CN101835890B - 有效建立诱导的多能干细胞的方法 - Google Patents
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Abstract
本发明提供了改善诱导的多能干(iPS)细胞建立效率的方法,其包括在体细胞核重编程步骤中抑制p53功能。p53功能的抑制通过使选自下述的物质与体细胞接触等来达到:(1)p53的化学抑制剂、(2)p53的显性失活突变体和其编码核酸、(3)针对p53的siRNA和shRNA以及其编码DNA、和(4)p53途径抑制剂。本发明还提供了用于改善iPS细胞建立效率的试剂,所述试剂包含p53功能抑制剂,特别是(1)p53的化学抑制剂、(2)p53的显性失活突变体和其编码核酸、(3)针对p53的siRNA和shRNA以及其编码DNA、和(4)p53途径抑制剂。本发明进一步提供了产生iPS细胞的方法,其包括使核重编程物质和p53功能抑制剂与体细胞接触。
Description
发明技术领域
本发明涉及改善诱导的多能干细胞(下文称为iPS)建立效率的方法和为此的药物。更具体而言,本发明涉及通过在体细胞核重编程步骤中抑制p53功能来改善iPS细胞建立效率的方法,和利用p53功能抑制剂作为活性成分的用于改善iPS细胞建立效率的试剂。
发明背景
近年来,小鼠和人iPS细胞已相继建立。Yamanaka等人通过下列诱导iPS细胞:将Oct3/4、Sox2、Klf4和c-Myc基因引入得自报道小鼠(reportermouse)的成纤维细胞内,其中将新霉素抗性基因敲入Fbx15基因座内,并且迫使细胞表达该基因(1,2)。Okita等人(3)通过下列成功地建立了iPS细胞(Nanog iPS细胞),所述iPS细胞显示与胚胎干(ES)细胞中的那些几乎相同的基因表达和后生修饰:产生转基因小鼠,其中绿色荧光蛋白(GFP)和嘌呤霉素抗性基因整合到Nanog的基因座内,它们的表达比Fbx15表达更集中于多能细胞中,迫使得自小鼠的成纤维细胞表达上述4种基因,以及选择嘌呤霉素抗性和GFP阳性的细胞。相似结果也由其他小组确认(4,5)。其后,揭示iPS细胞也可以利用除c-Myc基因之外的3种因子来产生(6)。
此外,Yamanaka等人通过将与小鼠中所使用的那些相同的4种基因引入人皮肤成纤维细胞内成功建立了iPS细胞(1,7)。另一方面,Thomson等人的小组使用Nanog和Lin28代替Klf4和c-Myc产生了人iPS细胞(8,9)。Park等人(10)使用TERT和SV40大T抗原加上4种因子Oct3/4、Sox2、Klf4和c-Myc产生了人iPS细胞,其中所述TERT被认为是人细胞无限增殖基因。因此,已证实在通过将所限定的因子引入到体细胞内在人和小鼠二者中所能够产生的多能性方面,iPS细胞与ES细胞相当。
然而,iPS细胞建立效率低至小于1%。特别地,当通过引入除c-Myc外的3种因子(Oct3/4、Sox2和Klf4)而产生它们时,会出现极低效率的iPS细胞建立的问题,其中,担心c-Myc在组织中会引起肿瘤发生或由iPS细胞分化的个体成为体细胞。
提及的参考文献:
1.WO 2007/069666 A1
2.Takahashi,K.和Yamanaka,S.,Cell,126:663-676(2006)
3.Okita,K.等人,Nature,448:313-317(2007)
4.Wernig,M.等人,Nature,448:318-324(2007)
5.Maherali,N.等人,Cell Stem Cell,1:55-70(2007)
6.Nakagawa,M.等人,Nat.Biotethnol.,26:101-106(2008)
7.Takahashi,K.等人,Cell,131:861-872(2007)
8.WO 2008/118820 A2
9.Yu,J.等人,Science,318:1917-1920(2007)
10.Park,I.H.等人,Nature,451:141-146(2008)
发明概述
本发明的目的是提供改善iPS细胞建立效率的手段;本发明的另一个目的是提供使用该手段有效产生iPS细胞的方法。
本发明人以实现上述目的为目标进行了广泛研究,并且发现通过在体细胞核重编程步骤中抑制p53功能,可以显著增加iPS细胞建立效率。该效应在人细胞中特别显著。此外,通过抑制p53功能,即使用3种因子,也获得了比常规方法更接近于用4种因子的效率的建立效率。此外,本发明人通过缺失p53功能成功地容易地建立了iPS细胞,即使对于其照惯例认为难以建立iPS细胞的T淋巴细胞,并且已开发了本发明。
因此,本发明提供了:
[1]改善iPS细胞建立效率的方法,其包括在体细胞核重编程(nuclear reprogramming)步骤中抑制p53功能。
[2]根据上述[1]的方法,其中通过使p53的化学抑制剂与体细胞接触而抑制所述p53功能。
[3]根据上述[1]的方法,其中通过使p53的显性失活突变体(dominant negative mutant)或其编码核酸与体细胞接触而抑制所述p53功能。
[4]根据上述[1]的方法,其中通过使选自针对p53的siRNA和shRNA以及其编码DNA的核酸与体细胞接触而抑制所述p53功能。
[5]根据上述[1]的方法,其中通过使p53途径抑制剂与体细胞接触而抑制所述p53功能。
[6]用于改善iPS细胞建立效率的试剂,所述试剂包含p53功能抑制剂。
[7]根据上述[6]的试剂,其中所述抑制剂是化学抑制剂。
[8]根据上述[6]的试剂,其中所述抑制剂是p53的显性失活突变体或其编码核酸。
[9]根据上述[6]的试剂,其中所述抑制剂是选自针对p53的siRNA和shRNA以及其编码DNA的核酸。
[10]根据上述[6]的试剂,其中所述抑制剂是p53途径抑制剂。
[11]产生iPS细胞的方法,其包括使核重编程物质和p53功能抑制剂与体细胞接触。
[12]根据上述[11]的方法,其中所述抑制剂是化学抑制剂。
[13]根据上述[11]的方法,其中所述抑制剂是p53的显性失活突变体或其编码核酸。
[14]根据上述[11]的方法,其中所述抑制剂是选自针对p53的siRNA和shRNA以及其编码DNA的核酸。
[15]根据上述[11]的方法,其中所述抑制剂是p53途径抑制剂。
[16]根据上述[11]的方法,其中所述核重编程物质是Oct3/4、Klf4和Sox2,或其编码核酸。
[17]根据上述[11]的方法,其中所述核重编程物质是Oct3/4、Klf4、Sox2和c-Myc,或其编码核酸。
[18]根据上述[11]的方法,其中所述体细胞是T细胞。
[19]iPS细胞,其中T细胞抗原受体(TCR)基因得到重排,通过使T细胞重编程而获得所述iPS细胞。
[20]iPS细胞,其包含p53的显性失活突变体或编码针对p53的siRNA或shRNA的外源核酸。
因为p53功能抑制剂使得能够显著增加iPS细胞建立效率,所以这在借助于除c-Myc外的3种因子诱导iPS细胞中特别有用,对于这3种因子通常其建立效率极低。因为担心c-Myc在被再活化时引起肿瘤发生,所以使用这3种因子,iPS细胞建立效率的改善在将iPS细胞应用于再生医学中具有首要效用。
因为得自最终分化的T细胞的iPS细胞在其中具有已重排的TCR,所以iPS细胞可以用作T细胞免疫治疗剂,前提是iPS细胞由识别呈递特定抗原的细胞(例如,癌细胞、受感染细胞等),大量扩增,并且允许再分化成细胞毒性T细胞(CTL)的T细胞诱导。
附图简述
图1显示p53缺陷对iPS细胞建立的作用的检查结果。图1(A)和图1(B)分别显示引入4种因子(Oct3/4、Sox2、Klf4、c-Myc)诱导iPS细胞的结果,和引入3种因子(Oct3/4、Sox2、Klf4)诱导iPS细胞的结果。在该图中,“p53+/-”显示关于p53-杂合-缺陷(hetro-deficient)细胞(对照)的结果;“p53-/-”显示关于p53-纯合-缺陷(homo-deficient)细胞的结果。在该图中,纵坐标轴指示GFP阳性集落数目。每个图显示总共3次实验。
图2是显示得自用4种因子(Oct3/4,Sox2,Klf4,c-Myc)感染的p53-纯合-缺陷细胞的GFP阳性集落皮下注射给免疫缺陷小鼠并且形成畸胎瘤的照片。
图3(A)-(D)显示引入p53的显性失活突变体(p53P275S)对iPS细胞建立的作用的检查结果。图3(A)显示实验操作的概要。图3(B)和图3(C)分别显示引入4种因子和引入3种因子的结果。在该图中,“P275S”显示引入p53P275S的结果。在该图中,“DsRed”和“p53”显示分别引入DsRedExpress和无突变野生型p53代替p53P275S的结果。纵坐标轴指示GFP阳性集落数目。图3(D)显示与分别结果对应的集落照片。
图3(E)-(G)显示将p53引入p53-纯合-缺陷小鼠内对iPS细胞建立的作用的检查结果。图3(E)和图3(F)分别显示引入4种因子和3种因子的结果。在该图中,“DsRed”显示引入DsRedExpress的结果;“p53”显示引入野生型p53的结果。纵坐标轴指示GFP阳性集落数目。图3(G)显示与各自结果对应的集落照片。
图4显示用Pifithrinp——p53抑制剂处理对iPS细胞建立的作用的检查结果。图4(A)显示实验操作的概要;图4(B)显示实验结果。在图4(B)中,“DMSO”显示用DMSO(对照)处理的结果;“Pifithrinα,环状,对硝基”显示用Pifithrin处理的结果。纵坐标轴指示GFP阳性集落数目。
图5是显示起因于T细胞的ES样细胞是GFP阳性的照片图示,所述T细胞得自用4种因子(Oct3/4、Sox2、Klf4、c-Myc)感染的Nanog-GFP/Trp53-/-小鼠。左图:相差图像,右图:GFP阳性集落图像。在该图中,408E2、408E7和408E8指示集落编号。
图6是显示起因于T细胞的ES样细胞表达ES细胞特异性基因的RT-PCR的照片图示,所述T细胞得自用4种因子(Oct3/4、Sox2、Klf4、c-Myc)感染的Nanog-GFP/Trp53-/-小鼠。在该图中,Oct3/4至Zfp296是ES细胞标记,并且FasL至Ifhg是T细胞标记。Nat1和Trim28是阳性对照,并且Oct3/4Tg至c-Myc Tg证实引入的4种因子的表达。在该图中,“CD90+T”和“Spleen”指示分别充当用于iPS细胞引入的细胞来源的T细胞和脾;7B3和38D2分别指示Fbx15 iPS细胞(Nature 448,313-317(2007))和Nanog iPS细胞(Nature 448,313-317(2007))。
图7是显示起因于T细胞的ES样细胞对于ES细胞标记SSEA1和碱性磷酸酶阳性的照片图示,所述T细胞得自用4种因子(Oct3/4、Sox2、Klf4、c-Myc)感染的Nanog-GFP/Trp53-/-小鼠。
图8是显示通过使用AFP、GATA4、α-SMA、结蛋白、βIII-微管蛋白和GFAP抗体染色,证实起因于T细胞的ES样细胞具有分化成3个胚层的潜力的结果的照片图示,所述T细胞得自用4种因子(Oct3/4、Sox2、Klf4、c-Myc)感染的Nanog-GFP/Trp53-/-小鼠。
图9是执行DNA微阵列分析以测定在从p53-纯合-缺陷小鼠中分离的MEF和从普通、非p53-缺陷小鼠中分离的MEF之间在表达模式中是否存在差异的结果的图示。(A)检测出所有基因,(B)检测出仅在ES细胞中特异性表达的基因,(C)检测出仅在成纤维细胞(MEF)中特异性表达的基因。
图10显示起因于ES样细胞的成年嵌合小鼠,所述ES样细胞得自用4种因子(Oct3/4、Sox2、Klf4、c-Myc)感染的Nanog-GFP/Trp53-/-小鼠的T细胞。
图11是显示通过基因组PCR证实Tcrβ基因的V-(D)-J DNA重排的结果的照片图示。在该图中,“GL”指示种系(germline)条带。
图12显示通过4种或3种因子来自p53-无效MEF的iPS产生。(a)iPS细胞通过3种因子由Nanog-GFP报道MEF产生,所述MEF是p53野生型、杂合或纯合的。在逆转录病毒转导后,通过流式细胞仪收集5000个活细胞。在转导后28天计数GFP阳性集落,并且显示在图的顶部。显示了3次独立实验的数据。(b)iPS细胞通过3种因子由在96孔板的孔中的单次分选细胞产生。在转导后28天计数GFP阳性集落。显示了来自3次独立实验的数据。(c)iPS细胞通过4种因子包括c-Myc由在96孔板的孔中的单次分选细胞产生。在转导后21天计数GFP阳性集落。显示了来自3次独立实验的数据。
图13显示通过与野生型或突变型p53共转导的3种因子由p53杂合或纯合MEF产生的iPS细胞。(a)表达显性失活p53突变型(P275S)或野生型的逆转录病毒与3种因子一起共转导到Oct3/4-GFP、p53杂合MEF内。逆转录病毒转导后,收集5000个细胞,并且在转导后28天计数GFP阳性集落。显示了3次独立实验的数据。(b)表达野生型或突变型p53的逆转录病毒与3种因子一起共转导到Nanog-GFP、p53纯合MEF内。逆转录病毒转导后,收集5000个细胞,并且在转导后28天计数GFP阳性集落。显示了2次独立实验的数据。
图14-16显示得自p53杂合或纯合MEF的iPS细胞的表征。
图14显示通过3种或4种因子得自Nanog-GFP、p53-无效MEF的iPS细胞的相差图像(上面)和荧光图像(下面)。条指示100μm。
图15显示ES细胞标记基因、p53和4种因子的表达的RT-PCR分析。通过使用特定引物组,区分总表达、内源表达和4种因子的转基因表达。
图16显示得自具有3种(a)或4种(b)因子的p53-无效iPS细胞的畸胎瘤的组织学检查。(a)显示的是神经组织(左上)、软骨(右上)、肌肉(左下)和肠样上皮组织(右下)的苏木精-伊红染色。(b)显示的是未分化的细胞(上面)和神经组织(下面)的苏木精-伊红染色。
图17-20显示通过p53抑制增加的人iPS细胞产生效率。
图17显示突变型p53共转导对通过4种或3种因子来自HDF的iPS产生的作用。将表达P275S或DD的逆转录病毒连同4种或3种重编程因子一起转导到HDF内。显示的是通过4种因子((a)来自5x103HDF)和通过3种因子((b),来自4x104HDF)的iPS细胞集落数目。图17c显示得自人iPS细胞的畸胎瘤,所述人iPS细胞由3种重编程因子和p53DD突变型产生。显示的是神经组织(左上)、软骨(右上)、肌肉(左下)和肠样上皮组织(右下)的苏木精-伊红染色。
图18显示通过p53shRNA抑制p53产生。将关于p53shRNA或对照RNA的逆转录病毒载体转导到HDF内。转导后6天,通过Western印迹分析测定p53蛋白质水平。
图19显示p53shRNA共转导对通过4种因子来自HDF的iPS产生的作用。将表达p53shRNA或对照RNA的逆转录病毒载体连同4种重编程因子一起转导到HDF内。为了援救RNAi介导的敲降(knockdown),共引入关于小鼠p53的逆转录病毒载体。显示的是在4次实验中的iPS集落数目。
图20显示p53shRNA共转导对通过3种因子来自HDF的iPS产生的作用。将表达p53shRNA或对照RNA的逆转录病毒载体连同3种重编程因子一起转导到HDF内。为了援救RNAi介导的敲降,共引入关于小鼠p53的逆转录病毒载体。显示的是在2次实验中来自5x104HDF(a)或5x105HDF(b)的iPS集落数目。
图21显示MDM2共转导对通过4种或3种重编程因子来自HDF的iPS产生的作用。将表达MDM2、p53shRNA或RB shRNA的逆转录病毒载体,或对照载体连同4种因子(a)或3种因子(b)一起转导到HDF内。显示的是来自5x104HDF细胞的iPS集落数目。
图22显示证实在由iPS集落分化的细胞中内胚层(AFP)、中胚层(α-SMA)和外胚层(βIII-微管蛋白)分化标记的表达的照片。
图23显示在未分化细胞中(U)和在分化细胞中在胚状体形成后(D)的标记基因表达。“模拟”显示空载体(pMKO.1-puro)与3种重编程因子的共转导。“p53shRNA-2”显示p53shRNA-2与3种重编程因子的共转导。
图24显示p53抑制对p21和Myc的作用。将通过p53抑制调节的基因(4种增加的和7种减少的)连同4种重编程因子(a)或4种重编程因子和p53shRNA(b)一起引入HDF内。在转导后第24天(a)或第28天(b)时,计数iPS细胞集落数目。**;与对照DsRed(n=3)比较p<0.01。将包含p53或Myc的应答元件的萤光素酶报道物,或由聚合酶II启动子驱动的那种引入HDF内,连同模拟逆转录病毒载体、p53shRNA、4种重编程因子或缺乏Myc的3种因子。2天后,测定萤光素酶活性(c)。**;与模拟对照(n=3)比较p<0.01,*;p<0.05。
图25a显示实验操作的概要。图25b显示引入4种因子(Oct3/4,Sox2,K1f4,c-Myc)的结果。在该图中,“+/+”显示关于野生型细胞(对照)的结果;“-/-”显示关于p53-纯合-缺陷细胞的结果。在该图中,纵坐标轴指示GFP阳性集落数目。图25c显示质粒DNA整合到基因组内的检查结果(上图:基因组PCR;下图:Southern印迹分析)。图25d显示获得的细胞的照片(左上:相差图像,右上:GFP阳性集落图像,左下:相差图像和GFP阳性集落图像的合并)和起因于获得的ES样细胞的嵌合小鼠(右下图)。
发明详述
本发明提供了通过在体细胞核重编程步骤中抑制p53功能来改善iPS细胞建立效率的方法。抑制p53功能的手段的选择并无具体限制;优选地,可以提及其中使p53功能抑制剂与体细胞接触的方法。
如本文所提及的,“p53功能抑制剂”可以是任何物质,只要它能够抑制(a)p53蛋白质的功能或(b)p53基因的表达。即,不仅直接作用于p53蛋白质以抑制其功能的物质和直接作用于p53基因以抑制其表达的物质,而且作用于参与p53信号转导的因子以导致p53蛋白质功能或p53基因表达的抑制的物质,也包括在如本文提及的“p53功能抑制剂”的范围中。
抑制p53蛋白质功能的物质的例子包括但不限于,p53的化学抑制剂、p53的显性失活突变体或其编码核酸、抗p53拮抗性抗体或其编码核酸、包含p53应答元件的共有序列的诱饵(decoy)核酸、抑制p53途径的物质等。优选地,可以提及p53的化学抑制剂、p53的显性失活突变体或其编码核酸、和p53途径抑制剂。
(a1)p53的化学抑制剂
p53的化学抑制剂的例子包括但不限于,由公开于WO 00/44364中的pifithrin(PFT)-α和-β代表的p53抑制剂,公开于Storm等人(Nat.Chem.Biol.2,474(2006))中的PFT-μ、其类似物及其盐(例如,酸加成盐例如盐酸化物和氢溴化物等)等。其中,PFT-α及其类似物[2-(2-亚氨基-4,5,6,7-四氢苯并噻唑-3-基)-1-对甲苯乙酮,HBr(产品名:Pifithrin-α)和1-(4-硝基苯基)-2-(4,5,6,7-四氢-2-亚氨基3(2H)-苯并噻唑基)乙酮,HBr(产品名:Pifithrin-α,对硝基)],PFT-β及其类似物[2-(4-甲基苯基)咪唑并[2,1-b]-5,6,7,8-四氢苯并噻唑,HBr(产品名:Pifithrin-α,环状)和2-(4-硝基苯基)亚氨基[2,1-b]-5,6,7,8-四氢苯并噻唑(产品名:Pifithrin-α,对硝基,环状)]和PFT-μ[苯基乙炔基氨苯磺胺(产品名:Pifithrin-μ)]可从Merck商购获得。
p53的化学抑制剂与体细胞的接触可以这样执行:使抑制剂以合适浓度溶解于含水或非水溶剂中,将抑制剂的溶液加入适合于培养从人或小鼠中分离的体细胞的培养基中(例如,补充有约5-20%胎牛血清的最小必需培养基(MEM)、达尔贝科改良伊格尔培养基(DMEM)、RPMl1640培养基、199培养基、F12培养基等),从而使得抑制剂浓度将落入到完全抑制p53功能并且不引起细胞毒性的范围中,并且使细胞培养给定时间段。抑制剂浓度依赖所使用的抑制剂种类而变,并且适当时选择为约0.1nM-约100nM。接触的持续时间并无具体限制,只要它足以达到细胞的核重编程;通常,抑制剂可以允许在培养基中共存直至出现多能性标记阳性集落。
已知p53基因为肿瘤抑制基因;p53功能的持续抑制潜在增加癌发生的风险。p53的化学抑制剂非常有用,不仅因为仅通过加入培养基允许引入细胞内的优点,还因为在iPS细胞诱导后通过去除包含抑制剂的培养基,容易且快速地终止p53功能的抑制的能力。
(a2)p53的显性失活突变体
p53的显性失活突变体的选择并无具体限制,只要突变体能够针对在体细胞中内源表达的野生型p53蛋白质竞争作用,以抑制其功能;例如,可以提及p53P275S,起因于位于小鼠p53的DNA结合区的第275位(在人的情况下,第278位)处的脯氨酸至丝氨酸的点突变(deVries,A.,Proc.Natl.Acad.Sci.USA,99,2948-2953(2002));p53DD,起因于在小鼠p53的第14-301位(在人p53中,与第11-304位对应)处的氨基酸的缺失(Bowman,T.,Genes Develop.,10,826-835(1996))等。其他已知突变体包括例如,p53S58A,起因于在小鼠p53的第58位(在人的情况下,第61位)处的丝氨酸至丙氨酸的点突变;p53C135Y,起因于在人p53的第135位(在小鼠的情况下,第132位)处的半胱氨酸至酪氨酸的点突变;p53A135V,起因于在小鼠p53的第135位(在人的情况下,第138位)处的丙氨酸至缬氨酸的点突变;p53R172H,起因于在小鼠p53的第172位(在人的情况下,第175位)处的精氨酸至组氨酸的点突变;p53R270H,起因于在第270位(在人的情况下,第273位)处的精氨酸至组氨酸的点突变;p53D278N,起因于在小鼠p53的第278位(在人的情况下,第281位)处的天冬氨酸至天冬酰胺的点突变;这些可以以相同方式使用。
