CN100422317C - 用于细胞核转移的静止细胞群体 - Google Patents
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Abstract
重组动物胚的方法涉及将静止的供体细胞的核转移至适当的受体细胞中。该供体细胞为静止的,原因在于使其在G1期退出生长和分裂周期并停顿于G0状态。可以通过细胞融合进行核转移。然后该重组胚可以产生一个或多个动物。本发明可用于具有高遗传价值的转基因以及非转基因动物的生产。
Description
本申请是分案申请,原申请的申请日为1996年8月30日,申请号为961978910,发明名称为“用于细胞核转移的静止细胞群体”。
技术领域
本发明涉及产生包括(但不限于)进行遗传选择地和/遗传修饰的动物在内的动物。
技术背景
通过将供体胚的细胞核转移至一个去核卵母细胞或一个细胞合子中重组哺乳动物胚,使生产遗传相同的个体。这显然既有利于研究(即作为生物学控制),也有利于商业应用(即有遗传价值的家畜的繁殖、肉产品的均一性、动物管理)。使用早期胚作为细胞核供体的一个问题是,可以由单个胚产生的后代数受细胞(32-64细胞期的胚最广泛地用于农畜物种中)数和细胞核转移方案效率这两者的限制。
与采用胚作为细胞核供体相反,由可以培养维持的细胞进行细胞核转移产生活后代的能力,是动物育种家一段时间来追求的目标。与使用早期胚相比,由培养的细胞系产生克隆后代的能力能够提供许多优点。这些优点包括:长期生产大量相同的后代(培养细胞可以冷冻和贮存),在胚重组前进行遗传修饰和/选择具有所需基因型(例如性)的细胞群体的能力。一种用于这些方法的潜在细胞类型为胚干(ES)细胞。已经在小鼠中分离出ES细胞,然而在用于细胞核转移后尚未有发育至足月的报道。目前在猪中有一个ES样细胞的报道,该细胞在注射到体内产生的胚囊的囊胚腔后有助于发育(Wheeler,Reprod Fertil Dev 6563-568(1994)),但在其它农畜物种中没有嵌合现象的报道,在任何哺乳动物物种中,也没有在由建立的细胞系进行细胞核转移后发育至足月的报道。
使用ES细胞系有几个选择的方法;其中一个是寻找用于细胞核转移时能够启动发育的其它细胞群体。几个报道已经表明原生殖细胞是适宜的候选细胞;然而尚未报道发育至足月。尽管在羊中已经报道由较长时间培养的早期传代细胞进行核转移可以引起发育(Campbell等,Therio 43 181(1995)),但由早期胚建立的细胞系已经表明,采用常规细胞核转移的方案不能引起发育(Campbell等,J Abstract Series(5)31(1995))。
为了在细胞核转移后能够发育至足月,必须重新设置转移细胞核的发育钟。为此,必须阻止转录的发生,然后以发育调节模式重新开始。现有报道已经表明,在母羊、羊、猪、兔和小鼠中可以由多种细胞类型达到发育至胚囊期。然而,在所有这些报道中,都没有报道发育至足月。先前已经报道了,由9天大羊胚的胚盘(ED)建立的初级细胞系(至多第3代,包括第3代)进行细胞核转移后,分娩了活羔羊(Campbell等,Therio 43 181(1995))。然而,采用常规细胞核转移方案,在随后的培养中没有得到发育至足月(第6代和第11代)(Campbell等,JReprod.Fertil Abstract Series(5)31(1995))。这些结果可以以多种方式解释;第一,可以假定在早期培养期间获得的所有得自ED的细胞都能够启动发育。然而,在建立细胞系的长时间培养过程中,当这些细胞用作细胞核供体以将细胞核转移至上述论文所指的“通用受体”中时,这些细胞改变,由此变得不能控制发育。或者,可以假定在早期培养期间,一个细胞亚群体保持启动发育的能力,这可以解释在这些早期传代期间进行细胞核转移后产生活的后代。现有研究已经强调供体细胞核和受体胞质细胞周期的协调在通过细胞核转移重组的胚发育中的作用。(Campbell等,Biol Reprod 49 933-942(1993)和Biol.Reprod50 1385-1393(1994))。
