(5百万个细胞/位点或1千万个细胞/位点)到5只猪中,这5只猪中,有3只具有RAG2双等位基因改变的猪,2只具有RAG2单等位基因改变的猪作为对照。通过分散酶(StemCell Technologies)和刮擦,分离培养的iPSC。离心(200×g, 5 min)之后,使用0.1 ml mTeSR1培养基重悬细胞沉淀块,并且与相同体积的50% Matrigel混合。然后,将细胞在冰上冷冻,并且加载到1ml注射器(BD, 富兰克林湖(Franklin Lakes), NJ)中,通过22号针头注射到每只猪的两个位点(一个位点是耳朵和一个位点是侧腹)中。随后解剖取出肿瘤,并且固定在10%(v/v)中性缓冲福尔马林中。对经石蜡包埋的组织进行切片,然后使用苏木精和伊红(H&E)进行染色。将猪iPSC(iTR)产生的表达滋养层表型的猪细胞移植到RAG2双等位基因突变体猪的其中一只中。通过TrypLE和刮擦,分离iTR亚型品系(p29)的1千万个细胞。制备作为人类iPSC的细胞悬浮液,并且皮下注射到左耳中。以盲转方式进行细胞移植程序,其中进行该程序的个体不知道猪的基因状态。实施例12:畸胎瘤的免疫组织化学分析对于免疫组织化学(IHC),使组织在含10%福尔马林的中性缓冲液(Fisher, 99-909-14)中固定,包埋在石蜡中,并且将制备的切片(5 μm)放置的玻璃载玻片上。首先,通过在3%过氧化氢酶中对组织切片处理1小时,阻断内源性过氧化物酶活性。然后,使用Borg Decloaker(Biocare Medical, CA)溶液预处理样品用于抗原修复,然后在Background Sniper(Biocare Medical, CA)溶液中进行封闭。冲洗之后,将样品与初级抗体(表4)孵育。孵育之后,冲洗样品,并且与辣根过氧化物酶(HRP)缀合的二级抗体孵育。利用EnVisionTM+系统(Dako, 卡平特里亚(Carpinteria),CA)进行检测。使用3, 3-二氨基联苯胺(DAB)或Romulin AEC色原(Romulin AECChromogen)(Biocare Medical, 康科德(Concord), CA)使信号可视化。还使用IP FLX苏木精对样品进行染色,以提供背景。使用配备有Olympus DP70高分辨率数字显微照像机(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。Borg、Sniper、Romulin红和IP FLX苏木精都购自Biocare(Concord, CA)。实施例13:畸胎瘤的来源为了鉴定畸胎瘤的来源,使用PCR扩增人类特异的线粒体融合蛋白1的基因(MFN1)。使用DNeasy血液和组织试剂盒(DNeasyBlood and Tissue kit)(Qiagen, USA),从人类iPSC、畸胎瘤和携带畸胎瘤的RAG2突变体猪的尾巴的血液中分离基因组DNA。PCR扩增的条件是:在96℃进行初始变性2 min,然后进行32个由在95℃变性30 sec、52℃退火30 sec和72℃延伸30 min组成的循环。将PCR产物加载在2.5%的琼脂糖凝胶上。PCR产物的预期大小是236bp。用于分析的引物是:F:GCTGGCTAAGAAGGCGATTA(SEQ ID NO. 2) 和R: TCCCCTTCTGGAGGTTAGAAA(SEQ ID NO. 3)。
【表1】
表1示出用于对TALEN诱导的RAG2突变体进行基因分型的引物。
【表2】
表2示出了用于鉴定猪RAG2的脱靶的引物对。
【表3】
表3示出本发明的核移植效率。【表4】
表4是用于IHC的抗体。[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:KCTC 12496
保藏日期:20131002
Claims (4)
1.一种具有重组激活基因(RAG)2的双等位基因突变的重症综合性免疫缺陷(SCID)样小型猪的生产方法,所述生产方法包括:
使用转录激活样效应因子核酸酶(TALEN)处理猪的染色体2上的由SEQ ID NO:1所示的TALEN识别序列区域,来诱导双等位基因突变;以及
使用具有诱导的双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。
2.根据权利要求1所述的生产方法,其中,所述生产方法通过使用在编码TAL效应因子DNA核酸酶的载体类型中的TALEN转染相关细胞来进行。
3.一种具有诱导的双等位基因突变的细胞,其中,所述双等位基因突变是通过使用转录激活样效应因子核酸酶(TALEN)处理猪的染色体2上的由SEQ ID NO:1所示的TALEN识别序列区域来诱导的。
4.根据权利要求3所述的细胞,其中,所述细胞是RAG2敲除的小型猪的成纤维细胞系KCTC12496。
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