p53的显性失活突变体可以通过例如下文描述的技术获得。首先,基于由SEQ ID NO:1或3显示的小鼠或人p53cDNA序列信息合成作为探针或引物的合适寡核苷酸,并且由得自小鼠或人细胞或组织的mRNA、cDNA或cDNA文库使用杂交法或(RT)-PCR法克隆小鼠或人p53cDNA,并且亚克隆到合适质粒内。以其中待引入突变的位点的密码子(例如,在p53P275S的情况下,在由SEQ ID NO:1显示的核苷酸序列中由核苷酸编号951-953显示的cct)由编码另一种所需氨基酸的密码子(例如,在p53P275S的情况下,tct)替换的形式,合成包含该位点的引物,并且使用这种引物与掺入p53cDNA的质粒作为模板执行逆转录PCR,由此获得编码所需显性失活突变体的核酸。在缺失突变体如p53DD的情况下,可以设计在待缺失位点外的引物,并且如上所述执行反向PCR(inverse PCR)。通过将因此获得的编码显性失活突变体的核酸引入宿主细胞内,并且从培养细胞或其条件培养基中回收重组蛋白质,可以获得所需显性失活突变体。
显性失活突变体与体细胞的接触可以使用本身已知用于蛋白质转移到细胞内的方法来达到。此种方法包括例如,使用蛋白质转移试剂的方法,使用蛋白质转移结构域(PTD)-或细胞穿透肽(CPP)-融合蛋白的方法,显微注射法等。蛋白质转移试剂是商购可得的,包括基于阳离子脂质的BioPOTER Protein Delivery Reagent(Gene TherapySystems)、Pro-JectTM Protein Transfection Reagent(PIERCE)和ProVectin(IMGENEX);基于脂质的Profect-1(Targeting Systems);基于可透过膜的肽的Penetrain Peptide(Q biogene)和Chariot Kit(Active Motif),和基于HVJ包膜(灭活的仙台病毒)的GenomONE(Ishihara Sangyo)等。转移可以根据这些试剂所附的方案——如下所述的通用操作来达到。在合适溶剂(例如,缓冲溶液例如PBS或HEPES)中稀释p53的显性失活突变体,加入转移试剂,使混合物在室温下温育5-15分钟,以形成复合物,在将培养基更换成无血清培养基后,将这种复合物加入细胞中,并且使细胞在37℃下温育1至数小时。其后,去除培养基,并且替换成含血清培养基。
开发的PTD包括使用蛋白质的细胞穿透结构域的那些,例如果蝇衍生的AntP、HIV衍生的TAT(Frankel,A.等人,Cell 55,1189-93(1988)或Green,M.& Loewenstein,P.M.Cell 55,1179-88(1988))、穿膜肽(Penetratin)(Derossi,D.等人,J.Biol.Chem.269,10444-50(1994))、Buforin II(Park,C.B.等人Proc.Natl Acad.Sci.USA 97,8245-50(2000))、Transportan(Pooga,M.等人FASEB J.12,67-77(1998))、MAP(模型兼性肽)(Oehlke,J.等人Biochim.Biophys.Acta.1414,127-39(1998))、K-FGF(Lin,Y.Z.等人J.Biol.Chem.270,14255-14258(1995))、Ku70(Sawada,M.等人Nature Cell Biol.5,352-7(2003))、朊病毒(Lundberg,P.等人Biochem.Biophys.Res.Commun.299,85-90(2002))、pVEC(Elmquist,A.等人Exp.Cell Res.269,237-44(2001))、Pep-1(Morris,M.C.等人Nature Biotechnol.19,1173-6(2001))、Pep-7(Gao,C.等人Bioorg.Med.Chem.10,4057-65(2002))、SynBl(Rousselle,C.等人Mol.Pharmacol.57,679-86(2000))、HN-I(Hong,F.D.& Clayman,G L.Cancer Res.60,6551-6(2000))、和HSV衍生的VP22。得自PTD的CPP包括聚精氨酸例如11R(Cell Stem Cell,4:381-384(2009))和9R(Cell Stem Cell,doi:10.1016/j.stem.2009.05.005(2009))。制备掺入p53的显性失活突变体的cDNA和PTD或CPP序列的融合蛋白表达载体,以允许融合蛋白的重组表达,并且回收融合蛋白用于在转移中使用。这种转移可以如上所述达到,除不添加蛋白质转移试剂外。
在具有约1μm的尖端直径的玻璃针中放置蛋白质溶液并且将溶液注射到细胞内的方法——显微注射确保蛋白质转移到细胞内。
如上所述,p53功能的持续抑制潜在增加了癌发生的风险;然而,因为p53的显性失活突变体在转染细胞中经历经由蛋白酶的降解并且逐渐消失,相应恢复在细胞中内源发生的p53功能,所以突变蛋白质的使用在其中需要高安全性的情况下可以是合适的,如在其中获得的iPS细胞用于治疗目的的情况下。
(a3)编码p53的显性失活突变体的核酸
然而,考虑到容易引入体细胞内,p53的显性失活突变体可以以编码蛋白质的核酸,而不是蛋白质自身的形式使用。因此,在本发明的另一个优选实施方案的模式中,p53功能抑制剂是编码p53的显性失活突变体的核酸。核酸可以是DNA或RNA、或DNA/RNA嵌合体,并且优选DNA。核酸可以是双链或单链的。编码p53的显性失活突变体的cDNA可以通过上述就突变蛋白质制备而言描述的技术进行克隆。
将分离的cDNA插入合适的表达载体内,所述表达载体包含能够在靶体细胞中起作用的启动子。有用的表达载体包括例如病毒载体,例如逆转录病毒、慢病毒、腺病毒、腺伴随病毒、疱疹病毒和仙台病毒,用于在动物细胞中表达的质粒(例如,pAl-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo)等。适当时,所使用的各种载体可以根据获得的iPS细胞的预期用途进行选择。
在表达载体中所使用的有用的启动子的例子包括例如SRα启动子、SV40启动子、LTR启动子、CMV(巨细胞病毒)启动子、RSV(劳斯(Rous)肉瘤病毒)启动子、MoMuLV(莫洛尼(Moloney)小鼠白血病病毒)LTR、HSV-TK(单纯疱疹病毒胸苷激酶)启动子、EF-α启动子、CAG启动子等。优先考虑MoMuLV LTR、CMV启动子、SRα启动子、EF-α启动子、CAG启动子等。
需要时,除启动子外,表达载体还可以包含增强子、多腺苷酸化信号、可选标记基因和SV40复制起点等。可选标记基因的例子包括二氢叶酸还原酶基因、新霉素抗性基因、嘌呤霉素抗性基因等。
具有编码p53的显性失活突变体的核酸的表达载体可以通过本身已知的技术根据载体的种类引入细胞内。在病毒载体的情况下,例如将包含编码p53的显性失活突变体的核酸的质粒引入合适的包装细胞(例如,Plat-E细胞)或互补细胞系(例如,293细胞)内,回收在培养上清液中产生的病毒载体,并且通过适合于病毒载体的方法使细胞感染载体。例如,使用逆转录病毒载体作为载体的具体方法公开于WO2007/69666、Cell,126,663-676(2006)和Cell,131,861-872(2007)中;当慢病毒载体用作载体时,公开内容在Science,318,1917-1920(2007)中可获得。当iPS细胞用于治疗目的时,p53功能的持续抑制潜在增加了在由iPS细胞分化的组织和器官中的癌发生风险;因此,编码p53的显性失活突变体的核酸优选瞬时表达,而不整合到细胞的染色体内。根据这个观点,其整合到染色体内是罕见的腺病毒载体的使用是优选的。使用腺病毒载体的具体方法公开于Science,322,945-949(2008)中。因为腺伴随病毒在整合到染色体内的频率方面也很低,并且就细胞毒性和炎症促使活性而言低于腺病毒载体,所以它可以作为另一种优选载体提及。因为持续表达类型的仙台病毒载体能够稳定存在于染色体外,并且需要时可以使用siRNA进行降解且去除,所以它也是优选利用的。关于持续表达类型的仙台病毒载体,可以使用J.Biol.Chem.,282,27383-27391(2007)中描述的那种。
当使用逆转录病毒载体或慢病毒载体时,即使转基因的沉默已发生,它也可以变得再活化;因此,例如,优选可以使用其中编码p53的显性失活突变体的核酸当变得不需要时使用Cre/loxP系统切掉的方法。即,用预先排列在核酸的2个末端处的loxP序列,诱导iPS细胞,其后使用质粒载体或腺病毒载体允许Cre重组酶作用于细胞,并且可以切掉由loxP序列夹在中间的区域。因为LTR U3区域的增强子-启动子序列可能通过插入突变上调在其附近的宿主基因,所以更优选避免内源基因通过在基因组中未切掉的保留的loxP序列外的LTR的表达调节,使用通过这样制备的3′自身灭活的(SIN)LTR:缺失序列,或用例如SV40的多腺苷酸化序列置换序列。使用Cre-loxP系统和SIN LTR的具体方法公开于Chang等人,Stem Cells,27:1042-1049(2009)中。
同时,在其为非病毒载体的质粒载体的情况下,可以使用脂转染法、脂质体法、电穿孔法、磷酸钙共沉淀法、DEAE葡聚糖法、显微注射法、基因枪法等将载体引入细胞内。此外,当使用质粒载体时,它整合到染色体内是罕见的,转基因通过细胞中的DNA酶得到降解且去除;因此,当iPS细胞用于治疗目的时,质粒载体的使用可以是优选的实施方式的模式。使用质粒作为载体的具体方法在例如Science,322,949-953(2008)中描述。
另一种优选的非整合型载体是附加型载体,它可在染色体外自主复制。使用附加型载体的具体方法公开于Science,324,797-801(2009)中。
此外,当使用腺病毒或质粒时,转基因可以变得整合到染色体内;因此,最终需要通过Southern印迹或PCR证实不存在基因插入染色体内的情况。为此,如同上述Cre-loxP系统,使用其中转基因整合到染色体内其后去除基因的方法可以是有利的。在另一种优选的实施方式的模式中,可以使用其中转基因使用转座子整合到染色体内的方法,其后使用质粒载体或腺病毒载体允许转移酶作用于细胞,以便从染色体中完全消除转基因。作为优选转座子的例子,可以提及piggyBac,得自鳞翅目昆虫的转座子等。使用piggyBac转座子的具体方法公开于KajiK.等人,2009年3月的Nature提前在线出版物1(doi:10.1038/nature07864),Woltjen等人,2009年3月的Nature提前在线出版物1(doi:10.1038/nature07863)中。在另一个实施方案中,在启动子区中的四环素应答元件(Tet-OnR & Tet-Off R Gene ExpressionSystems,Clontech)可以用于切除转基因。
(a4)p53途径抑制剂
此处,术语p53途径用包括可以活化p53的所有上游信号级联和由活化p53介导的所有下游信号级联的含义使用。因此,p53途径抑制剂包括抑制上述信号转导途径中的任何一种的所有物质,但在优选实施方式的模式中,p53途径抑制剂是抑制其转录由p53活化的p21的表达或功能(Myc抑制剂活性)的物质;例如,可以提及针对p21的siRNA、shRNA、反义核酸、核酶等。抑制p21的表达的这些核酸可以以与下文描述的关于针对p53的siRNA、shRNA、反义核酸和核酶的方法相同的方式进行设计且合成,并且可以引入体细胞内。核酸可以以表达它们的载体的形式提供,载体可以以下文描述的用于表达针对p53的siRNA、shRNA、反义核酸或核酶的载体的方法相同的方式进行构建,并且引入体细胞内。
在另一个优选的实施方式的模式中,p53途径抑制剂是抑制ARF-MDM2-p53途径的物质;例如,作为ARF-MDM2-p53途径抑制剂,可以提及MDM2,其与p53直接结合以促进其核外转运或泛素化,或其编码核酸,p19ARF,其抑制MDM2对p53的作用,抑制ATM(毛细血管扩张性共济失调突变基因)的表达或功能的物质(例如,针对这些因子的siRNA和shRNA)等。
(a5)其他物质
作为抑制p53蛋白质功能的其他物质的例子,可以提及抗p53拮抗性抗体或其编码核酸。抗p53拮抗性抗体可以是多克隆抗体或单克隆抗体。抗体的同种型并无具体限制,并且优选IgG、IgM或IgA,特别优选IgG。除完整抗体分子外,抗体可以是例如片段,例如Fab、Fab’或F(ab’)2,由基因工程技术制备的缀合物分子,例如scFv、scFv-Fc、微型抗体或双抗体(diabody),或用具有蛋白质稳定作用的分子例如聚乙二醇(PEG)修饰的其衍生物。抗p53拮抗性抗体可以使用p53或其部分肽作为抗原来产生,通过本身已知的抗体或抗血清生产方法。作为已知的抗p53拮抗性抗体的例子,可以提及PAb 1801(OncogeneScience Ab-2)和DO-1(Oncogene Science Ab-6)(Gire和Wynford-Thomas,Mol.Cell.Biol.,18,1611-1621(1998))。编码抗p53拮抗性抗体的核酸可以通过常规方法从生产抗p53单克隆抗体的杂交瘤中分离。获得的H链和L链基因可以连接在一起,以制备编码单链抗体的核酸。优选地,这些抗体与上述PTD或CPP融合。
作为抑制p53蛋白质功能的另一种物质,可以提及抗p21拮抗性抗体或其编码核酸。抗p21拮抗性抗体或其编码核酸也可以与上述抗p53拮抗性抗体或其编码核酸一样制备。
抑制p53蛋白质功能的另外一种物质是包含p53应答元件的共有序列的诱饵核酸(例如,Pu-Pu-Pu-G-A/T-T/A-C-Py-Py-Py(Pu:嘌呤碱基,Py:嘧啶碱基);SEQ ID NO:27)此种核酸可以基于上述核苷酸序列信息使用自动化DNA/RNA合成仪进行合成。可替代地,此种诱饵核酸是商购可得的(例如,p53转录因子诱饵(GeneDetect.com))。
抗p53拮抗性抗体和抗p21拮抗性抗体、或编码抗体的核酸可以分别用在p53的显性失活突变体或编码突变体的核酸的陈述中描述的方法引入细胞内。上述诱饵核酸可以通过脂转染法等引入细胞内。
同时,作为抑制p53基因表达的物质的例子,可以提及针对p53的siRNA或shRNA、表达针对p53的siRNA或shRNA的载体、针对p53的反义核酸和针对p53的核酶等,并且针对p53的siRNA和shRNA以及表达siRNA或shRNA的载体是优选的。
(b1)针对p53的siRNA和shRNA
针对p53的siRNA可以基于由SEQ ID NO:1或3显示的小鼠或人p53cDNA序列信息进行设计,依照例如由Elbashir等人(Genes Dev.,15,188-200(2001))提出的规则。作为一般规则,关于siRNA的靶序列是AA+(N)19,但可以是AA+(N)21或NA+(N)21。有义链的5′末端无需是AA。尽管靶序列的位置并无具体限制,但希望靶序列选择在5’-UTR和距离起始密码子约50个碱基之间,以及来自除3’-UTR外的区域。靶序列的GC含量亦并无具体限制,但含量优选为约30-约50%;在GC分布中无不规则且仅具有少数重复的序列是希望的。当在设计下文(b2)表达siRNA或shRNA的载体中,polIII启动子用作启动子时,不应选择连续4个或更多T或A碱基的序列,以便防止聚合酶转录停止。
就与除靶外的mRNA中连续16-17个碱基的序列的同源性检查基于上述规则选择的靶序列候选物,使用同源性搜索软件程序例如BLAST(http://www.ncbi.nlm.nih.gov/BLAST/),以便证实选择的靶序列的特异性。对于其特异性已得到证实的靶序列,由在AA(或NA)后的19-21个碱基中具有TT或UU的3′末端突出端和与该19-21个碱基互补的序列的有义链,和具有TT或UU的3′末端突出端的反义链组成的双链RNA设计为siRNA。此外,可以这样设计shRNA,适当时选择能够形成环结构的任选选择的连接序列(例如,约8-25个碱基),并且经由连接序列使上述有义链和反义链连接。
siRNA和/或shRNA的序列可以使用无需成本在各种网站上可获得的搜索软件程序进行搜索。此种站点的例子包括但不限于,siRNA TargetFinder(http://www.ambion.com/jp/techlib/misc/siRNA_finder.html)和关于pSilencerTM Expression Vector的插入设计工具(http://www.ambion.com/jp/techlib/misc/psilencer_converter.html),两者都由Ambion提供,以及由RNAi Codex提供的GeneSeer(http://codex.cshl.edu/scripts/newsearchhairpin.cgi);并且相似搜索在QIAGEN、Takara Bio、SiSearch、Dharmacon、Whitehead Institute、Invitrogen、Promega等的网站上也是可能的
下文显示的是使用在Ambion(SEQ ID NO:5-24)和RNAi Codex(SEQ ID NO:25和26)的网站上可获得的软件程序设计的、针对小鼠p53的shRNA的序列。有下划线的序列是在用切酶(不包含3′-突出端“TT”)切割后产生的dsRNA的有义链(粗体字母)和反义链。小写字母指示错配或环。
[SEQ ID NO:5]
5’-TTTGACTGGATGACTGCCATGGttcaagagaCCATGGCAGTCAT CCAGTCTTTTTT-3’
[SEQ ID NO:6]
5’-TTTGATATCCTGCCATCACCTCttcaagagaGAGGTGATGGCAG GATATCTTTTTT-3’
[SEQ ID NO:7]
5’-TTTGGCCCAAGTGAAGCCCTCCttcaagagaGGAGGGCTTCAC TTGGGCCTTTTTT-3’
[SEQ ID NO:8]
5’-TTTGTGAAGCCCTCCGAGTGTCttcaagagaGACACTCGGAGG GCTTCACTTTTTT-3’
[SEQ ID NO:9]
5’-TTTGCCCTCCGAGTGTCAGGAGttcaagagaCTCCTGACACTCG GAGGGCTTTTTT-3’
[SEQ ID NO:10]
5’-TTTGTCTGTTATGTGCACGTACttcaagagaGTACGTGCACATAA CAGACTTTTTT-3’
[SEQ ID NO:11]
5’-TTTGTA CTCTCCTCCCCTCAATttcaagagaATTGAGGGGAGGA GAGTACTTTTTT-3’
[SEQ ID NO:12]
5’-TTTGCTATTCTGCCAGCTGGCGttcaagagaCGCCAGCTGGCAG AATAGCTTTTTT-3’
[SEQ ID NO:13]
5’-TTTGACGTGCCCTGTGCAGTTGttcaagagaCAACTGCACAGG GCACGTCTTTTTT-3’
[SEQ ID NO:14]
5’-TTTGAAGTCACAGCACATGACGttcaagagaCGTCATGTGCTGT GACTTCTTTTTT-3’
[SEQ ID NO:15]
5’-TTTGTCACAGCACATGACGGAGttcaagagaCTCCGTCATGTGC TGTGACTTTTTT-3’
[SEQ ID NO:16]
5’-TTTGGAAATTTGTATCCCGAGTttcaagagaACTCGGGATACAA ATTTCCTTTTTT-3’
[SEQ ID NO:17]
5’-TTTGTACATGTGTAATAGCTCCttcaagagaGGAGCTATTACACA TGTACTTTTTT-3’
[SEQ ID NO:18]
5’-TTTGACTCCAGTGGGAACCTTCttcaagagaGAAGGTTCCCACT GGAGTCTTTTTT-3’
[SEQ ID NO:19]
5’-TTTGTCCTTTGCCCTGAACTGCttcaagagaGCAGTTCAGGGCA AAGGACTTTTTT-3’
[SEQ ID NO:20]
5’-TTTGATCCGCGGGCGTAAACGCttcaagagaGCGTTTACGCCCG CGGATCTTTTTT-3’
[SEQ ID NO:21]
5’-TTTGACCAAGAAGGGCCAGTCTttcaagagaAGACTGGCCCTT CTTGGTCTTTTTT-3’
[SEQ ID NO:22]
5’-TTTGAAAGTGGGGCCTGACTCAttcaagagaTGAGTCAGGCCC CACTTTCTTTTTT-3’
[SEQ ID NO:23]
5’-TTTGTTGGGGAATAGGTTGATAttcaagagaTATCAACCTATTCC CCAACTTTTTT-3’
[SEQ ID NO:24]
5’-TTTGATTCTATCTTGGGCCCTCttcaagagaGAGGGCCCAAGAT AGAATCTTTTTT-3’
[SEQ ID NO:25]
5’-TTTGCAuTACAgGTACgTGTGTAgtgtgctgtccTACACATGTACT TGTAGTGTTTTTT-3’
[SEQ ID NO:26]
5’-TTTGCAGTuTACTTuCCGCCgTAgtgtgctgtccTATGGCGGGAAG TAGACTGTTTTTT-3’