采用现行细胞核转移方案,用分离的全能细胞系进行细胞核转移有如下两种两个可能的策略:
(1) 修改现有的细胞核转移方法;或
(2) 在细胞核转移前修饰供体细胞的染色质。
全能细胞可以指导整个动物的发育(当通过将细胞核从供体细胞转移至诸如去核卵母细胞之类的受体细中重组胚时,正是供体细胞的细胞核是全能的)。这包括指导胚外谱系(即胎盘)的发育。在该定义中,受精合子和某些物种中的个别分裂球也是全能的。相反,多能细胞(即胚干细胞)类型已经定义为在注射到囊胚腔后,在胎体/后代中可以形成所有组织的细胞类型。
在上面概述的细胞核转移策略(1)和(2)两者中,都需要一个方法,以允许对转移细胞核的基因表达进行重编程序:然后这一方法允许使用分化或部分分化的细胞作为细胞核供体,并可以“显示”它们的内在全能性。
现已发现,在动物胚的重组中,最好可以将静止细胞(也就是说不通过细胞周期活性增殖的细胞)用作细胞核供体。然后可以让这类胚发育至足月。这些细胞用作细胞核供体之前,通过使它们进入静止状态,似乎可诱导在胚重组后观察到的供体核变化,而该变化是有效核转移所需的。在本申请中利用了这一事实。
发明内容
按照本发明的第一个方面,提供一个重组动物胚的方法,该方法包括将静止供体细胞的细胞核转移至适宜的受体细胞中。
原则上,本发明可应用于所有动物,包括诸如家禽之类的乌类、两栖类物种和鱼物种。然而实际上,对于非人类动物,尤其是(非人类)哺乳动物,特别是有胎盘哺乳动物而言,它是目前想象的最具有商业用途的应用。对于有蹄类动物,特别是经济上重要的有蹄类动物,诸如牛、羊、山羊、水牛、骆驼和猪等,本发明作为克隆动物的方法和作为产生转基因或遗传修饰动物的方法,都可能是最有用的.大家也应该注意到,本发明也可以应用于其它经济上重要的动物物种,诸如马、驼羊或啮齿动物,例如大鼠和小鼠或兔。
本发明同样可应用于转基因以及非转基因动物的生产中。可以由遗传改变的供体细胞生产转基因动物。与常规生产转基因(即遗传修饰的)动物的方法相比,整个方法有几个优点,可以总结如下:
(1) 需要较少的受体;
(2) 可以采用克隆供体细胞产生多个同系基因建立者;
(3) 允许通过基因靶进行精细的遗传改变,
(4) 由于每个动物都得自单个细胞核,因此由通过本发明制备的胚生产的所有动物都应该通过种系传递相关的遗传修饰;相反,在通过胚囊注射或其它方法包含修饰的干细胞种群后,通过原核注射或嵌合现象进行转基因动物的生产,产生一部分嵌合动物,在后者中,不是所有的细胞都含有该修饰,产生的动物可能不通过种系传递该修饰;
(5)在产生整个动物之前,可以选择具有遗传修饰(例如整合)位点的细胞.
大家应该注意,与动物有关的术语“转基因”不应该有限地用来指种系中含有一个或多个另一物种基因的动物,尽管许多转基因动物含有这样的一个或多个基因.相反地,更广义地讲,该术语是指其种系为重组DNA技术进行技术介入的对象的任何动物.因此,本发明目的的转基因动物既是例如种系中缺失、复制、激活或修饰一个内源基因的动物,也是种系中加入外源DNA序列的动物.
在该动物为转基因的本发明实施方案中,对供体细胞核进行遗传修饰.供体细胞核可以含有一个或多个转基因,可以在细胞核转移和胚重组之前进行遗传修饰.尽管与注射到合子的雄性原核或雌性原核类似的微注射可以用作遗传修饰的方法,但本发明不限于该方法:也可以使用物质转化或转染技术,例如电穿孔、病毒转染或脂染(lipofection).
在上述本发明方法中,将细胞核从静止供体细胞转移至受体细胞中.该方法的使用不限于特殊的供体细胞类型.采用该技术,可以证明可以诱导进入静止状态或体内存在于静止状态的所有核型正常的细胞(包括胚体细胞、胎儿体细胞和成体体细胞)都是全能的.因此,本发明打算使用至少部分分化的细胞,包括全分化细胞。供体细胞可以(但不必)处于培养中.下面例举培养的牛初级成纤维细胞、得自胚的羊细胞系(TNT4)、得自6岁成年羊羊乳房上皮细胞的细胞系(OME)、得自胎羊组织的成纤维细胞细胞系(BLWF1)和得自9天大羊胚的上皮样细胞系(SEC1)。包括TNT4细胞系在内的可用于本发明的一类得自胚的细胞系也是共同未决的PCT专利申请PCT/GB95/02095(以WO96/07732公布)的对象.
为了用于本发明,供体细胞是静止的,也就是说它们不通过有丝分裂细胞周期进行活性增殖.有丝分裂细胞周期有四个不同的时期,为G、S、G2和M期.在细胞周期中称为起始(start)的开始现象发生在G1期,具有独特的功能。在起始时作出进行另一细胞周期的决定或保证。一旦细胞通过起始,则细胞通过余下的G1期,这是前DNA合成期.DNA合成时,为第二阶段S期.随后为G2期,为DNA合成与有丝分裂之间的时期。有丝分裂本身发生在M期.一般认为静止细胞(包括已经诱导静止的细胞以及天然静止的细胞,诸如某些完全分化的细胞)不处于该周期四个时期中的任一期内;通常描述它们处于G0状态,以便表示它们不能正常地通过该周期进行.静止G0细胞的细胞核具有二倍体DNA含量.
可以通过各种方法,包括化学处理、营养物缺乏、生长抑制或基因表达的操作,诱导培养的细胞进入静止状态.目前,降低培养基中的血清浓度已经成功地在羊和牛细胞系中用来诱导静止.如上面所解释的,在这种情况下细胞在G1期期间退出生长周期,并停顿于所谓的G0期.这类细胞可以保持处于该状态几天(可能更长时间,取决于该细胞),直至当它们重新进入生长周期时受到重新刺激.
停顿于G0状态的静止细胞为二倍体.G0状态为细胞周期中细胞能够由此进行分化的点.已经报道了静止时的大量新陈代谢变化,这些变化包括:组蛋白单磷酸化、中心粒纤毛化、所有蛋白质合成减少或完全停止、蛋白酶解增加、转录减少和RNA更新增加导致总细胞RNA减少、多核糖体解聚、失活80S核糖体累积和染色质浓缩(Whitfield等的综述,Control of Ammal Cell Proliferation,1 331-365(1985))。
这些特征中的许多特征是将细胞核转移至去核卵母细胞后需要产生的特征。G0状态与细胞分化有关的事实表明,可以提供的核/染色质结构更有利于由受体细胞胞质重定模式和/或者重编程序。这样,借助于处于静止状态的供体细胞核,供体细胞核的染色质可以在胚重组或重建前进行修饰,使得该细胞核能够指导发育。这与所有先前报道的细胞核转移方法的不同处在于,在使用细胞作为细胞核供体之前修饰供体细胞的细胞核.
由供体转移细胞核的受体细胞可以为卵母细胞或另一种合适的细胞.
可以使用不同发育时期的受体细胞,即从中期I通过中期II的卵母细胞至合子和双细胞胚.每种受体都有优点和缺点.使用受精卵确保有效的激活,而单性生殖激活需要用卵母细胞(参见下面).在某些物种中可能适于使用卵裂期胚的另一机制是需要基因表达重编程序的程度.在小鼠的第二细胞周期期间开始转录,直至胚囊期的双向电泳揭示,合成的蛋白质的性质没有主要变化(Howlett和Bolton,J EmbryolExp Morphol 87 175-206(1985)).但是在大多数情况下,受体细胞为卵母细胞.