针对p53的siRNA可以这样制备:使用自动化DNA/RNA合成仪分别合成如上所述设计的有义链寡核苷酸和反义链寡核苷酸,并且例如在合适的退火缓冲溶液中在约90-约95℃下使寡核苷酸变性1分钟,其后在约30-约70℃下使其退火约1-8小时。针对p53的shRNA可以这样制备:使用自动化DNA/RNA合成仪合成如上所述设计的具有shRNA序列的寡核苷酸,并且如上所述允许其自我退火。
尽管构成siRNA和shRNA的核苷酸分子可以是天然存在的RNA,但分子可以包含各种化学修饰,以便增加稳定性(化学和/或对于酶的)或特异性活性(对于mRNA的亲和力)。例如,为了防止经由水解酶例如核酸酶的降解,构成siRNA或shRNA的每个核苷酸的磷酸残基(磷酸酯)可以用例如化学修饰的磷酸残基例如磷硫酰(PS)、甲基膦酸酯或二硫代磷酸酯置换。在每个核苷酸的糖(核糖)的2′-位置处的羟基可以由-H或-OR(R表示例如CH3(2′-O-Me)、CH2CH2OCH3(2′-O-MOE)、CH2CH2NHC(NH)NH2、CH2CONHCH3、CH2CH2CN等)替换。此外,碱基部分(嘧啶、嘌呤)可以进行化学修饰;例如,可以提及将甲基或阳离子官能团引入嘧啶碱基的5′-位置处,用硫代羰基置换2-位置的羧基等。
关于RNA的糖部分的构象,2种类型是占优势的:C2′-内(S型)和C3′-内(N型);在单链RNA中,糖部分以两者的平衡存在,但当形成双链时,构象固定在N型上。因此,也可以优选使用BNA(LNA)(Imanishi,T.等人,Chem.Commun.,1653-9,2002;Jepsen,J.S.等人,Oligonucleotides,14,130-46,2004)和ENA(Morita,K.等人,Nucleosides Nucleotides Nucleic Acids,22,1619-21,2003),其为其中糖部分的构象固定在N型上的RNA衍生物,通过使2′氧和4′碳桥接以便赋予与靶RNA的强结合能力。
然而,因为用经修饰类型的分子替换在天然存在的RNA中的所有核糖核苷分子可以导致RNAi活性的丧失,所以必须引入允许RISC复合物起作用的修饰至最低限度可能程度的核苷。
针对p53的siRNA也可以购自,例如Ambion(例如,Ambion Cat#AM16708,siRNA ID#69659、69753、69843、187424、187425、187426),Santa Cruz(例如,Santa Cruz Cat# sc-29436、44219)等。
针对人p53的siRNA和shRNA也可以使用上述搜索软件程序之一进行设计且合成,通过输入由SEQ ID NO:3或Refseq.No.(NM_000546)显示的人p53cDNA序列作为查询,或还可以购自Ambion等。具体地,可以提及具有序列5’-GACTCCAGTGGTAATCTACTGCTCGAGCAGTAGATTACCACTGG AGTC-3’(SEQ ID NO:28;粗体字母指示关于p53的靶序列;有下划线的是其中形成dsRNA的部分)的针对人p53的shRNA,在Science,296,550-553(2002)中描述的针对p53的shRNA。
如在质粒DNA的情况下,针对p53的siRNA或shRNA与体细胞的接触可以通过将核酸引入细胞内来达到,使用脂质体法、聚胺法、电穿孔法、珠法等。使用阳离子脂质体的方法是最常见的,并且提供高转移效率。除常见转染试剂例如Lipofectamine2000和Oligofectamine(Invitrogen)外,例如,适合于引入siRNA的转移试剂,例如GeneEraserTM siRNA转移试剂(Stratagene),也是商购可得的。(b2)表达针对p53的siRNA或shRNA的载体
表达siRNA的载体可以串联类型和茎环(发夹)类型获得。前者是其中关于siRNA的有义链的表达盒和关于反义链的表达盒串联连接的类型,每条链在细胞中表达并且经历退火以形成双链siRNA(dsRNA)。同时,后者是其中关于shRNA的表达盒插入载体内的类型,shRNA在细胞中表达并且经历经由切酶的加工以形成dsRNA。尽管polII启动子(例如,CMV的立即早期启动子)可以用作启动子,但使用polIII启动子以便允许短RNA的精确转录是常规实践。作为polIII启动子,可以提及小鼠和人U6-snRNA启动子、人H1-RNA酶PRNA启动子、人缬氨酸-tRNA启动子等。作为转录终止信号,使用连续4个或更多T残基的序列。
随后将因此构建的siRNA或shRNA表达盒插入质粒载体或病毒载体内。作为此种载体,可以优选利用与就编码p53的显性失活突变体的核酸而言描述的那些相同的(病毒载体例如逆转录病毒、慢病毒、腺病毒、腺伴随病毒、疱疹病毒和仙台病毒;动物细胞表达质粒等)。适当时,所使用的载体可以根据获得的iPS细胞的预期用途进行选择,如在显性失活突变体的情况下一样。可替代地,作为编码针对p53的shRNA的表达载体,也可以使用基于商购可得的质粒(例如,从Addgene商购可得的pMKO.1-puro p53 shRNA2:#10672,等)制备的病毒载体例如逆转录病毒等。需要时,也可以使用上述Cre-loxP系统或piggyBac转座子系统。
表达针对p53的siRNA或shRNA的载体与体细胞的接触通过将如上所述制备的质粒载体或病毒载体引入细胞内来达到。这些基因的转移可以通过与就编码p53的显性失活突变体的核酸而言描述的相同技术来达到。
(b3)其他物质
作为抑制p53基因表达的其他物质,可以提及针对p53的反义核酸和核酶。
反义核酸可以是DNA或RNA、或DNA/RNA嵌合体。当反义核酸是DNA时,通过靶RNA和反义DNA形成的RNA:DNA杂交物能够由内源RNA酶H识别,以引起靶RNA的选择性降解。因此,在反义DNA待由RNA酶H降解的情况下,靶序列不仅可以是p53mRNA中的序列,还可以是p53基因的原始转录物的内含子区中的序列。关于反义核酸的靶区长度并无具体限制,只要反义核酸的杂交导致翻译成p53蛋白质的抑制;靶区可以是p53mRNA的整个序列或部分序列,并且可以是最短约15个碱基的序列,或最长mRNA或原始转录物的整个序列。考虑到容易合成、抗原性、在细胞和其他组织中的可转移性,由约15至约40个碱基,特别是约18至约30个碱基组成的寡核苷酸是优选的。靶序列的位置包括但不限于,5’-和3’-UTR,起始密码子附近等。
核酶指以狭义具有酶活性以切割核酸的RNA,并且在本文中应理解为作为包含DNA的概念使用,只要核酶具有序列特异性核酸切割活性的核酶。最通用的核酶之一是在感染性RNA例如类病毒和病毒样体中发现的自我剪接RNA,并且锤头类型、发夹类型等是已知的。锤头类型显示对于长度约40个碱基的酶活性,并且它可以特异性切割靶mRNA,通过使得在2个末端处与锤头结构部分连接的数个碱基(总共约10个碱基)成为与mRNA的所需切割位点互补的序列。
反义核酸或核酶可以使用自动化DNA/RNA合成仪进行合成。构成其的核苷酸分子也可以具有与关于siRNA的那些相同的修饰,以便增加稳定性、特异性活性等。
可替代地,反义核酸或核酶也可以以其编码核酸的形式使用,如在siRNA的情况下。
上述p53功能抑制剂需要以在体细胞核重编程步骤中足以抑制p53功能的方式与体细胞接触。此处,体细胞的核重编程可以通过使核重编程物质与体细胞接触来达到。
(c)核重编程物质
在本发明中,“核重编程物质”指能够由体细胞诱导iPS细胞的任何物质,例如蛋白质性质因子或其编码核酸(包括掺入载体中的形式)或低分子化合物。当核重编程物质是蛋白质性质因子或其编码核酸时,下述组合可以作为优选例子提及(在下文中,仅显示关于蛋白质性质因子的名称)。
(1)Oct3/4、Klf4、c-Myc
(2)Oct3/4、Klf4、c-Myc、Sox2(此处,Sox2可由Sox1、Sox3、Sox15、Sox17或Sox18代替。同样,Klf4可由Klf1、Klf2或Klf5代替。此外,c-Myc可由T58A(活性突变型)、N-Myc或L-Myc代替。)
(3)Oct3/4、Klf4、c-Myc、Sox2、Fbx 15、Nanog、Eras、ECAT15-2、TclI、β-连环蛋白(活性突变型S33Y)
(4)Oct3/4、Klf4、c-Myc、Sox2、TERT、SV40大T
(5)Oct3/4、Klf4、c-Myc、Sox2、TERT、HPV16E6
(6)Oct3/4、Klf4、c-Myc、Sox2、TERT、HPV16E7
(7)Oct3/4、Klf4、c-Myc、Sox2、TERT、HPV6E6、HPV16E7
(8)Oct3/4、Klf4、c-Myc、Sox2、TERT、Bmil
(所有上述全部,参见WO 2007/069666(然而,在上述组合(2)中,关于用Sox18代替Sox2和用Klf1或Klf5代替Klf4,参见NatureBiotechnology,26,101-106(2008))。关于“Oct3/4、Klf4、c-Myc、Sox2”的组合,还参见Cell,126,663-676(2006),Cell,131,861-872(2007)等。关于“Oct3/4、Klf2(或Klf5)、c-Myc、Sox2”的组合,还参见Nat.Cell Biol.,l1,197-203(2009)。关于“Oct3/4、Klf4、c-Myc、Sox2、hTERT、SV40大T”的组合,还参见Nature,451,141-146(2008)。)
(9)Oct3/4、Klf4、Sox2(参见Nature Biotechnology,26,101-106(2008))
(10)Oct3/4、Sox2、Nanog、Lin28(参见Science,318,1917-1920(2007))
(11)Oct3/4、Sox2、Nanog、Lin28、hTERT、SV40大T(参见Stem Cells,26,1998-2005(2008))
(12)Oct3/4、Klf4、c-Myc、Sox2、Nanog、Lin28(参见Cell Research(2008)600-603)
(13)Oct3/4、Klf4、c-Myc、Sox2、SV40大T(还参见Stem Cells,26,1998-2005(2008))
(14)Oct3/4、Klf4(参见Nature 454,646-650(2008),Cell StemCell,2:525-528(2008))
(15)Oct3/4、c-Myc(参见Nature 454,646-650(2008))
(16)Oct3/4、Sox2(参见Nature,451,141-146(2008),WO2008/118820)
(17)Oct3/4、Sox2、Nanog(参见WO2008/118820)
(18)Oct3/4、Sox2、Lin28(参见WO2008/118820)
(19)Oct3/4、Sox2、c-Myc、Esrrb(此处,Essrrb可由Esrrg代替;参见Nat.Cell Biol.,11,197-203(2009))
(20)Oct3/4、Sox2、Esrrb(参见Nat.Cell Biol.,11,197-203(2009))
(21)Oct3/4、Klf4、L-Myc
(22)Oct3/4、Nanog
(23)Oct3/4
(24)Oct3/4、Klf4、c-Myc、Sox2、Nanog、Lin28、SV40LT(参见Science,324:797-801(2009))
在上述(1)-(24)中,代替Oct3/4,还可以使用Oct家族的其他成员,例如,Oct1A、Oct6等。代替Sox2(或Sox1、Sox3、Sox15、Sox17、Sox18),还可以使用Sox家族的其他成员,例如,Sox7等。代替c-Myc,还可以使用Myc家族的其他成员,例如,L-Myc等。代替Lin28,还可以使用Lin家族的其他成员,例如,Lin28b等。
未落入上述(1)-(24)的范围内,但包含其任一项中的所有组成成分,并且进一步包含任选选择的其他物质的组合,也可以包括在本发明中的“核重编程物质”范围中。在其中其为核重编程对象的体细胞以足以确保核重编程的水平内源表达上述(1)-(24)中任一项的一种或多种组成成分的条件下,除去一种或多种组成成分仅其余组成成分的组合也可以包括在本发明中的“核重编程物质”范围中。
在这些组合中,作为优选核重编程物质的例子,可以提及选自Oct3/4、Sox2、Klf4、c-Myc、Nanog、Lin28和SV40LT的至少一种、优选2种或更多、更优选3种或更多。
对于获得的iPS细胞考虑用于治疗目的的用途,在这些组合中,优选3种因子Oct3/4、Sox2和Klf4的组合(即,上述的(9))。在本发明的方法中,仅用上述3种因子,可以以足够高的效率获得iPS细胞。即,即使仅用3种因子,也可以以与用4种因子(Oct3/4、Sox2、Klf4和c-Myc)的效率更接近的效率建立iPS细胞。同时,当并不考虑iPS细胞用于治疗目的的用途时(例如,用作研究工具用于药物开发筛选等),以及上述4种因子,5种因子Oct3/4、Klf4、c-Myc、Sox2和Lin28,或包括5种因子加上Nanog的6种因子(即,上述的(12))是优选的。在这些优选组合中,L-Myc也可以用于代替c-Myc。
通过参考WO 2007/069666(在该出版物中,Nanog由命名“ECAT4”提及;关于Lin28、Lin28b、Esrrb和Esrrgd的小鼠和人cDNA序列信息可以通过提及下述NCBI登记号分别获得)中提及的NCBI登记号,可获得关于上述蛋白质性质因子的小鼠和人cDNA序列的信息;本领域技术人员能够容易地分离这些cDNA。
基因名称 小鼠 人
Lin28 NM_145833 NM_024674
Lin28b NM_001031772 NM 001004317
Esrrb NM_011934 NM_004452
Esrrg NM_011935 NM_001438
当蛋白质性质因子其自身用作核重编程物质时,因子可以这样制备:将获得的cDNA插入合适的表达载体内,将载体引入宿主细胞内,培养细胞,并且从获得的培养物中回收重组蛋白质性质因子。同时,当编码蛋白质性质因子的核酸用作核重编程物质时,将获得的cDNA插入病毒或质粒载体内,以构建表达载体,如在上述编码p53的显性失活突变体的核酸的情况下,并且对载体实施核重编程步骤。需要时,可以利用上述Cre-loxP系统或piggyBac转座子系统。当编码2种或更多蛋白质性质因子的核酸引入细胞内作为核重编程物质时,核酸可以由分别的载体携带,并且多个核酸可以串联连接,以获得多顺反子载体。在后面一种情况下,为了使得能够有效的多顺反子表达,希望口蹄疫病毒的2A自身切割肽(参见Science,322,949-953,2008等)、IRES序列等,优选2A序列连接在各个核酸之间。
当p53功能被抑制时,经由逆转录病毒或慢病毒载体整合到染色体内的转基因趋于对基因沉默有抗性。因此,质粒载体的使用对于防止外源核重编程物质不必要的持续表达是有利的。
(a)当物质是蛋白质性质因子时,核重编程物质与体细胞的接触可以与上述p53的显性失活突变体一样达到;(b)当物质是编码蛋白质性质因子的核酸时,与上述编码p53的显性失活突变体的核酸一样达到;和(c)当物质是低分子化合物时,与上述p53的化学抑制剂一样达到。
如上所述,p53功能抑制剂需要以在体细胞核重编程步骤中足以抑制p53功能的方式与体细胞接触。只要满足这个需求,核重编程物质和p53功能抑制剂可以同时与体细胞接触,或任何一种可以预先接触。在实施方式的模式中,例如,当核重编程物质是编码蛋白质性质因子的核酸,和p53功能抑制剂是化学抑制剂时,前者涉及从转染处理到蛋白质性质因子的大量表达的给定延时长度,而后者能够快速抑制p53功能,从而使得在转染处理后细胞培养给定时间长度后,p53的化学抑制剂可以加入培养基中。在另一个实施方式的模式中,例如,当核重编程物质和p53功能抑制剂都以病毒载体或质粒载体形式使用时,两者可以同时引入细胞内。
将腺病毒或非病毒表达载体引入体细胞内的操作重复次数并无具体限制,转染可以执行一次或更多任选选择的次数(例如,1-10次、1-5次等)。当2个或更多种类的腺病毒或非病毒表达载体引入体细胞内时,这些所有这些种类的腺病毒或非病毒表达载体同时引入体细胞内是优选的;然而,即使在这种情况下,转染也可以执行一次或更多任选选择的次数(例如,1-10次、1-5次等),优选地转染可以重复执行2次或更多次(例如,3次或4次)。
(d)iPS细胞建立效率改进剂
除p53功能抑制剂外,通过使另一种公众已知的iPS细胞建立效率改进剂与体细胞接触,预期iPS细胞建立效率增加更多。iPS细胞建立效率改进剂的例子包括但不限于,组蛋白脱乙酰基酶(HDAC)抑制剂[例如,丙戊酸(VPA)(Nat.Biotechnol.,26(7):795-797(2008)),低分子抑制剂例如曲古抑菌素A、丁酸钠、MC 1293和M344,基于核酸的表达抑制剂例如针对HDAC的siRNA和shRNA(例如,HDAC1siRNA(Millipore)、针对HDACl的HuSH 29聚体shRNA构建体(OriGene)等),等]、DNA甲基转移酶抑制剂(例如,5′-氮胞苷)[Nat.Biotechnol.,26(7):795-797(2008)],G9a组蛋白甲基转移酶抑制剂[例如,低分子抑制剂例如BIX-01294(Cell Stem Cell,2:525-528(2008)),基于核酸的表达抑制剂例如针对G9a的siRNA和shRNA(例如,G9a siRNA(人)(Santa Cruz Biotechnology)等),等],L-通道钙激动剂(例如,Bayk8644)(Cell Stem Cell,3,568-574(2008)),UTF1(Cell Stem Cell,3,475-479(2008)),Wnt信号(例如,可溶性Wnt3a)(Cell Stem Cell,3,132-135(2008)),2i/LIF(2i是丝裂原活化蛋白激酶信号和糖原合酶激酶-3的抑制剂,PloS Biology,6(10),2237-2247(2008))等。如上所述,基于核酸的表达抑制剂可以为具有编码siRNA或shRNA的DNA的表达载体形式。
在上述核重编程物质的组成成分中,例如SV40大T也可以包括在iPS细胞建立效率改进剂的范围中,因为它们是对于体细胞的核重编程非必需的辅助因子。虽然体细胞核重编程的机制仍不明了,但除核重编程必需的因子外,辅助因子是视为核重编程物质,还是视为iPS细胞建立效率改进剂并不重要。因此,因为体细胞核重编程过程设想为起因于核重编程物质和iPS细胞建立效率改进剂与体细胞接触的总体事件,所以似乎本领域技术人员无需总是区分两者。
这些iPS细胞建立效率改进剂与体细胞的接触可以如同就p53功能抑制剂而言分别如上所述达到,当改进剂是(a)蛋白质性质因子,(b)编码蛋白质性质因子的核酸,或(c)低分子化合物时。
(e)体细胞来源
在本发明中可以用作原材料用于制备iPS细胞的体细胞可以是得自哺乳动物(例如小鼠、人)除生殖细胞外的任何细胞;例如,可以提及角质化上皮细胞(例如,角质化的表皮细胞)、粘膜上皮细胞(例如,舌浅层的上皮细胞)、外分泌腺上皮细胞(例如,乳腺细胞)、激素分泌细胞(例如,肾上腺髓质细胞)、用于代谢或贮藏的细胞(例如,肝细胞)、构成界面的内膜上皮细胞(例如,I型肺泡细胞)、闭膜管的内膜上皮细胞(例如,血管内皮细胞)、含具有运输能力的纤毛的细胞(例如,气道上皮细胞)、用于细胞外基质分泌的细胞(例如,成纤维细胞)、收缩细胞(例如,平滑肌细胞)、血液和免疫系统细胞(例如,T淋巴细胞)、感觉相关细胞(例如,杆状细胞)、自主神经系统神经元(例如,胆碱能神经元)、感觉器官和外周神经元的支持细胞(例如,卫星细胞)、中枢神经系统的神经细胞和神经胶质细胞(例如,星形胶质细胞)、色素细胞(例如,视网膜色素上皮细胞)、及其祖细胞(组织祖细胞)等。关于细胞分化程度并无限制;即使未分化的祖细胞(包括体干细胞)和终末分化的成熟细胞也同样可以在本发明中用作体细胞来源。此处,作为未分化的祖细胞的例子,可以提及组织干细胞(体干细胞),例如神经干细胞、造血干细胞、间质干细胞和牙髓干细胞。
根据本发明的方法,iPS细胞甚至可以由终末分化的体细胞有效获得,对于其据报告一般难以建立iPS细胞。在本发明的优选实施方式的模式中,T细胞用作体细胞。T细胞可以是CD4阳性或CD8阳性的,并且可以是CD4/CD8双阳性分化阶段中的细胞。可以通过本身已知的方法例如流式细胞术,使用针对细胞表面标记例如CD4、CD8或CD3的抗体,和细胞分选器,从脾、淋巴结、外周血、脐带血等中分离T细胞。在小鼠的情况下,优选从其中T细胞的含量比很高的脾或淋巴结中收集体细胞;然而,在人的情况下,从低侵入性和易于制备的观点来看,希望T细胞由外周血、脐带血等制备。
充当收集的体细胞来源的哺乳动物选择并无具体限制;然而,当获得的iPS细胞用于在人中的再生医学时,从不发生移植物排斥的观点来看,特别优选的是体细胞从患者或具有相同HLA类型的其他人收集。当IPS细胞并非施用于(移植给)人,而是用作细胞来源例如用于筛选评估患者的药物敏感性或不良反应的存在或不存在时,可能必须收集来自患者或具有与药物敏感性或不良反应相关的相同遗传多态性的其他人的体细胞,。