受体最好去核.尽管一般假定在细胞核转移方法中受体卵母细胞的去核是必需的,但该判断没有公开的实验证实.最初描述用于有蹄类动物的方法涉及将该细胞分裂为两半,其中一半可以去核(WilladsenNature 320(6)63-65(1986)).该方法的缺点是,未知的另一半仍具有中期器,并且胞质体积减小相信促进新胚的分化模式(Eviskov等,Development 109 322-328(1990)).
新近,已经使用不同的方法试图除去染色质,同时除去最小量的胞质.我们发现吸出第一极体和周围的胞质除去67%羊卵母细胞中的中期II器(Smith & Wilmut Biol Reprod 40 1027-1035(1989))。只有使用DNA特异性荧光染料(Hoechst 33342)提供的一个方法去核,才可以保证最小限度地减少胞质体积(Tsunoda等,J Rprod Fertil 82 173(1988))。在家畜物种中,这可能是一个目前常规使用的方法(Prather &First J Rprod Fertil Suppl 41 125(1990)),Westhusin等,Biol Reprod(Suppl)42 176(1990))。
在哺乳动物中有非常少的非侵入性去核方法的报道,而在两栖类动物中,将用紫外光辐射用作常规方法(Gurdon Q J Microsc Soc 101299-311(1960))。尽管在使用DNA特异性荧光染料期间,注意到小鼠卵母细胞暴露于紫外光超过30秒,将减小该细胞发育的潜力(Tsunoda等,J Rprod Fertil 82 173(1988)),但在哺乳动物中没有使用该方法的详细报道.
供体细胞核转移至的受体宿主细胞最好为去核中期II卵母细胞、去核未激活卵母细胞或去核预激活卵母细胞。至少当受体为去核中期II卵母细胞时,可以在转移时进行激活.或者,至少当受体为去核未激活中期II卵母细胞时,可以同时进行激活.如上所述,可以通过实际除去核、原核或中期板(取决于受体细胞)进行物理性去核,或者诸如通过应用紫外光辐射或另一种去核影响,进行功能性去核.
三种适当的胞质体(去核卵母细胞)受体为:
1 在我们今天申请的共同未决的PCT专利申请PCT/GB96/02098(由GB 9517779 6要求优先权)中描述的“MAGIC受体”(中期停顿的G1/G0接纳胞质体MetaphaseArrested G1/G0 AcceptIng Cytoplast).
2 “GOAT”(G0/G1 Activation and Transfer)-激活时为MII(中期II)的卵母细胞(Campbell等,Biol Rprod 49 933-942(1993)).
3 “通用受体”(Campbell等,(Campbell等,Biol Rprod 49 933-942(1993),Biol Rprod 50 1385-1393(1994)).
当在重组胚中使用处于G0的供体细胞核时,所有这三种受体都产生常规倍性.然而,最近的报道表明,一部分来自静止细胞的细胞核当置于S期胞质而不经历核膜分解时,不能进入DNA合成期(Leno和Munshi,J Cell Biol 127(1)5-14(1994))。因此,尽管使用“通用受体”时一部分胚发育,但假设使用含有高农度MPF(M期启动因子或成熟启动因子)的MII卵母细胞作为胞质体受体,通过方法1或者方法2将导致较高频率的发育。
一旦鉴定出合适的供体和受体,则必须将前者的细胞核转移至后者中.更好是通过融合进行细胞核转移。
已经建立的三种诱导融合的方法为:
(1)将细胞暴露于促进融合的化学药品,诸如聚乙二醇;
(2)使用失活病毒,诸如仙台病毒;
(3)使用电刺激.
将细胞暴露于促进融合的化学药品(诸如聚乙二醇或其它二醇)为体细胞融合的常规方法,但它尚未广泛地用于胚。由于聚乙二醇有毒,必需将细胞进行最短时间的暴露,需要能够快速除去化学药品迫使除去透明带(Kanka等,Mol Rprod Dev 29 110-116(1991)).在用小鼠胚的实验中,失活仙台病毒提供一个融合卵裂期胚的细胞的有效方法(Graham Wistar Inst Symp.Monogr 919(1969)),其另一实验优点是不诱导激活。在有蹄类动物中,通常通过用诱导单性生殖激活的相同电刺激达到融合(Willadsen Nature 320(6)63-65(1986),Prather等,BiolRprod 37 859-866(1987)).在这些物种中,仙台病毒在一部分情况下诱导融合,但不能用作常规应用(Willadsen Nature 320(6)63-65(1986))。
尽管细胞-细胞融合为进行细胞核转移的推荐方法,但它并不是可以使用的唯一方法.其它适宜的技术包括微注射(Ritchie和Campbell,JReproduction and Fertility Abstract Series No 15,第60页).