从小鼠或人中分离的体细胞可以使用本身已知适合于其培养的培养基进行预培养,依赖于细胞的种类。此种培养基的例子包括但不限于,包含约5-20%胎牛血清的最小必需培养基(MEM)、达尔贝科改良伊格尔培养基(DMEM)、RPMI1640培养基、199培养基、F12培养基等。可获得通过在5%或更少的低血清浓度下进行预培养,改善iPS细胞建立效率的报告(例如,WO 2009/006997)。当T细胞用作体细胞时,希望细胞使用包含细胞因子例如白介素(IL)-2、IL-7、干细胞因子(SCF)或Fit3配体的培养基进行预培养。当在与核重编程物质和p53功能抑制剂(和改善iPS细胞建立效率的另一种物质)的接触中使用例如转染试剂,例如阳离子脂质时,有时优选先用无血清培养基替换培养基,以防止转染效率中的减少。在接触核重编程物质后,可以在适合于培养例如ES细胞的条件下培养细胞。在小鼠细胞的情况下,通过向普通培养基中添加作为分化抑制因子的白血病抑制因子(LIF)进行培养是优选的。同时,在人细胞的情况下,希望加入碱性成纤维细胞生长因子(bFGF)和/或干细胞因子(SCF)代替LIF。通常,在作为饲养细胞的胚胎小鼠衍生的成纤维细胞(MEF)的共存在下培养细胞,所述MEF用辐射或抗生素处理,以终止细胞分裂。作为MEF,通常STO细胞等是常用的,但对于诱导iPS细胞,SNL细胞(McMahon,A.P.&Bradley,A.Cell 62,1073-1085(1990))等是常用的。与饲养细胞的共培养可以在核重编程物质接触前、接触时或接触后(例如,10天后)开始。
iPS细胞的候选集落可以通过用药物抗性和报道物活性作为指示剂的方法,以及通过基于形态目测的方法进行选择。作为前者的例子,使用重组体细胞选择对于药物抗性和/或报道物活性阳性的集落,在所述重组体细胞中药物抗性基因和/或报道基因靶向在多能细胞中高度特异性表达的基因的基因座(例如,Fbx15、Nanog、Oct3/4等,优选Nanog或Oct3/4)。作为此种重组细胞的例子,可以提及其中βgeo(其编码β-半乳糖苷酶和新霉素磷酸转移酶的融合蛋白)基因敲入Fbx15基因座中的小鼠衍生的MEF(Takahashi & Yamanaka,Cell,126,663-676(2006))、或其中绿色荧光蛋白(GFP)基因和嘌呤霉素抗性基因整合在Nanog基因座中的转基因小鼠衍生的MEF(Okita等人,Nature,448,313-317(2007))等。同时,形态目测的方法包括例如由Takahashi在Cell,131,861-872(2007)中描述的方法。尽管使用报道细胞的该方法是方便和有效的,但从安全性的观点来看,当iPS细胞制备用于人治疗目的时,希望经由目测的集落选择。当3种因子Oct3/4、Klf4和Sox2用作核重编程物质时,建立的集落数目有时减少,但所得到的集落大多数是其质量与ES细胞一样高的iPS细胞;因此,甚至无需使用报道细胞也可以有效建立iPS细胞。
选择的集落细胞作为iPS细胞的同一性可以这样加以证实:通过对Nanog(或Oct3/4、Fbx15)报道物的阳性应答(GFP阳性、β-半乳糖苷酶阳性等)和对选择标记的阳性应答(嘌呤霉素抗性、G418抗性等),以及通过可见ES细胞样集落的形成,如上所述;然而,为了增加准确度,可以执行诸如分析各种ES细胞特异性基因的表达且将选择的细胞移植给小鼠且证实畸胎瘤形成的测试。这些测试方法对于本领域技术人员是显而易见的,代表性证实测试在下文实施例中描述。
因此建立的iPS细胞可以用于各种目的。例如,通过利用就ES细胞而言报告的分化诱导方法,可以诱导由iPS细胞分化成各种细胞(例如,心肌细胞、血细胞、神经细胞、血管内皮细胞、胰岛素分泌细胞等)。因此,使用从患者或具有相同HLA类型的其他人收集的体细胞诱导iPS细胞将使得通过自体或同种异体移植的干细胞治疗成为可能,其中iPS细胞分化成所需细胞(即,患者的受影响器官的细胞、对疾病具有疗效的细胞等),将所需细胞移植给患者。此外,因为由iPS细胞分化的功能细胞(例如,肝细胞)被认为比相应的现有细胞系更好地反映功能细胞在体内的实际状态,所以它们还可以适合于用于就药物候选化合物的有效性和毒性的体外筛选等。
当以其中p53的显性失活突变体或编码针对p53的siRNA或shRNA的核酸等引入体细胞内且在其中强迫表达的方式达到p53的抑制时,因为外源核酸的污染,获得的iPS细胞是不同于常规已知的iPS细胞的新型细胞。特别地,当外源核酸使用逆转录病毒、慢病毒等引入体细胞内时,外源核酸通常整合到获得的iPS细胞的基因组中,从而使得包含外源核酸的表型稳定保留。当外源核酸使用持续的仙台病毒载体引入体细胞内时,外源核酸可以稳定存在于获得的iPS细胞的细胞质中,从而使得包含外源核酸的表型同样可以稳定保留。
本发明还提供了其中TCR基因已重排的iPS细胞,通过使T细胞重编程获得的细胞。作为T细胞重编程的方法,可以提及其中在如上所述用于抑制p53功能的条件下使T细胞与核重编程物质接触的方法。在由T细胞诱导的iPS细胞(T-iPS细胞)中,它由其衍生的T细胞中的TCR重排是保守的。尽管迄今为止已存在其中iPS细胞由B细胞诱导的某些情况,但未呈现关于得自T细胞的iPS细胞建立的报告。因为TCR的重排即使在由T-iPS细胞分化的细胞中也是保守的,所以可以大量产生能够特异性损伤呈递上述肽之一的细胞(例如,癌细胞、受感染细胞等)的T细胞,通过例如由能够特异性识别抗原呈递细胞(例如,巨噬细胞、树突状细胞等)的T细胞克隆建立T-iPS细胞,所述抗原呈递细胞用癌抗原肽或得自病原体例如病毒的细胞表面抗原的肽脉冲,使用本发明的方法在体外扩增iPS细胞至大量,随后诱导其分化成T细胞,并且可以制备为T细胞免疫治疗剂与常规T细胞免疫治疗一样。
本发明在下文通过提及实施例更详细地说明,所述实施例不应解释为限制性的。
实施例
[实施例1:p53缺陷的作用]
具有Nanog报道物的p53纯合-缺陷小鼠或杂合-缺陷小鼠用作实验系统。p53是在几乎所有细胞中表达的基因,并且在损伤细胞的修复过程中控制细胞周期终止或凋亡诱导。这些小鼠已通过用新霉素抗性基因替换外显子5而丧失p53基因的功能(Lawrence A.Donehower(1992).Nature 356,215-221)。已报告p53纯合-缺陷小鼠正常出生但通常患有肿瘤发生。通过将绿色荧光蛋白(EGFP)和嘌呤霉素抗性基因插入购自BACPAC Resources的BAC(细菌人工染色体)的Nanog基因座内制备Nanog报道物(Okita K.等人,Nature 448,313-317(2007))。小鼠Nanog基因是在多能细胞例如ES细胞和早期胚胎中特异性表达的基因。已变得对这种报道物阳性的小鼠iPS细胞已知具有与ES细胞几乎等价的分化潜能。通过制备具有这种Nanog报道物的Nanog报道小鼠(Okita K.等人,Nature 448,313-317(2007)),并且使这种小鼠与p53缺陷小鼠交配,制备具有Nanog报道物的p53纯合-缺陷小鼠和杂合-缺陷小鼠。
通过将分别的逆转录病毒表达载体(pMXs-Oct3/4、pMXs-Sox2、pMXs-Klf4、pMXs-cMyc:Cell,126,663-676(2006))引入Plat-E细胞(Morita,S.等人,Gene Ther.7,1063-1066)内,所述Plat-E细胞在前一天已以0.6x106细胞/孔接种至6孔培养板(Falcon),制备用于重编程的逆转录病毒。所使用的培养肉汤是DMEM/10%FCS(补充有10%胎牛血清的DMEM(Nacalai Tesque)),并且在37℃和5%CO2下培养细胞。对于载体转移,将4.5μL FuGene6转染试剂(Roche)置于100μL Opti-MEM I Reduced-Serum Medium(Invitrogen)中,并且允许这种混合物在室温下静置5分钟。其后,加入1.5μg每种表达载体,并且进一步允许混合物在室温下静置15分钟,并且随后加入Plat-E培养肉汤。在第2天时,用新鲜供应的培养基替换Plat-E上清液;在第3天时,回收培养上清液并且过滤通过0.45μm无菌滤器(Whatman),加入聚凝胺(Nacalai)以获得4μg/mL的浓度,并且这用作病毒液体。
从具有小鼠Nanog报道物的胎儿p53纯合-缺陷小鼠(受精后13.5天)中分离成纤维细胞(MEF)。因为不存在Nanog基因表达的情况,所以MEF不表达EGFP,并且不发出绿色荧光。因为也不存在嘌呤霉素抗性基因表达的情况,所以MEF对嘌呤霉素——抗生素敏感。像这样,将MEF以1x105/孔接种至用0.1%明胶(Sigma)包被的6孔培养板(Falcon)。所使用的培养肉汤是DMEM/10%FCS,并且在37℃和5%CO2下培养细胞。第二天,加入逆转录病毒液体以引起过夜感染以引入基因。
在病毒感染后第3天开始,使用补充LIF的ES细胞培养基(通过向DMEM(Nacalai Tesque)中加入15%胎牛血清、2mM L-谷氨酰胺(Invitrogen)、100μM非必需氨基酸(Invitrogen)、100μM 2-巯基乙醇(Invitrogen)、50U/mL青霉素(Invitrogen)和50mg/mL链霉素(Invitrogen)制备)培养细胞。在感染后第5天时,回收MEF的培养基,并且通过加入1mL PBS洗涤细胞。在去除PBS后,加入0.25%胰蛋白酶/1mM EDTA(Invitrogen),并且允许反应在37℃下进行约5分钟。细胞浮起后,通过加入ES细胞培养基使细胞悬浮,并且将5x103细胞接种至先前在其上接种饲养细胞的100-mm皿。所使用的饲养细胞是已用丝裂霉素C处理以终止细胞分裂的SNL细胞(McMahon,A.P.&Bradley,A.Cell 62,1073-1085(1990))。随后,每2天用新鲜供应更换ES细胞培养基,直至可观察到集落。对于用4种因子(Oct3/4、Sox2、Klf4、c-Myc)感染的细胞,在第13天时开始执行用嘌呤霉素(1.5μg/mL)的选择,并且对于用3种因子(Oct3/4、Sox2、Klf4)感染的细胞,在第19天时开始。对于4种因子集落在约第10天时可见,并且对于3种因子在约第20天时可见,并且逐渐变得GFP阳性。
对于4种因子在第21天时计数GFP阳性集落,并且对于3种因子在第28天时。3次实验的总结果显示于图1中。与构成对照组的p53杂合-缺陷小鼠比较,在p53纯合-缺陷小鼠中,对于4种因子GFP阳性集落增加约2倍,并且对于3种因子增加约9倍。
接下来,对于4种因子在第21天时收集GFP阳性集落,并且对于3种因子在第28天时,并且在胰蛋白酶消化后,在饲养细胞上继续培养(这个时间点作为次培养代1)。当将1x106细胞皮下注射到免疫缺陷小鼠时,2周后开始观察到畸胎瘤形成;无论引入3种因子还是4种因子,所得到的GFP阳性集落被鉴定为iPS细胞。关于4种因子的结果显示于图2中。
根据上述结果,证实通过缺失(敲除)p53,增加iPS细胞建立效率。
[实施例2:用显性失活突变体的研究]
使用p53的显性失活突变体抑制内源p53的功能,并且研究它对iPS细胞建立效率的影响。所使用的显性失活突变体是p53P275S,通过引起位于p53的基因组结合区的第275位处的脯氨酸至丝氨酸的点突变制备(Annemieke de Vries(2002).PNAS 99,2948-2953)。
通过将逆转录病毒表达载体(pMXs-Oct3/4、pMXs-Sox2、pMXs-Klf4、pMXs-cMyc、pMXs-p53P275S)引入Plat-E细胞内,所述Plat-E细胞在前一天已以0.6x106细胞/孔接种至6孔培养板(Falcon),制备用于重编程的逆转录病毒(关于如何制备pMXs-p53P275S,参见下文实施例6)。所使用的培养肉汤是DMEM/10%FCS(补充有10%胎牛血清的DMEM(Nacalai Tesque)),并且在37℃和5%CO2下进行培养。对于载体转移,将4.5μL FuGene6转染试剂(Roche)置于100μLOpti-MEM I Reduced-Serum Medium(Invitrogen)中,并且允许这种混合物在室温下静置5分钟。其后,加入1.5μg每种表达载体,并且进一步允许混合物在室温下静置15分钟,并且随后加入Plat-E培养肉汤。在第2天时,用新鲜供应的培养基替换Plat-E上清液;在第3天时,回收培养上清液并且过滤通过0.45μm无菌滤器(Whatman),加入聚凝胺(Nacalai)以获得4μg/mL的浓度,并且这用作病毒液体。
从具有Nanog报道物的胎儿p53杂合-缺陷小鼠(受精后13.5天)中分离成纤维细胞(MEF)。提供先前在其上接种饲养细胞的6孔培养板,并且对于4种因子MEF以4x103细胞/孔接种,并且对于3种因子以2x104细胞/孔接种。所使用的饲养细胞是已用丝裂霉素C处理以终止细胞分裂的SNL细胞。所使用的培养肉汤是DMEM/10%FCS,并且在37℃和5%CO2下进行培养。第二天,细胞在从Plat-E回收的病毒液体中培养过夜以引入基因。在病毒感染后第3天开始,使用补充LIF的ES细胞培养基培养细胞。随后,每2天用新鲜供应更换ES细胞培养基,直至可见到集落。对于用4种因子(Oct3/4、Sox2、Klf4、c-Myc)和P275S感染的细胞,在感染后第13天时开始执行用嘌呤霉素(1.5μg/mL)的选择,并且对于用3种因子(Oct3/4、Sox2、Klf4)和P275S感染的细胞,在第19天时开始。对于4种因子集落在约第10天时可见,并且对于3种因子在约第20天时可见,并且逐渐变得GFP阳性。
对于4种因子在第21天时计数GFP阳性集落,并且对于3种因子在第28天时。实验操作的概要显示于图3(A)中,并且实验结果显示于图3(B)-(D)中。与掺入无突变p53或红色荧光蛋白(DsRedExpress)代替p53P275S的细胞比较,在掺入p53P275S的细胞中,对于4种因子GFP阳性集落增加约4倍,并且对于3种因子增加约3倍。
关于上述3种因子或4种因子和p53或DsRedExpress引入从胎儿p53纯合-缺陷小鼠中分离的MEF内的结果显示于图3(E)-(G)中。与掺入对照DsRedExpress的细胞比较,在掺入p53的细胞中,GFP阳性集落数目减少。
根据上述结果,证实通过抑制内源p53功能,增加iPS细胞建立效率。
[实施例3:使用p53抑制试剂的研究]
检查了p53抑制剂对iPS细胞建立效率的作用。通过将逆转录病毒表达载体(pMXs-Oct3/4、pMXs-Sox2、pMXs-Klf4、pMXs-cMyc)引入Plat-E细胞内,所述Plat-E细胞在前一天已以0.6x106细胞/孔接种至6孔培养板(Falcon),制备用于重编程的逆转录病毒。所使用的培养肉汤是DMEM/10%FCS(补充有10%胎牛血清的DMEM(NacalaiTesque)),并且在37℃和5%CO2下进行培养。对于载体转移,将4.5μLFuGene6转染试剂(Roche)置于100μL Opti-MEM I Reduced-SerumMedium(Invitrogen)中,并且允许这种混合物在室温下静置5分钟。其后,加入1.5μg每种表达载体,并且进一步允许混合物在室温下静置15分钟,并且随后加入Plat-E培养肉汤。在第2天时,用新鲜供应的培养基替换Plat-E上清液;在第3天时,回收培养上清液并且过滤通过0.45μm无菌滤器(Whatman),加入聚凝胺(Nacalai)以获得4μg/mL的浓度,并且这用作病毒液体。
从具有Nanog报道物的胎儿p53杂合-缺陷小鼠(受精后13.5天)中分离成纤维细胞(MEF)。将这些MEF以1x105细胞/孔接种至用0.1%明胶(Sigma)包被的6孔培养板(Falcon)。所使用的培养肉汤是DMEM/10%FCS,并且在37℃和5%CO2下培养细胞。第二天,细胞用从Plat-E回收的病毒液体感染过夜以引入基因。在病毒感染后第3天开始,使用补充LIF的ES细胞培养基培养细胞。在感染后第5天时,回收MEF的培养基,并且通过加入1mL PBS洗涤细胞。在去除PBS后,加入0.25%胰蛋白酶/1mM EDTA(Invitrogen),并且允许反应在37℃下进行约5分钟。细胞浮起后,通过加入ES细胞培养基使细胞悬浮,并且将8x102细胞接种至先前在其上接种饲养细胞的6孔培养板。在第6天时开始,将Pifithrinα,对硝基,环状(MERCK),6nM加入培养基中,并且继续培养。Pifithrin以在DMSO中的溶液使用。在感染后第16天时开始,执行用嘌呤霉素(1.5μg/mL)的选择。集落在约第10天时可见,并且逐渐变得GFP阳性。在第20天时计数GFP阳性集落。结果显示于图4中。与用DMSO处理的细胞比较,在用Pifithrin处理的细胞中,GFP阳性集落数目增加约4倍。根据上述结果,证实通过抑制p53功能,增加iPS细胞建立效率。
[实施例4:在来自T细胞的iPS建立中p53缺陷的作用]
对Nanog报道小鼠(下文,Nanog-GFP Tg小鼠:17周龄雄性)和具有Nanog报道物的p53纯合-缺陷小鼠(下文,Nanog-GFP/Trp53-/-小鼠:24周零雌性)实施安乐死,这之后摘除脾。组织进行机械磨碎后,使用CD90微珠(Miltenyi biotec)和MACS系统获得CD90阳性细胞。使1x106细胞悬浮于T细胞培养基(DMEM、10%FBS、10μl/1x106细胞CD3/CD28T细胞扩增剂(Invitrogen)、10单位/mlIL-2)中,并且接种至用retronectin(50μg/ml,Takara)包被的24孔板(1x106细胞/孔)。
第二天,用包含具有4种因子(Oct3/4、Sox2、Klf4、c-Myc)的逆转录病毒(以与实施例1相同的方式制备,补充有8μg/ml聚凝胺和10单位/mlIL-2的含病毒上清液)的1ml培养基替换培养基,离心在3000rpm下执行30分钟(旋转感染法),并且在37℃下培养细胞过夜。第二天,用T细胞培养基更换培养基,并且其后每2天执行培养基更换。
在T细胞建立后第12天时,将细胞再接种到丝裂霉素处理的SNL-PH细胞上(5x104细胞/100-mm皿)。在第二天时开始,用补充有1.5μg/ml嘌呤霉素的ES培养基培养细胞。
在T细胞建立后第17天时,观察到11个GFP阳性集落,挑选所述GFP阳性集落并且接种至24孔SNL-PH平板。因此,获得3个ES样细胞克隆(408E2、E7、E8)。这些克隆显示小鼠ES细胞样形态并且对于Nanog-GFP是阳性的(图5)。通过RT-PCR证实ES细胞特异性基因(Oct3/4、Sox2、Nanog、Cripto、Dax1、ERas、Fgf4、Esg1、Rex1)的表达(图6)。掺入的外源基因的沉默是不完全的(图6)。这些克隆无一表达T细胞标记(FasL、GzmA、GzmB、Ifng)(使用ReverTra Ace kit,Takara)。用抗SSEA1抗体(Santacruz)的染色证实其表达,并且克隆还对于碱性磷酸酶活性是阳性的(图7)。此外,由于通过胚状体形成的体外分化诱导,使用AFP(R&D systems)、GATA4(Santacruz)、a-SMA(DAKO)、结蛋白(Neomarker)、bIII-微管蛋白(Chemicon)、和GFAP(DAKO)抗体的染色证实其表达;发现克隆具有分化成3个胚层的潜力(图8)。还证实克隆还促成嵌合小鼠的发生(数据未显示)。因此,所得到的GFP阳性集落被鉴定为iPS细胞。
当执行相同实验但使用Nanog-GFP Tg小鼠衍生的T细胞时,完全不产生GFP阳性集落。根据上述实验结果,证实通过缺失(灭活)p53,促进iPS细胞建立,并且通过灭活p53,可以由终末分化的T细胞建立iPS细胞。
通过将如上所述由T细胞建立的iPS细胞显微注射到ICR小鼠衍生的胚泡内,产生成年嵌合小鼠(图10)。然而,所有嵌合小鼠在7周内经历肿瘤发生。
接下来,通过PCR就T细胞受体基因(Tcrβ基因的V-(D)-J DNA)的重排检查这些建立的iPS细胞、嵌合体的各种组织和嵌合体的肿瘤。具体地,依照Curr Biol11(19),1553(2001)中描述的方法执行这个检查。简言之,通过使用引物组Dβ2(GTAGGCACCTGTGGGGAAGAAACT;SEQ ID NO:29)和Jβ2(TGAGAGCTGTCTCCTACTATCGATT;SEQ ID NO:30)对基因组DNA进行PCR扩增,并且在1.2%琼脂糖凝胶上对所得到的PCR产物进行电泳,进行检测Tcrβ基因的Dβ2-Jβ2重排的尝试。因此,检测出重排的T细胞受体的条带;iPS细胞被鉴定为得自T细胞。嵌合体肿瘤中的重排条带与iPS细胞一样密集,暗示肿瘤可能得自iPS细胞(图11)。
[实施例5:微阵列分析]
为了测定在从p53纯合-缺陷小鼠中分离的MEF和从普通、非p53-缺陷小鼠中分离的MEF之间在表达模式中是否存在差异,执行DNA微阵列分析。使用得自从每只小鼠分离的MEF的总RNA执行分析,通过Cell,131,861-872(2007)中描述的技术。结果显示于图9中。进行检测在ES细胞中特异性表达的基因的尝试(与在成纤维细胞中比较,基因在ES细胞中以10倍或更高的水平表达);在得自p53缺陷小鼠的MEF中,与野生型MEF比较,在ES细胞中特异性表达的这些基因表达得更多(图9(B))。相反,进行检测在成纤维细胞中特异性表达的基因的尝试(与在ES细胞中比较,一组基因在成纤维细胞中以10倍或更高的水平表达);在得自p53缺陷小鼠的MEF中,与野生型MEF比较,在成纤维细胞中特异性表达的这些基因的表达极低(图9(C))。根据上述结果,显示通过缺失p53,产生与ES细胞接近的状态。
[实施例6:p53缺陷和显性失活突变体的引入的作用]
使用Nanog-GFP或Oct3/4-GFP报道系统用于iPS细胞的灵敏性和特异性鉴定(Okita K.等人Nature 448,313(2007))。还包含Nanog-GFP报道物的野生型、p53+/-或p53-/-MEF以6孔板的1x105细胞/孔接种。第二天(第0天)用由Plat-E制备的逆转录病毒感染细胞。在第5天时,细胞通过细胞分选器(FACS Aria,Beckton Dekinson)以96孔板的1个细胞/孔或5000个细胞/100mm皿再接种。在4因子方案中在第13天时并且在3因子方案中在第19天时起始嘌呤霉素选择(1.5μg/ml)。对于4因子方案在第21天时并且在3因子方案中在第28天时测定GFP阳性集落数目。结果显示于图12中。
当缺乏Myc的3种因子引入Nanog-GFP、p53-野生型MEF内时,由5000个转导的成纤维细胞获得1~18个GFP阳性集落(图12a)。由Nanog-GFP、p53-杂合突变型MEF,观察到7~81个GFP阳性集落。相比之下,由Nanog-GFP、p53-无效成纤维细胞,出现38~478个GFP阳性集落。
接下来尝试由单个成纤维细胞产生iPS细胞。通过使用流式细胞仪,将1个Nanog-GFP细胞(p53野生型、杂合突变型或纯合突变型)铺平板到96孔板的孔内,所述细胞在再铺平板前5天用3种因子进行转导。再铺平板后23天,观察到在具有p53野生型成纤维细胞的每块96孔板的零或1个孔中的GFP阳性集落(图12b)。通过极大对照,观察到分别在具有p53-杂合成纤维细胞和p53-无效成纤维细胞的每块96孔板的~2和~10个孔中的GFP阳性集落。这些数据显示p53功能的丧失显著增加直接重编程的效率,并且用缺乏Myc的3种因子,高达10%的转导细胞可以变成iPS细胞。
用包括c-Myc的4种因子执行相同实验。观察到在具有p53野生型成纤维细胞的每块96孔板的零或1个孔中的GFP阳性集落(图12c)。通过极大对照,观察到分别在具有p53-杂合成纤维细胞和p53-无效成纤维细胞的每块96孔板的~9和~25个孔中的GFP阳性集落。这些数据显示Myc逆转录病毒的添加使直接重编程的效率进一步增加直至20%。
为了证实p53在iPS细胞产生中的抑制作用,执行2组实验。首先,测试p53的显性失活突变体对iPS细胞产生的作用。通过RT-PCR用p53-1S(CAC CAG GAT GAC TGC CAT GGA GGA GTC;SEQ ID NO:31)和p53-1223AS(gtg tct cag ccc tga agt cat aa;SEQ ID NO:32)扩增小鼠p53基因的互补DNA,并且亚克隆到pENTER-D-TOPO(Invitrogen)内。测序验证后,通过Gateway克隆技术(Invitrogen)将cDNA转移给pMXs-gw。通过2步PCR产生关于p53突变型的逆转录病毒载体。对于P275S突变型(Annemieke de Vries(2002),同上),用2个引物组——p53-P275S-S(tgt ttg tgc ctg ctc tgg gag aga ccg c;SEQID NO:33)和p53-1223AS,以及p53-P275S-AS(gcg gtc tct ccc aga gcaggc aca aac a;SEQ ID NO:34)和p53-1S执行第一次PCR。对于D278N突变型(Shinmura K.等人Oncogene 26(20),2939(2007)),用2个引物组——p53-D278N-S(tgc cct ggg aga aac cgc cgt aca gaa;SEQ IDNO:35)和p53-1223AS,以及p53-D278N-AS(ttc tgt acg gcg gtt tct cccagg gca;SEQ ID NO:36)和p53-1S执行第一次PCR。对于S58A突变型(Cecchinelli B.等人Cell Death Differ 13(11),1994(2006)),用2个引物组——p53-S58A-S(ttt gaa ggc cca GCt gaa gcc ctc cga;SEQID NO:37)和p53-1223AS,以及p53-S58A-AS(tcg gag ggc ttc aGC tgggcc ttc aaa;SEQ ID NO:38)和p53-1S执行第一次PCR。使每种第一次PCR的2种PCR产物混合,并且用作模板用于用引物组p53-1S和p53-1223AS的第二次PCR。将这些突变型克隆到pENTR-D-TOPO内,并且随后通过Gateway克隆技术转移给pMXs-gw。获得的表达p53突变型的逆转录病毒与3种因子一起共转导到Oct4-GFP、p53+/-或p53-/-MEF内。结果显示于图13中。
当显性失活突变体P275S引入Oct4-GFP、p53-杂合MEF内时,观察到GFP阳性集落数目中的大量增加(图13a)。相比之下,野生型p53减少iPS细胞产生效率。
其次,将编码野生型p53或反式激活阴性突变体(D278N或S58A)的cDNA置入pMXs逆转录病毒载体(Morita S.等人Gene Ther 7(12),1063(2000))内,并且将其连同关于3种重编程因子的逆转录病毒一起引入Nanog-GFP、p53-无效MEF内。发现野生型p53显著减少GFP阳性集落数目(图13b)。相比之下,反式激活阴性p53突变型显示比野生型蛋白质更弱的作用。这些数据证实p53的丧失负责在直接重编程的效率中观察到的增加。
接下来,随机扩增通过3种因子产生的、GFP阳性、p53-无效细胞的10个克隆。所有克隆显示与小鼠ES细胞相似的形态(图14,左)。由3种因子产生的iPS细胞以与ES细胞中相当的水平下表达ES细胞标记基因(图15,左)。3种转基因的表达得到有效沉默。当移植到裸鼠内,它们引起包含各种组织的畸胎瘤(图16a)。这些数据证实通过3种因子由p53-无效MEF产生的iPS细胞的多能性。
发现通过包括c-Myc的4种因子产生的iPS细胞也显示与ES细胞无法区别的形态(图14,右)。然而,ES细胞标记的表达在这些细胞中低于ES细胞中(图15,右)。此外,4种因子的转基因表达在这些细胞中仍是活性的。与这个观察一致,在裸鼠中得自这些细胞的肿瘤主要由未分化细胞组成,仅具有小区域的分化组织(图16b)。因此,在p53-无效背景中,c-Myc抑制逆转录病毒沉默且抑制分化。
[实施例7:p53抑制对人iPS细胞建立的作用]
(A)随后检查p53缺失是否增加人iPS细胞产生效率。为此,将p53的显性失活突变体(p275S或DD(Bowman T.等人Genes Dev 10(7),826(1996)))连同3种或4种重编程因子一起引入成年人表皮成纤维细胞(HDF)内。关于P275S突变型的逆转录病毒载体如实施例6中所述产生。关于DD突变型的逆转录病毒载体通过2步PCR产生。用2个引物组——p53-DD-S(cgg ata tca gcc tca aga gag cgc tgc c;SEQ ID NO:39)和p53-1223AS,以及p53-DD-AS(ggc agc gct ctc ttgagg ctg ata tcc g;SEQ ID NO:40)和p53-1S执行第一次PCR。在PLAT-E细胞中产生关于显性失活突变体、以及4种或3种重编程因子的逆转录病毒。对于iPS细胞产生,使包含每种逆转录病毒的等量PLAT-E上清液混合,并且转导给HDF。
发现用2种独立的p53的显性失活突变体,显著增加了iPS细胞集落数目(图17a,b)。当移植到SCID小鼠的睾丸内时,由3种因子和p53DD突变型产生的人iPS细胞产生包含3个胚层的各种组织的畸胎瘤(图17c)。
(B)在另一个实验中,检查针对人p53的shRNA的作用(shRNA2;Stewart S.A.等人RNA 9(4),493(2003))。用于shRNA表达的逆转录病毒载体,pMKO.1-puro(Addgene#8452)、pMKO.1-puro p53 shRNAl(Addgene #10671)和pMKO.1-puro p53shRNA2(Addgene #10672;shRNA序列显示于SEQ ID NO:28中)得自Addgene。在PLAT-E细胞中产生关于shRNA、以及4种或3种重编程因子的逆转录病毒。对于iPS细胞产生,使包含每种逆转录病毒的等量PLAT-E上清液混合,并且转导给HDF。
证实p53shRNA2有效抑制HDF中的p53蛋白质水平(图18)。当与4种重编程因子共转导时,p53shRNA显著增加人iPS细胞集落数目(图19)。在反义序列中包含1个核苷酸缺失的对照shRNA(shRNA1)不显示此种作用(图18和图19)。小鼠p53的共引入抑制shRNA2的作用。当与3种重编程因子共引入时,获得相似结果(图20)。这些数据证实p53不仅在小鼠中还在人中抑制直接重编程。
[实施例8:p53经由MDM2的功能抑制]
检查了与p53结合且抑制p53的MDM2的作用。具体地,使用逆转录病毒载体使人MDM2基因(SEQ ID NO:41)与4种或3种重编程因子共引入HDF内,并且以与实施例7中相同的方式形成iPS集落(n=4)。
因此,MDM2基因的共引入改善iPS细胞建立效率(图21)。虽然MDM2还与另一种肿瘤抑制物成视网膜细胞瘤(Rb)相互作用且抑制其,但经由shRNA抑制Rb不增加iPS细胞产生效率。这些数据暗示MDM2的作用通过p53功能的抑制引起。
[实施例9:体外分化诱导和免疫染色]
接下来,本发明人证实在实施例7(B)中用3种重编程因子和p53shRNA产生的细胞通过体外分化是否是多能性的。为了形成胚状体,收获细胞并且转移至聚羟乙基异丁烯酸酯(HEMA)包被的皿,并且温育8天。漂浮培养后,将形成的胚状体铺平板到明胶包被的板上并且温育另外6天。温育后,用4%多聚甲醛固定细胞,并且进行渗透化处理,并且用包含5%正常山羊血清、1%BSA和0.2%TritonX-100的PBS封闭。通过免疫细胞化学检查分化标记(AFP、α-SMA、βIII-微管蛋白)的表达。作为初级抗体,使用抗甲胎蛋白(AFP)(1∶100,R&D systems)、抗α-平滑肌肌动蛋白(α-SMA)(1∶500,DAKO)和抗-βIII-微管蛋白(1∶100,Chemicon)。Cy3标记的抗小鼠IgG(1∶500,Chemicon)用作次级抗体。核用Hoechst 33342(Invitrogen)进行染色。结果显示于图22中。iPS细胞分化成3个胚层,例如内胚层(AFP)、中胚层(α-SMA)和外胚层(βIII-微管蛋白)。在iPS克隆之间未发现分化潜力中的显著差异。
[实施例10:未分化标记和分化标记的RT-PCR分析]
使用Rever Tra Ace Kit(Takara)通过RT-PCR分析在实施例7(B)中获得的人iPS细胞中和在实施例9中获得分化细胞中的干细胞标记(Oct3/4、Sox2、Nanog)和分化标记(FoxA2和Sox 17(内胚层)、Msx1(中胚层)、Map2和Pax6(外胚层))的表达。用于扩增标记的引物对显示于表1中。
表1
干细胞标记
表1(续)
分化标记
内部标准
因此,用3种重编程因子和p53shRNA产生的细胞以与ES细胞中相当的水平表达Nanog、内源Oct3/4和内源Sox2(图23,泳道U)。在胚状体介导的分化后,这些细胞表达3个胚层的标记基因(图23,泳道D)。这些数据证实p53不仅在小鼠中还在人中抑制直接重编程。
[实施例11:p21通过p53抑制在改善iPS细胞建立效率中的作用]
为了阐明负责观察到的iPS细胞产生增强的p53靶基因,通过DNA微阵列比较在p53野生型MEF和p53-无效MEF之间,以及在对照HDF和p53敲降HDF之间的基因表达。在MEF中,在p53-无效MEF中1590种基因增加,并且1485种基因减少>5倍。在HDF中,通过p53shRNA,290种基因增加,并且430种基因减少>5倍。在小鼠和人中,8种增加的基因是共同的,包括v-myb成髓细胞血症病毒癌基因同系物(MYB)和RAS癌基因家族RAB39B(表2)。27种减少的基因在2个物种是共同的,包括p53,细胞周期蛋白依赖激酶抑制剂1A(p21、Cip1),BTG家族、成员2(BTG2),锌指,基质蛋白3型(ZMAT3)和MDM2。
表2
增加的基因
登记号 | 基因名称 |
NM_000809 | 智人γ-氨基丁酸(GABA)A受体,α4(GABRA4) |
NM_182767 | 智人溶质载体家族6(中性氨基酸转运蛋白),成员15(SLC6A 15),转录变体1 |
NM_014264 | 智人polo样激酶4(果蝇)(PLK4) |
NM_198391 | 智人纤连蛋白富含亮氨酸的跨膜蛋白质3(FLRT3),转录变体2 |
NM_171998 | 智人RAB39B,成员RAS癌基因家族(RAB39B) |
NM_001482 | 智人甘氨酸脒基转移酶(L-精氨酸:甘氨酸脒基转移酶)(GATM),核基因编码 |
NM_001130173 | 智人v-myb成髓细胞血症病毒癌基因同系物(禽类)(MYB),转录变体1 |
NM_004004 | 智人间隙连接蛋白,β2,26kDa(GJB2) |
减少的基因
登记号 | 基因名称 |
NM_000546 | 智人肿瘤蛋白质p53(TP53),转录变体1 |
NM_000389 | 智人细胞周期蛋白依赖激酶抑制剂1A(p21、Cip1)(CDKN1A) |
NM_001461 | 智人含黄素单加氧酶5(FMO5) |
NM_002474 | 智人肌球蛋白,重链11,平滑肌(MYH11),转录变体SM1A |
NM_006763 | 智人BTG家族,成员2(BTG2) |
NM_004455 | 智人外生骨疣(多个)-样1(EXTL1) |
NM_022470 | 智人锌指,基质蛋白3型(ZMAT3),转录变体1 |
NM_006536 | 智人CLCA家族成员2,氯化物通道调节剂(CLCA2) |
NM_001017915 | 智人多磷酸肌醇-5-磷酸酶,145kDa(INPP5D),转录变体1 |
NM_006879 | 智人Mdm2,转化的3T3细胞双重minute 2,p53结合蛋白(小鼠)(MDM2),转录变体 |
NM_004469 | 智人c-fos诱导的生长因子(血管内皮生长因子D)(FIGF) |
NM_016352 | 智人羧肽酶A4(CPA4) |
NM_024817 | 智人凝血酶敏感蛋白,1型,含结构域4(THSD4) |
NM_000526 | 智人角蛋白14(KRT14) |
NM_000846 | 智人谷胱甘肽S-转移酶A2(GSTA2) |
NM_000198 | 智人羟基-δ-5-类固醇脱氢酶,3β和类固醇δ-异构酶2(HSD3B2) |
NM_032866 | 智人扣带素(cingulin)-样1(CGNL1) |
NM_000230 | 智人瘦素(LEP) |
NM_020405 | 智人含plexin结构域1(PLXDC1) |
NM_004975 | 智人钾电压门控通道,Shab-相关亚家族,成员1(KCNB1) |
NM_001128310 | 智人SPARC-样1(hevin)(SPARCL1),转录变体1 |
NM_032588 | 智人含三重基序63(TRIM63) |
NM_022047 | 智人在FDCP 6同系物(小鼠)(DEF6)中差异表达 |
的 | |
NM_003322 | 智人肥胖样蛋白质1(TULP1) |
NM_003012 | 智人分泌的frizzled相关蛋白质1(SFRP1) |
NM_002164 | 智人吲哚胺-吡咯2,3二加氧酶(INDO) |
NM_004060 | 智人细胞周期蛋白G1(CCNG1),转录变体1 |
在这些中,通过逆转录病毒将4种增加的基因和7种减少的基因转导到HDF内,连同单独的4种重编程因子,或连同4种因子和p53shRNA。推论如果这些靶基因中的某些负责观察到的iPS细胞产生增强,那么在野生型成纤维细胞中增加基因的强迫表达将模拟p53抑制的作用,而减少基因和p53shRNA的共表达将抵消p53抑制的作用。在4种增加的基因中,无一模拟经由MDM2的强迫表达的p53抑制作用,所述MDM2与p53蛋白质结合且使p53蛋白质降解(图24a)。在7种减少的基因中,仅得自小鼠的p53和p21有效抵消p53shRNA的作用(图24b)。此外,p21的强迫表达显著减少来自p53-无效MEF的iPS细胞产生。这些数据突出在小鼠和人中在iPS细胞产生过程中p21作为p53靶的重要性。
p53-p21的抑制导致Rb的灭活。然而,经由shRNA抑制Rb不增加iPS细胞产生效率(图21)。这些数据暗示p53-p21抑制对iPS细胞产生效率的作用至少部分可归于除Rb调节外的机制。
p21蛋白质与Myc结合且抑制其转录活性(Kitaura,H.等人,J Biol.Chem.,275(14),10477-10483(2000))。这可能促成通过p53-p21抑制增加的iPS细胞产生效率。通过引入由p53应答元件或Myc应答元件驱动的萤光素酶报道物,在HDF中评估Myc活性(图24c)。证实p53活性经由shRNA减少。在这些敲降细胞中,Myc活性显著增强。效应强于由引入4种因子包括c-Myc观察到的效应。这些数据暗示Myc的活化归于通过p53-p21抑制增强的iPS细胞产生。
[实施例12:通过质粒引入建立iPS细胞]
根据Science,322,第949-953页(2008)制备表达小鼠Oct3/4、Klf4和Sox2的表达载体pCX-2A-Ms-OKS,和表达小鼠c-Myc的表达载体pCX-Ms-cMyc。
从具有小鼠Nanog报道物的胎儿p53纯合-缺陷小鼠(受精后13.5天)和野生型小鼠胎儿(受精后13.5天)中分离成纤维细胞(MEF)。将MEF以1.3x105/孔接种至用0.1%明胶(Sigma)包被的6孔培养板(Falcon)。所使用的培养肉汤是DMEM/10%FBS(补充有10%胎牛血清的DMEM(Nacalai tesque)),并且在37℃和5%CO2下培养细胞。第二天,使用FuGene6转染试剂(Roche)或Lipofectamin LTX(Invitrogen)且根据与试剂附着的方案,立刻引入pCX-2A-Ms-OKS和pCX-Ms-cMyc(第0天)。每天重复引入直至距离引入的第7天。在距离引入的第4天时,将培养基更换成补充LIF的ES细胞培养基(通过向DMEM(Nacalai Tesque)中加入15%胎牛血清、2mM L-谷氨酰胺(Invitrogen)、100μM非必需氨基酸(Invitrogen)、100μM 2-巯基乙醇(Invitrogen)、50U/mL青霉素(Invitrogen)和50μg/mL链霉素(Invitrogen)制备)。随后,每2天用新鲜供应更换补充LIF的ES细胞培养基,直至可观察到集落到。从引入后第21天起执行用嘌呤霉素(1.5μg/mL)的选择,并且在转导后第33天时计数GFP阳性集落。时间表的概括显示于图25a,并且获得的GFP阳性集落数目显示于图25b中。当4种基因通过质粒引入野生型MEF内时(在图25b中+/+),未形成GFP阳性集落。当4种基因通过质粒引入p53纯合-缺陷ME内时(在图25b中-/-),获得许多GFP阳性集落。
为了检查质粒整合到基因组内,设计了能够扩增质粒的每个部分的16种PCR引物(参见Science,322,第949-953页(2008),图2A),并且其中的11种引物用于基因组PCR。结果显示于图25c中。在获得的12个GFP阳性克隆中的6个克隆中未观察到外源DNA的扩增(图25c:上图)。此外,通过使用Oct3/4、Sox2、Klf4和c-Myc作为探针的Southern印迹分析,在这些克隆中未检测出外源基因的整合(图25c:下图)。上述结果揭示这些iPS细胞不将pCX-2A-Ms-OKS和pCX-Ms-cMyc质粒整合到宿主基因组内。
获得的细胞的照片显示于图25d中(左上:相差图像,右上:GFP阳性集落图像,左下:相差图像和GFP阳性集落图像的合并)。因为细胞具有在形态上与小鼠ES细胞无法区分的形式,并且是GFP阳性的,所以证实iPS细胞的建立。此外,将不含质粒整合的iPS克隆注射给ICR小鼠衍生的胚泡。结果显示于图25d中,右下图。根据毛发颜色判断,成年嵌合体可以由iPS克隆产生。结果证实不含质粒整合的iPS细胞具有多能性。
虽然本发明已着重于优选实施方案进行描述,但对于本领域技术人员显而易见的是,优选实施方案可以进行修改。本发明意欲本发明可以通过除本说明书中详细描述的那些外的方法来实现。因此,本发明包含了在所附“权利要求”的要旨和范围中包含的所有修改。
本文引用的所有出版物包括专利和专利申请中公开的内容在此整体引入作为参考,其程度与它们已在本文中公开一样。
本申请基于美国临时专利申请号61/076,487、61/095,573、61/194,700、61/200,307和61/209,686,所述美国临时专利申请的内容在此引入作为参考。
序列表
<110>Kyoto University
<120>有效建立诱导的多能干细胞的方法
<130>091382
<150>US 61/076,487
<151>2008-06-27
<150>US 61/095,573
<151>2008-09-09
<150>US 61/194,700
<151>2008-09-30
<150>US 61/200,307
<151>2008-11-25
<150>US 61/209,686
<151>2009-03-10
<160>60
<170>PatentIn version 3.5
<210>1
<211>1782
<212>DNA
<213>小鼠(Mus musculus)
<220>
<221>CDS
<222>(129)..(1301)
<400>1
aagttctgta gcttcagttc attgggacca tcctggctgt aggtagcgac tacagttagg 60
gggcacctag cattcaggcc ctcatcctcc tccttcccag cagggtgtca cgcttctccg 120
aagactgg atg act gcc atg gag gag tca cag tcg gat atc agc ctc gag 170
Met Thr Ala Met Glu Glu Ser Gln Ser Asp Ile Ser Leu Glu
1 5 10
ctc cct ctg agc cag gag aca ttt tca ggc tta tgg aaa cta ctt cct 218
Leu Pro Leu Ser Gln Glu Thr Phe Ser Gly Leu Trp Lys Leu Leu Pro