如果该细胞为卵母细胞,那么在细胞核转移前或(最好)在核转移后(或在某些情况下至少与细胞核转移同时),一般必须通过单性生殖激活刺激受体细胞进入发育.最近的实验已经表明,单性生殖激活的需求比想象的更复杂。已经假定 激活为全或无现象,能够诱导原核形成的多种处理全都引起“激活”.然而,将兔卵母细胞暴露于重复的电脉冲表明,仅选择适当系列的脉冲并控制Ca2+能够促进二倍体化卵母细胞发育至妊娠中期(Ozil Development 109 117-127(1990)).在受精期间,胞内钙浓度重复地瞬时提高(Cutbertson & Cobbold Nature 316541-542(1985)),相信电脉冲引起类似的钙浓度的增加.有证据表明,钙的瞬变模式随物种而变化,预期电脉冲的最佳模式以类似方式变化.在兔中,脉冲之间的间隔大约为4分钟(Ozil Development 109117-127(1990)),在小鼠中,为10-20分钟(Cutbertson & Cobbold Nature316 541-542(1985)),而在母牛中的初步观察到该间隔大约为20-30分钟(Robl等,Symposium on Cloning Mammals by Nuclear transplantation(seidel ed),Colorado State University,24-27(1992)).在大多数建立的实验中,用单个电脉冲诱导激活,但新的观察表明,应用多个脉冲,将增加发育的重组胚比例(Collas & Robl Biol Reprod 43 877-884(1990)).在任一个别情况下,可以进行常规调整,以优化脉冲数、场强度和脉冲持续时间以及培养基的钙浓度.
按照本发明的第二个方面,提供用前述方法制备的重组动物胚.
按照本发明的第三个方面,提供制备动物的方法,该方法包括:
(a) 按上述方法重组动物胚;
(b) 使动物由胚发育至足月;
(c) 可选地由如此形成的动物进行繁殖。
上面已经深入地描述了步骤(a).
本发明该方面方法中的第二步-步骤(b)是使动物由胚发育至足月.这可以直接或间接进行.在直接发育中,简单地让步骤(a)的重组胚进行发育,除允许进行发育可能必需的介入外,不用进一步的介入.然而在间接发育中,该胚在完全发育前可以进一步进行操作.例如,为了增加产量,可以使胚分裂,克隆细胞增加。
或者或另外,借助于本发明,通过克隆扩大培养供体和/或如果采用系列(细胞核)转移方法,可以增加能活胚的产量。由于大多数胚不“重编程序”(尽管可接受数量的胚“重编程序”),可能引起目前得到的胚囊形成速率的限制.如果是这样,那么可以如下提高该速率,每个自身发育的胚(诸如桑椹期或在32-64细胞期)可以用作细胞核供体;另一方法是,可以使用胚囊期的内细胞群细胞.得自这些随后转移的胚本身也可以用作潜在的细胞核供体,以进一步提高效率。如果这些胚实际上确实反映出已经具有重编程序的基因表达并且那些细胞核事实上发生重编程序(看上去可能是),这样每个发育中的胚的繁殖可能有赖于细胞核转移方法的效率.可能达到的增加程度取决于细胞类型.在羊中,通过将16细胞胚的单个分裂球转移至预激活的“通用受体”卵母细胞中,可以容易地获得55%的胚囊期胚.因此,可以这样假设,由单个细胞发育的每个胚可以产生8个16细胞期的胚.尽管这些数字只是大致的指导,但很明显,在较迟的发育阶段时,收益程度取决于该阶段时该方法的效率.
我们也预计,由按前述形成的胚或产生的胎儿或成体,可以产生作为细胞核供体细胞源的新细胞系.