15 20 25 30
cca gaa gat atc ctg cca tca cct cac tgc atg gac gat ctg ttg ctg 266
Pro Glu Asp Ile Leu Pro Ser Pro His Cys Met Asp Asp Leu Leu Leu
35 40 45
ccc cag gat gtt gag gag ttt ttt gaa ggc cca agt gaa gcc ctc cga 314
Pro Gln Asp Val Glu Glu Phe Phe Glu Gly Pro Ser Glu Ala Leu Arg
50 55 60
gtg tca gga gct cct gca gca cag gac cct gtc acc gag acc cct ggg 362
Val Ser Gly Ala Pro Ala Ala Gln Asp Pro Val Thr Glu Thr Pro Gly
65 70 75
cca gtg gcc cct gcc cca gcc act cca tgg ccc ctg tca tct ttt gtc 410
Pro Val Ala Pro Ala Pro Ala Thr Pro Trp Pro Leu Ser Ser Phe Val
80 85 90
cct tct caa aaa act tac cag ggc aac tat ggc ttc cac ctg ggc ttc 458
Pro Ser Gln Lys Thr Tyr Gln Gly Asn Tyr Gly Phe His Leu Gly Phe
95 100 105 110
ctg cag tct ggg aca gcc aag tct gtt atg tgc acg tac tct cct ccc 506
Leu Gln Ser Gly Thr Ala Lys Ser Val Met Cys Thr Tyr Ser Pro Pro
115 120 125
ctc aat aag cta ttc tgc cag ctg gcg aag acg tgc cct gtg cag ttg 554
Leu Asn Lys Leu Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu
130 135 140
tgg gtc agc gcc aca cct cca gct ggg agc cgt gtc cgc gcc atg gcc 602
Trp Val Ser Ala Thr Pro Pro Ala Gly Ser Arg Val Arg Ala Met Ala
145 150 155
atc tac aag aag tca cag cac atg acg gag gtc gtg aga cgc tgc ccc 650
Ile Tyr Lys Lys Ser Gln His Met Thr Glu Val Val Arg Arg Cys Pro
160 165 170
cac cat gag cgc tgc tcc gat ggt gat ggc ctg gct cct ccc cag cat 698
His His Glu Arg Cys Ser Asp Gly Asp Gly Leu Ala Pro Pro Gln His
175 180 185 190
ctt atc cgg gtg gaa gga aat ttg tat ccc gag tat ctg gaa gac agg 746
Leu Ile Arg Val Glu Gly Asn Leu Tyr Pro Glu Tyr Leu Glu Asp Arg
195 200 205
cag act ttt cgc cac agc gtg gtg gta cct tat gag cca ccc gag gcc 794
Gln Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Ala
210 215 220
ggc tct gag tat acc acc atc cac tac aag tac atg tgt aat agc tcc 842
Gly Ser Glu Tyr Thr Thr Ile His Tyr Lys Tyr Met Cys Asn Ser Ser
225 230 235
tgc atg ggg ggc atg aac cgc cga cct atc ctt acc atc atc aca ctg 890
Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu
240 245 250
gaa gac tcc agt ggg aac ctt ctg gga cgg gac agc ttt gag gtt cgt 938
Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asp Ser Phe Glu Val Arg
255 260 265 270
gtt tgt gcc tgc cct ggg aga gac cgc cgt aca gaa gaa gaa aat ttc 986
Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn Phe
275 280 285
cgc aaa aag gaa gtc ctt tgc cct gaa ctg ccc cca ggg agc gca aag 1034
Arg Lys Lys Glu Val Leu Cys Pro Glu Leu Pro Pro Gly Ser Ala Lys
290 295 300
aga gcg ctg ccc acc tgc aca agc gcc tct ccc ccg caa aag aaa aaa 1082
Arg Ala Leu Pro Thr Cys Thr Ser Ala Ser Pro Pro Gln Lys Lys Lys
305 310 315
cca ctt gat gga gag tat ttc acc ctc aag atc cgc ggg cgt aaa cgc 1130
Pro Leu Asp Gly Glu Tyr Phe Thr Leu Lys Ile Arg Gly Arg Lys Arg
320 325 330
ttc gag atg ttc cgg gag ctg aat gag gcc tta gag tta aag gat gcc 1178
Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala
335 340 345 350
cat gct aca gag gag tct gga gac agc agg gct cac tcc agc tac ctg 1226
His Ala Thr Glu Glu Ser Gly Asp Ser Arg Ala His Ser Ser Tyr Leu
355 360 365
aag acc aag aag ggc cag tct act tcc cgc cat aaa aaa aca atg gtc 1274
Lys Thr Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Thr Met Val
370 375 380
aag aaa gtg ggg cct gac tca gac tga ctgcctctgc atcccgtccc 1321
Lys Lys Val Gly Pro Asp Ser Asp
385 390
catcaccagc ctccccctct ccttgctgtc ttatgacttc agggctgaga cacaatcctc 1381
ccggtccctt ctgctgcctt ttttaccttg tagctagggc tcagccccct ctctgagtag 1441
tggttcctgg cccaagttgg ggaataggtt gatagttgtc aggtctctgc tggcccagcg 1501
aaattctatc cagccagttg ttggaccctg gcacctacaa tgaaatctca ccctacccca 1561
caccctgtaa gattctatct tgggccctca tagggtccat atcctccagg gcctactttc 1621
cttccattct gcaaagcctg tctgcattta tccacccccc accctgtctc cctctttttt 1681
ttttttttac ccctttttat atatcaattt cctattttac aataaaattt tgttatcact 1741
taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1782
<210>2
<211>390
<212>PRT
<213>小鼠
<400>2
Met Thr Ala Met Glu Glu Ser Gln Ser Asp Ile Ser Leu Glu Leu Pro
1 5 10 15
Leu Ser Gln Glu Thr Phe Ser Gly Leu Trp Lys Leu Leu Pro Pro Glu
20 25 30
Asp Ile Leu Pro Ser Pro His Cys Met Asp Asp Leu Leu Leu Pro Gln
35 40 45
Asp Val Glu Glu Phe Phe Glu Gly Pro Ser Glu Ala Leu Arg Val Ser
50 55 60
Gly Ala Pro Ala Ala Gln Asp Pro Val Thr Glu Thr Pro Gly Pro Val
65 70 75 80
Ala Pro Ala Pro Ala Thr Pro Trp Pro Leu Ser Ser Phe Val Pro Ser
85 90 95
Gln Lys Thr Tyr Gln Gly Asn Tyr Gly Phe His Leu Gly Phe Leu Gln
100 105 110
Ser Gly Thr Ala Lys Ser Val Met Cys Thr Tyr Ser Pro Pro Leu Asn
115 120 125
Lys Leu Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu Trp Val
130 135 140
Ser Ala Thr Pro Pro Ala Gly Ser Arg Val Arg Ala Met Ala Ile Tyr
145 150 155 160
Lys Lys Ser Gln His Met Thr Glu Val Val Arg Arg Cys Pro His His
165 170 175
Glu Arg Cys Ser Asp Gly Asp Gly Leu Ala Pro Pro Gln His Leu Ile
180 185 190
Arg Val Glu Gly Asn Leu Tyr Pro Glu Tyr Leu Glu Asp Arg Gln Thr
195 200 205
Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Ala Gly Ser
210 215 220
Glu Tyr Thr Thr Ile His Tyr Lys Tyr Met Cys Asn Ser Ser Cys Met
225 230 235 240
Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu Asp
245 250 255
Ser Ser Gly Asn Leu Leu Gly Arg Asp Ser Phe Glu Val Arg Val Cys
260 265 270
Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn Phe Arg Lys
275 280 285
Lys Glu Val Leu Cys Pro Glu Leu Pro Pro Gly Ser Ala Lys Arg Ala
290 295 300
Leu Pro Thr Cys Thr Ser Ala Ser Pro Pro Gln Lys Lys Lys Pro Leu
305 310 315 320
Asp Gly Glu Tyr Phe Thr Leu Lys Ile Arg Gly Arg Lys Arg Phe Glu
325 330 335
Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala His Ala
340 345 350
Thr Glu Glu Ser Gly Asp Ser Arg Ala His Ser Ser Tyr Leu Lys Thr
355 360 365
Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Thr Met Val Lys Lys
370 375 380
Val Gly Pro Asp Ser Asp
385 390
<210>3
<211>2586
<212>DNA
<213>智人(Homo sapiens)
<220>
<221>CDS
<222>(198)..(1379)
<400>3
gattggggtt ttcccctccc atgtgctcaa gactggcgct aaaagttttg agcttctcaa 60
aagtctagag ccaccgtcca gggagcaggt agctgctggg ctccggggac actttgcgtt 120
cgggctggga gcgtgctttc cacgacggtg acacgcttcc ctggattggc agccagactg 180
ccttccgggt cactgcc atg gag gag ccg cag tca gat cct agc gtc gag 230
Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu
1 5 10
ccc cct ctg agt cag gaa aca ttt tca gac cta tgg aaa cta ctt cct 278
Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro
15 20 25
gaa aac aac gtt ctg tcc ccc ttg ccg tcc caa gca atg gat gat ttg 326
Glu Asn Asn Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu
30 35 40
atg ctg tcc ccg gac gat att gaa caa tgg ttc act gaa gac cca ggt 374
Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly
45 50 55
cca gat gaa gct ccc aga atg cca gag gct gct ccc ccc gtg gcc cct 422
Pro Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
60 65 70 75
gca cca gca gct cct aca ccg gcg gcc cct gca cca gcc ccc tcc tgg 470
Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp
80 85 90
ccc ctg tca tct tct gtc cct tcc cag aaa acc tac cag ggc agc tac 518
Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr
95 100 105
ggt ttc cgt ctg ggc ttc ttg cat tct ggg aca gcc aag tct gtg act 566
Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr
110 115 120
tgc acg tac tcc cct gcc ctc aac aag atg ttt tgc caa ctg gcc aag 614
Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys
125 130 135
acc tgc cct gtg cag ctg tgg gtt gat tcc aca ccc ccg ccc ggc acc 662
Thr Cys Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr
140 145 150 155
cgc gtc cgc gcc atg gcc atc tac aag cag tca cag cac atg acg gag 710
Arg Val Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu
160 165 170
gtt gtg agg cgc tgc ccc cac cat gag cgc tgc tca gat agc gat ggt 758
Val Val Arg Arg Cys Pro His His Glu Arg Cys Ser Asp Ser Asp Gly
175 180 185
ctg gcc cct cct cag cat ctt atc cga gtg gaa gga aat ttg cgt gtg 806
Leu Ala Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn Leu Arg Val
190 195 200
gag tat ttg gat gac aga aac act ttt cga cat agt gtg gtg gtg ccc 854
Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val Val Val Pro
205 210 215
tat gag ccg cct gag gtt ggc tct gac tgt acc acc atc cac tac aac 902
Tyr Glu Pro Pro Glu Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn
220 225 230 235
tac atg tgt aac agt tcc tgc atg ggc ggc atg aac cgg agg ccc atc 950
Tyr Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile
240 245 250
ctc acc atc atc aca ctg gaa gac tcc agt ggt aat cta ctg gga cgg 998
Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg
255 260 265
aac agc ttt gag gtg cgt gtt tgt gcc tgt cct ggg aga gac cgg cgc 1046
Asn Ser Phe Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg
270 275 280
aca gag gaa gag aat ctc cgc aag aaa ggg gag cct cac cac gag ctg 1094
Thr Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu
285 290 295
ccc cca ggg agc act aag cga gca ctg ccc aac aac acc agc tcc tct 1142
Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
300 305 310 315
ccc cag cca aag aag aaa cca ctg gat gga gaa tat ttc acc ctt cag 1190
Pro Gln Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln
320 325 330
atc cgt ggg cgt gag cgc ttc gag atg ttc cga gag ctg aat gag gcc 1238
Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala
335 340 345
ttg gaa ctc aag gat gcc cag gct ggg aag gag cca ggg ggg agc agg 1286
Leu Glu LeuLys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg
350 355 360
gct cac tcc agc cac ctg aag tcc aaa aag ggt cag tct acc tcc cgc 1334
Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg
365 370 375
cat aaa aaa ctc atg ttc aag aca gaa ggg cct gac tca gac tga 1379
His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp
380 385 390
cattctccac ttcttgttcc ccactgacag cctcccaccc ccatctctcc ctcccctgcc 1439
attttgggtt ttgggtcttt gaacccttgc ttgcaatagg tgtgcgtcag aagcacccag 1499
gacttccatt tgctttgtcc cggggctcca ctgaacaagt tggcctgcac tggtgttttg 1559
ttgtggggag gaggatgggg agtaggacat accagcttag attttaaggt ttttactgtg 1619
agggatgttt gggagatgta agaaatgttc ttgcagttaa gggttagttt acaatcagcc 1679
acattctagg taggggccca cttcaccgta ctaaccaggg aagctgtccc tcactgttga 1739
attttctcta acttcaaggc ccatatctgt gaaatgctgg catttgcacc tacctcacag 1799
agtgcattgt gagggttaat gaaataatgt acatctggcc ttgaaaccac cttttattac 1859
atggggtcta gaacttgacc cccttgaggg tgcttgttcc ctctccctgt tggtcggtgg 1919
gttggtagtt tctacagttg ggcagctggt taggtagagg gagttgtcaa gtctctgctg 1979
gcccagccaa accctgtctg acaacctctt ggtgaacctt agtacctaaa aggaaatctc 2039
accccatccc acaccctgga ggatttcatc tcttgtatat gatgatctgg atccaccaag 2099
acttgtttta tgctcagggt caatttcttt tttctttttt tttttttttt ttctttttct 2159
ttgagactgg gtctcgcttt gttgcccagg ctggagtgga gtggcgtgat cttggcttac 2219
tgcagccttt gcctccccgg ctcgagcagt cctgcctcag cctccggagt agctgggacc 2279
acaggttcat gccaccatgg ccagccaact tttgcatgtt ttgtagagat ggggtctcac 2339
agtgttgccc aggctggtct caaactcctg ggctcaggcg atccacctgt ctcagcctcc 2399
cagagtgctg ggattacaat tgtgagccac cacgtccagc tggaagggtc aacatctttt 2459
acattctgca agcacatctg cattttcacc ccacccttcc cctccttctc cctttttata 2519
tcccattttt atatcgatct cttattttac aataaaactt tgctgccacc tgtgtgtctg 2579
aggggtg 2586
<210>4
<211>393
<212>PRT
<213>智人
<400>4
Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln
1 5 10 15
Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu
20 25 30
Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp
35 40 45
Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro
50 55 60
Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro
65 70 75 80
Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser
85 90 95
Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly
100 105 110
Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro
115 120 125
Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln
130 135 140
Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met
145 150 155 160
Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys
165 170 175
Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln
180 185 190
His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp
195 200 205
Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu
210 215 220
Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
225 230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr
245 250 255
Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val
260 265 270
Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn
275 280 285
Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr
290 295 300
Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys
305 310 315 320
Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu
325 330 335
Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp
340 345 350
Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His
355 360 365
Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met
370 375 380
Phe Lys Thr Glu Gly Pro Asp Ser Asp
385 390
<210>5
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>5
tttgactgga tgactgccat ggttcaagag accatggcag tcatccagtc tttttt 56
<210>6
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>6
tttgatatcc tgccatcacc tcttcaagag agaggtgatg gcaggatatc tttttt 56
<210>7
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>7
tttggcccaa gtgaagccct ccttcaagag aggagggctt cacttgggcc tttttt 56
<210>8
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>8
tttgtgaagc cctccgagtg tcttcaagag agacactcgg agggcttcac tttttt 56
<210>9
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>9
tttgccctcc gagtgtcagg agttcaagag actcctgaca ctcggagggc tttttt 56
<210>10
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>10
tttgtctgtt atgtgcacgt acttcaagag agtacgtgca cataacagac tttttt 56
<210>11
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>11
tttgtactct cctcccctca atttcaagag aattgagggg aggagagtac tttttt 56
<210>12
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>12
tttgctattc tgccagctgg cgttcaagag acgccagctg gcagaatagc tttttt 56
<210>13
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>13
tttgacgtgc cctgtgcagt tgttcaagag acaactgcac agggcacgtc tttttt 56
<210>14
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>14
tttgaagtca cagcacatga cgttcaagag acgtcatgtg ctgtgacttc tttttt 56
<210>15
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>15
tttgtcacag cacatgacgg agttcaagag actccgtcat gtgctgtgac tttttt 56
<210>16
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>16
tttggaaatt tgtatcccga gtttcaagag aactcgggat acaaatttcc tttttt 56
<210>17
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>17
tttgtacatg tgtaatagct ccttcaagag aggagctatt acacatgtac tttttt 56
<210>18
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>18
tttgactcca gtgggaacct tcttcaagag agaaggttcc cactggagtc tttttt 56
<210>19
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>19
tttgtccttt gccctgaact gcttcaagag agcagttcag ggcaaaggac tttttt 56
<210>20
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>20
tttgatccgc gggcgtaaac gcttcaagag agcgtttacg cccgcggatc tttttt 56
<210>21
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>21
tttgaccaag aagggccagt ctttcaagag aagactggcc cttcttggtc tttttt 56
<210>22
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>22
tttgaaagtg gggcctgact cattcaagag atgagtcagg ccccactttc tttttt 56
<210>23
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>23
tttgttgggg aataggttga tattcaagag atatcaacct attccccaac tttttt 56
<210>24
<211>56
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>24
tttgattcta tcttgggccc tcttcaagag agagggccca agatagaatc tttttt 56
<210>25
<211>59
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>25
tttgcautac aggtacgtgt gtagtgtgct gtcctacaca tgtacttgta gtgtttttt 59
<210>26
<211>59
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>26
tttgcagtut acttuccgcc gtagtgtgct gtcctatggc gggaagtaga ctgtttttt 59
<210>27
<211>10
<212>DNA
<213>人工序列
<220>
<223>p53应答元件
<400>27
rrrgwwcyyy 10
<210>28
<211>48
<212>DNA
<213>人工序列
<220>
<223>针对p53的shRNA
<400>28
gactccagtg gtaatctact gctcgagcag tagattacca ctggagtc 48
<210>29
<211>24
<212>DNA
<213>人工序列
<220>
<223>引物
<400>29
gtaggcacct gtggggaaga aact 24
<210>30
<211>25
<212>DNA
<213>人工序列
<220>
<223>引物
<400>30
tgagagctgt ctcctactat cgatt 25
<210>31
<211>27
<212>DNA
<213>人工序列
<220>
<223>引物
<400>31
caccaggatg actgccatgg aggagtc 27
<210>32
<211>23
<212>DNA
<213>人工序列
<220>
<223>引物
<400>32
gtgtctcagc cctgaagtca taa 23
<210>33
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>33
tgtttgtgcc tgctctggga gagaccgc 28
<210>34
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>34
gcggtctctc ccagagcagg cacaaaca 28
<210>35
<211>27
<212>DNA
<213>人工序列
<220>
<223>引物
<400>35
tgccctggga gaaaccgccg tacagaa 27
<210>36
<211>27
<212>DNA
<213>人工序列
<220>
<223>引物
<400>36
ttctgtacgg cggtttctcc cagggca 27
<210>37
<211>27
<212>DNA
<213>人工序列
<220>
<223>引物
<400>37
tttgaaggcc cagctgaagc cctccga 27
<210>38
<211>27
<212>DNA
<213>人工序列
<220>
<223>引物
<400>38
tcggagggct tcagctgggc cttcaaa 27
<210>39
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>39
cggatatcag cctcaagaga gcgctgcc 28
<210>40
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>40
ggcagcgctc tcttgaggct gatatccg 28
<210>41
<211>2357
<212>DNA
<213>智人
<220>
<221>CDS
<222>(279)..(1772)
<400>41
cgagcttggc tgcttctggg gcctgtgtgg ccctgtgtgt cggaaagatg gagcaagaag 60
ccgagcccga ggggcggccg cgacccctct gaccgagatc ctgctgcttt cgcagccagg 120
agcaccgtcc ctccccggat tagtgcgtac gagcgcccag tgccctggcc cggagagtgg 180
aatgatcccc gaggcccagg gcgtcgtgct tccgcgcgcc ccgtgaagga aactggggag 240
tcttgaggga cccccgactc caagcgcgaa aaccccgg atg gtg agg agc agg caa 296
Met Val Arg Ser Arg Gln
1 5
atg tgc aat acc aac atg tct gta cct act gat ggt gct gta acc acc 344
Met Cys Asn Thr Asn Met Ser Val Pro Thr Asp Gly Ala Val Thr Thr
10 15 20
tca cag att cca gct tcg gaa caa gag acc ctg gtt aga cca aag cca 392
Ser Gln Ile Pro Ala Ser Glu Gln Glu Thr Leu Val Arg Pro Lys Pro
25 30 35
ttg ctt ttg aag tta tta aag tct gtt ggt gca caa aaa gac act tat 440
Leu Leu Leu Lys Leu Leu Lys Ser Val Gly Ala Gln Lys Asp Thr Tyr
40 45 50
act atg aaa gag gtt ctt ttt tat ctt ggc cag tat att atg act aaa 488
Thr Met Lys Glu Val Leu Phe Tyr Leu Gly Gln Tyr Ile Met Thr Lys
55 60 65 70
cga tta tat gat gag aag caa caa cat att gta tat tgt tca aat gat 536
Arg Leu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser Asn Asp
75 80 85
ctt cta gga gat ttg ttt ggc gtg cca agc ttc tct gtg aaa gag cac 584
Leu Leu Gly Asp Leu Phe Gly Val Pro Ser Phe Ser Val Lys Glu His
90 95 100
agg aaa ata tat acc atg atc tac agg aac ttg gta gta gtc aat cag 632
Arg Lys Ile Tyr Thr Met Ile Tyr Arg Asn Leu Val Val Val Asn Gln
105 110 115
cag gaa tca tcg gac tca ggt aca tct gtg agt gag aac agg tgt cac 680
Gln Glu Ser Ser Asp Ser Gly Thr Ser Val Ser Glu Asn Arg Cys His
120 125 130
ctt gaa ggt ggg agt gat caa aag gac ctt gta caa gag ctt cag gaa 728
Leu Glu Gly Gly Ser Asp Gln Lys Asp Leu Val Gln Glu Leu Gln Glu
135 140 145 150
gag aaa cct tca tct tca cat ttg gtt tct aga cca tct acc tca tct 776