在某些情况下,在重组胚发育至足月期间可能有一些限制,最好可以产生由得自自然形成的胚和通过细胞核转移重组的胚的细胞形成的嵌合动物.采用自然胚的部分细胞和至胚囊期的任何时期的重组胚的部分细胞,通过聚集或注射,可以形成这一嵌合体.细胞的比例为50 50,或为达到形成发育至足月的胚的另一合适的比例.在这些情况下,认为正常细胞的存在有助于解救重组胚,并允许成功地发育至足月并分娩活体。
除提高产量的权宜之计的问题外,重组胚还可以体内或体外培养至胚囊。
经验表明,通过细胞核转移得到的胚与正常胚不同,有时得益于体内培养条件或甚至需要体内培养条件,而不是胚通常培养的条件(至少在体内).不知道其原因.在牛胚常规增殖中,已经将重组胚(一次多个胚)在羊输卵管中培养5-6天(如Willadsen在Mammalian EggTransfer(Adams,EE,ed)185 CRC Press,Boca Raton,Florida(1982)中所述).但是在本发明实施中,为了保护该胚,在转移前最好应该将该胚包埋在保护性培养基(诸如琼脂)中,然后在从临时受体回收后从琼脂中解剖出来.保护性琼脂或其它培养基的功能有两个;第一,它起该胚的结构辅助器的作用,使透明带保持在一起;第二,它对受体动物的免疫系统细胞起屏障的作用。尽管该方法提高形成胚囊的胚的比例,但其缺点是可能损失大量的胚.
如果使用体外培养条件,那么本领域采用的那些常规条件是相当可接受的.
在胚囊期,可以筛选该胚发育至足月的适应性.通常,这在该胚为转基因和进行筛选,并且已经选择稳定的整合体时进行.在该阶段也可以筛选非转基因的遗传标记.然而,因为本发明方法允许早期筛选供体,因此一般最好进行筛选.
如果进行筛选,那么在筛选后,让胚囊胚发育至足月.这一般在体内进行.如果在体外发育至胚囊,那么在该阶段转移到最终受体动物中.如果在体内进行胚囊发育,那么尽管原则上可以让胚囊在胚囊前宿主中发育至足月,但实际上通常从(临时)胚囊前受体中取出胚囊,在从保护性培养基中解剖出来后,将其转移至(永久)胚囊后受体中.
在本发明该方面的可选步骤(c)中,可以由用先前步骤制备的动物繁殖动物.这样,可以用一个动物来建立具有所需遗传特性的一群动物.
通过转移来自遗传相同细胞的细胞核生产的动物共享相同的细胞核,但严格来讲这些动物是不相同的,因为它们得自不同的卵母细胞.不清楚这种不同来源的重要性,但它可能影响商业品质。最近在Iowa State University Breed Herd的奶牛中进行的线粒体DNA分析表明与牛奶和生殖性能有关(Freeman和Beitz,Symposium on CloningMammals by Nuclear Transplantation(Seidel,G E Jr,ed)17-20,Colorado State University,Colorado(1992))。仍有待于证实在整个牛群体中存在相似的作用,并且考虑是否可能或必需在特定情况下考虑卵母细胞的选择.在牛育种领域中,由高遗传价值供体生产大量胚的能力在通过国家牧群传播遗传改良方面可能具有相当大的潜在价值。应用范围取决于每个胚的成本和能够发育至足月的转移胚的比例.
通过说明和概述,以下流程陈述了可以用来制备转基因和非转基因动物的一般方法。该方法包括5个步骤:
(1) 选择和分离适当的供体细胞,可能包括评价核型、诱导静止(停顿于G0)和/或诱导发育;
(2) 应用适当的分子生物学技术生产遗传修饰的细胞群体.这类技术可能包括基因加入、基因打出(knock-out)、基因打入(knock-in)和其它基因修饰.也可以可选地通过转染具有或不具有选择标记的适当构建物进行转基因;
(3) 可选地筛选和选择所需基因型/表型的修饰细胞群体(即稳定的整合体);
(4) 在修饰细胞群体中诱导静止;
(5) 通过细胞核转移进行胚重组;
(6) 体内或体外培养至胚囊;
(6a)可选地筛选和选择稳定的整合体(如果在(3)进行筛选,则省略此步骤)或其它所需的特性;
(7) 必要时转移至最终受体中.