Glu Lys Pro Ser Ser Ser His Leu Val Ser Arg Pro Ser Thr Ser Ser
155 160 165
aga agg aga gca att agt gag aca gaa gaa aat tca gat gaa tta tct 824
Arg Arg Arg Ala Ile Ser Glu Thr Glu Glu Asn Ser Asp Glu Leu Ser
170 175 180
ggt gaa cga caa aga aaa cgc cac aaa tct gat agt att tcc ctt tcc 872
Gly Glu Arg Gln Arg Lys Arg His Lys Ser Asp Ser Ile Ser Leu Ser
185 190 195
ttt gat gaa agc ctg gct ctg tgt gta ata agg gag ata tgt tgt gaa 920
Phe Asp Glu Ser Leu Ala Leu Cys ValIle Arg Glu Ile Cys Cys Glu
200 205 210
aga agc agt agc agt gaa tct aca ggg acg cca tcg aat ccg gat ctt 968
Arg Ser Ser Ser Ser Glu Ser Thr Gly Thr Pro Ser Asn Pro Asp Leu
215 220 225 230
gat gct ggt gta agt gaa cat tca ggt gat tgg ttg gat cag gat tca 1016
Asp Ala Gly Val Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser
235 240 245
gtt tca gat cag ttt agt gta gaa ttt gaa gtt gaa tct ctc gac tca 1064
Val Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu Asp Ser
250 255 260
gaa gat tat agc ctt agt gaa gaa gga caa gaa ctc tca gat gaa gat 1112
Glu Asp Tyr Ser Leu Ser Glu Glu Gly Gln Glu Leu Ser Asp Glu Asp
265 270 275
gat gag gta tat caa gtt act gtg tat cag gca ggg gag agt gat aca 1160
Asp Glu Val Tyr Gln Val Thr Val Tyr Gln Ala Gly Glu Ser Asp Thr
280 285 290
gat tca ttt gaa gaa gat cct gaa att tcc tta gct gac tat tgg aaa 1208
Asp Ser Phe Glu Glu Asp Pro Glu Ile Ser Leu Ala Asp Tyr Trp Lys
295 300 305 310
tgc act tca tgc aat gaa atg aat ccc ccc ctt cca tca cat tgc aac 1256
Cys Thr Ser Cys Asn Glu Met AsnPro Pro Leu Pro Ser His Cys Asn
315 320 325
aga tgt tgg gcc ctt cgt gag aat tgg ctt cct gaa gat aaa ggg aaa 1304
Arg Cys Trp Ala Leu Arg Glu Asn Trp Leu Pro Glu Asp Lys Gly Lys
330 335 340
gat aaa ggg gaa atc tct gag aaa gcc aaa ctg gaa aac tca aca caa 1352
Asp Lys Gly Glu Ile Ser Glu Lys Ala Lys Leu Glu Asn Ser Thr Gln
345 350 355
gct gaa gag ggc ttt gat gtt cct gat tgt aaa aaa act ata gtg aat 1400
Ala Glu Glu Gly Phe Asp Val Pro Asp Cys Lys Lys Thr Ile Val Asn
360 365 370
gat tcc aga gag tca tgt gtt gag gaa aat gat gat aaa att aca caa 1448
Asp Ser Arg Glu Ser Cys Val Glu Glu Asn Asp Asp Lys Ile Thr Gln
375 380 385 390
gct tca caa tca caa gaa agt gaa gac tat tct cag cca tca act tct 1496
Ala Ser Gln Ser Gln Glu Ser Glu Asp Tyr Ser Gln Pro Ser Thr Ser
395 400 405
agt agc att att tat agc agc caa gaa gat gtg aaa gag ttt gaa agg 1544
Ser Ser Ile Ile Tyr Ser Ser Gln Glu Asp Val Lys Glu Phe Glu Arg
410 415 420
gaa gaa acc caa gac aaa gaa gag agt gtg gaa tct agt ttg ccc ctt 1592
Glu Glu Thr Gln Asp Lys Glu Glu Ser Val Glu Ser Ser Leu Pro Leu
425 430 435
aat gcc att gaa cct tgt gtg att tgt caa ggt cga cct aaa aat ggt 1640
Asn Ala Ile Glu Pro Cys ValIle Cys Gln Gly Arg Pro Lys Asn Gly
440 445 450
tgc att gtc cat ggc aaa aca gga cat ctt atg gcc tgc ttt aca tgt 1688
Cys Ile Val His Gly Lys Thr Gly His Leu Met Ala Cys Phe Thr Cys
455 460 465 470
gca aag aag cta aag aaa agg aat aag ccc tgc cca gta tgt aga caa 1736
Ala Lys Lys Leu Lys Lys Arg Asn Lys Pro Cys Pro Val Cys Arg Gln
475 480 485
cca att caa atg att gtg cta act tat ttc ccc tag ttgacctgtc 1782
Pro Ile Gln Met Ile Val Leu Thr Tyr Phe Pro
490 495
tataagagaa ttatatattt ctaactatat aaccctagga atttagacaa cctgaaattt 1842
attcacatat atcaaagtga gaaaatgcct caattcacat agatttcttc tctttagtat 1902
aattgaccta ctttggtagt ggaatagtga atacttacta taatttgact tgaatatgta 1962
gctcatcctt tacaccaact cctaatttta aataatttct actctgtctt aaatgagaag 2022
tacttggttt ttttttttct taaatatgta tatgacattt aaatgtaact tattattttt 2082
tttgagaccg agtcttgctc tgttacccag gctggagtgc agtggcgtga tcttggctca 2142
ctgcaagctc tgcctcccgg gttcgcacca ttctcctgcc tcagcctccc aattagcttg 2202
gcctacagtc atctgccacc acacctggct aattttttgt acttttagta gagacagggt 2262
ttcaccgtgt tagccaggat ggtctcgatc tcctgacctc gtgatccgcc cacctcggcc 2322
tcccaaagtg ctgggattac aggcatgagc caccg 2357
<210>42
<211>497
<212>PRT
<213>智人
<400>42
Met Val Arg Ser Arg Gln Met Cys Asn Thr Asn Met Ser Val Pro Thr
1 5 10 15
Asp Gly Ala Val Thr Thr Ser Gln Ile Pro Ala Ser Glu Gln Glu Thr
20 25 30
Leu Val Arg Pro Lys Pro Leu Leu Leu Lys Leu Leu Lys Ser Val Gly
35 40 45
Ala Gln Lys Asp Thr Tyr Thr Met Lys Glu Val Leu Phe Tyr Leu Gly
50 55 60
Gln Tyr Ile Met Thr Lys Arg Leu Tyr Asp Glu Lys Gln Gln His Ile
65 70 75 80
Val Tyr Cys Ser Asn Asp Leu Leu Gly Asp Leu Phe Gly Val Pro Ser
85 90 95
Phe Ser Val Lys Glu His Arg Lys Ile Tyr Thr Met Ile Tyr Arg Asn
100 105 110
Leu Val Val Val Asn Gln Gln Glu Ser Ser Asp Ser Gly Thr Ser Val
115 120 125
Ser Glu Asn Arg Cys His Leu Glu Gly Gly Ser Asp Gln Lys Asp Leu
130 135 140
Val Gln Glu Leu Gln Glu Glu Lys Pro Ser Ser Ser His Leu Val Ser
145 150 155 160
Arg Pro Ser Thr Ser Ser Arg Arg Arg Ala Ile Ser Glu Thr Glu Glu
165 170 175
Asn Ser Asp Glu Leu Ser Gly Glu Arg Gln Arg Lys Arg His Lys Ser
180 185 190
Asp Ser Ile Ser Leu Ser Phe Asp Glu Ser Leu Ala Leu Cys Val Ile
195 200 205
Arg Glu Ile Cys Cys Glu Arg Ser Ser Ser Ser Glu Ser Thr Gly Thr
210 215 220
Pro Ser Asn Pro Asp Leu Asp Ala Gly Val Ser Glu His Ser Gly Asp
225 230 235 240
Trp Leu Asp Gln Asp Ser Val Ser Asp Gln Phe Ser Val Glu Phe Glu
245 250 255
Val Glu Ser Leu Asp Ser Glu Asp Tyr Ser Leu Ser Glu Glu Gly Gln
260 265 270
Glu Leu Ser Asp Glu Asp Asp Glu Val Tyr Gln Val Thr Val Tyr Gln
275 280 285
Ala Gly Glu Ser Asp Thr Asp Ser Phe Glu Glu Asp Pro Glu Ile Ser
290 295 300
Leu Ala Asp Tyr Trp Lys Cys Thr Ser Cys Asn Glu Met Asn Pro Pro
305 310 315 320
Leu Pro Ser His Cys Asn Arg Cys Trp Ala Leu Arg Glu Asn Trp Leu
325 330 335
Pro Glu Asp Lys Gly Lys Asp Lys Gly Glu Ile Ser Glu Lys Ala Lys
340 345 350
Leu Glu Asn Ser Thr Gln Ala Glu Glu Gly Phe Asp Val Pro Asp Cys
355 360 365
Lys Lys Thr Ile Val Asn Asp Ser Arg Glu Ser Cys Val Glu Glu Asn
370 375 380
Asp Asp Lys Ile Thr Gln Ala Ser Gln Ser Gln Glu Ser Glu Asp Tyr
385 390 395 400
Ser Gln Pro Ser Thr Ser Ser Ser Ile Ile Tyr Ser Ser Gln Glu Asp
405 410 415
Val Lys Glu Phe Glu Arg Glu Glu Thr Gln Asp Lys Glu Glu Ser Val
420 425 430
Glu Ser Ser Leu Pro Leu Asn Ala Ile Glu Pro Cys Val Ile Cys Gln
435 440 445
Gly Arg Pro Lys Asn Gly Cys Ile Val His Gly Lys Thr Gly His Leu
450 455 460
Met Ala Cys Phe Thr Cys Ala Lys Lys Leu Lys Lys Arg Asn Lys Pro
465 470 475 480
Cys Pro Val Cys Arg Gln Pro Ile Gln Met Ile Val Leu Thr Tyr Phe
485 490 495
Pro
<210>43
<211>24
<212>DNA
<213>人工序列
<220>
<223>引物
<400>43
gacaggggga ggggaggagc tagg 24
<210>44
<211>26
<212>DNA
<213>人工序列
<220>
<223>引物
<400>44
cttccctcca accagttgcc ccaaac 26
<210>45
<211>26
<212>DNA
<213>人工序列
<220>
<223>引物
<400>45
gggaaatggg aggggtgcaa aagagg 26
<210>46
<211>26
<212>DNA
<213>人工序列
<220>
<223>引物
<400>46
ttgcgtgagt gtggatggga ttggtg 26
<210>47
<211>25
<212>DNA
<213>人工序列
<220>
<223>引物
<400>47
cagccccgat tcttccacca gtccc 25
<210>48
<211>25
<212>DNA
<213>人工序列
<220>
<223>引物
<400>48
cggaagattc ccagtcgggt tcacc 25
<210>49
<211>26
<212>DNA
<213>人工序列
<220>
<223>引物
<400>49
tgggagcggt gaagatggaa gggcac 26
<210>50
<211>26
<212>DNA
<213>人工序列
<220>
<223>引物
<400>50
tcatgccagc gcccacgtac gacgac 26
<210>51
<211>26
<212>DNA
<213>人工序列
<220>
<223>引物
<400>51
cgctttcatg gtgtgggcta aggacg 26
<210>52
<211>26
<212>DNA
<213>人工序列
<220>
<223>引物
<400>52
tagttggggt ggtcctgcat gtgctg 26
<210>53
<211>24
<212>DNA
<213>人工序列
<220>
<223>引物
<400>53
cgagaggacc ccgtggatgc agag 24
<210>54
<211>24
<212>DNA
<213>人工序列
<220>
<223>引物
<400>54
ggcggccatc ttcagcttct ccag 24
<210>55
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>55
acccattatc cagatgtgtt tgcccgag 28
<210>56
<211>26
<212>DNA
<213>人工序列
<220>
<223>引物
<400>56
atggtgaagc tgggcatagg cggcag 26
<210>57
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>57
caggtggcgg acgtgtgaaa attgagagtg 30
<210>58
<211>26
<212>DNA
<213>人工序列
<220>
<223>引物
<400>58
cacgctggat ctgcctgggg actgtg 26
<210>59
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>59
attcttcgtt gtcaagccgc caaagtggag 30
<210>60
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>60
agttgtttgc tgcggagttg tcatctcgtc 30
Claims (5)
1.一种改善iPS细胞建立效率的方法,其包括在体细胞核重编程步骤中抑制p53功能,其中所述体细胞核重编程通过使包含下述的核重编程物质接触体细胞来进行:
(1)Oct3/4和Sox2,或其编码核酸,
(2)Oct3/4、Sox2和Klf4,或其编码核酸,或
(3)Oct3/4、Sox2、Klf4和c-Myc,或其编码核酸,
其中通过使下列物质与体细胞接触而抑制所述p53功能:Pifithrin-α,对硝基,环状;MDM2;p53的显性失活突变体或其编码核酸;或选自针对p53的siRNA和shRNA以及其编码DNA的核酸。
2.权利要求1的方法,其中p53的显性失活突变体是p53P275S或p53DD。
3.一种用于改善iPS细胞建立效率的试剂,所述试剂包含选自下列的p53功能抑制剂:Pifithrin-α,对硝基,环状;MDM2;p53的显性失活突变体或其编码核酸;或选自针对p53的siRNA和shRNA以及其编码DNA的核酸。
4.一种体外产生iPS细胞的方法,其包括使核重编程物质和p53功能抑制剂与体细胞接触,其中所述核重编程物质包含:
(1)Oct3/4和Sox2,或其编码核酸,
(2)Oct3/4、Sox2和Klf4,或其编码核酸,或
(3)Oct3/4、Sox2、Klf4和c-Myc,或其编码核酸;
其中所述p53功能抑制剂选自下列:Pifithrin-α,对硝基,环状;MDM2;p53的显性失活突变体或其编码核酸;或选自针对p53的siRNA和shRNA以及其编码DNA的核酸;
其中所述方法不用于克隆人或被克隆的人。
5.权利要求4的方法,其中所述体细胞是T细胞。
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CN107290543A (zh) * | 2017-05-02 | 2017-10-24 | 南方医科大学 | 一种检测细胞内与p53转录激活域相互作用蛋白的方法 |
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EP1692113B1 (en) | 2003-11-14 | 2017-09-27 | Lorus Therapeutics Inc. | Aryl imidazoles and their use as anti-cancer agents |
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JP2011522520A (ja) * | 2008-05-06 | 2011-08-04 | エージェンシー フォー サイエンス,テクノロジー アンド リサーチ | 細胞の脱分化を行う方法 |
US8530238B2 (en) | 2008-06-27 | 2013-09-10 | Kyoto University | Method of efficiently establishing induced pluripotent stem cells |
CA2697621C (en) * | 2008-07-30 | 2017-01-17 | Kyoto University | Method of efficiently establishing induced pluripotent stem cells |
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