按照本发明的第四个方面,提供按上述方法制备的动物.
本发明每个方面的推荐特征是对其它方面而言的,可予以必需的修改.
现在参考所附的实施例描述本发明,这些实施例的提供是为了说明本发明,不要解释为限制本发明。
具体实施方式
实施例
实施例1:在供体细胞中诱导静止
已经表明在培养的细胞系中诱导静止的各种方法,包括:接触抑制法或血清饥饿法(Whitfield等的综述,Contril of Animal CellProliferation,1 331-365(1985))。我们认为诱导静止的方法不重要,重要的步骤是细胞退出细胞周期,停顿于G0状态,同时具有二倍体DNA含量并保持有活力。在实施例3和实施例4中,采用牛初级成纤维细胞的血清饥饿法、由胚囊体内产生的7天大的内细胞群团块建立的牛细胞系诱导静止,使细胞停顿于细胞周期的G0期。同样地,将血清饥饿法用来诱导实施例5中描述的供体静止。
实施例2:分离卵母细胞和细胞核转移
可以通过(i)屠宰场材料的体外成熟,或经阴道进行滤泡穿刺;(ii)体内成熟和手术回收;或(iii)任何其它适当的方法获得卵母细胞。应该通过在含有10%胎牛血清(FCS)的无钙镁磷酸盐缓冲液(PBS)中冲洗输卵管,收获所有体内成熟的卵母细胞,将其转移至含有10%FCS的无钙M2(Whittingham和Wales,Aust J.Biol.Sci 22 1065-1068(1969))中。剥下卵丘细胞的卵母细胞,按前述方法去核(Campbell等,Biol.Reprod.49 933-942(1993)和Biol Reprod 50 1385-1393(1994)),只是所有步骤都使用无钙培养基。融合步骤修改了先前报道的那些方法(Campbell等,1993,1994 loc cit),并且按照下面相关节描述的方法;或者可以通过将供体细胞注射到去核卵母细胞中导入该细胞核(Ritchie和Campbell,J Reprod Fertil Abstract Series(5)60(1995))。这些活性的定时取决于物种,以下两个方案概述了体内成熟的羊卵母细胞和体外成熟的牛卵母细胞的用法。
实施例3.羊的细胞核转移
31 供体母羊的超刺激和卵母细胞的回收
将苏格兰黑脸母羊用孕激素(progestagen)海绵同步化14天(VeramixTM,Upjohn,UK),通过连续两天单次注射30mg/天(总共60mg)的羊促滤泡素(FSH)(OvagenTM,Immuno-chemical Products Ltd,NewZealand),诱导超数排卵。在第二次注射FSH后24小时,用单次剂量为8mg的促性腺素释放激素类似物(GnRH ReceptalTM,Hoechst,UK)诱导排卵.
在GnRH注射后24-29小时时,用含有10%胎牛血清(FCS)Dulbecco’s磷酸盐缓冲液(保持37℃直至使用)冲洗输卵管,回收未受精的中期II卵母细胞.
32 卵母细胞的操作
将回收的卵母细胞保持在37℃下,在含有10%FCS的PBS中洗涤,于37℃将其转移至含有10%胎牛血清(FCS)的无钙M2培养基上.为了除去染色体,于37℃下将(去核)卵母细胞在含有1.0%FCS、75μg ml细胞松弛素B(Sigma)和50μg/ml Hoechst 33342(Sigma)的无钙M2中放置20分钟.用20μM玻璃吸管,直接从第一极体下面吸出少量胞质.将吸出的胞质部分置于UV光下并检查中期板的存在以证实去核.
33 胚重组
将多组10-20个卵母细胞去核,在矿物油(SIGMA)下 于37℃、5%CO2下,将其置于20μl无钙M2培养基小滴上。将以下三个方案(a)、(b)和(c)中的每个方案用于胚重组.
(a)“MAGIC”(中期停顿的G1/G0接纳胞质体Metaphase Arrested G1/G0 AcceptIng Cytoplast)
用玻璃吸管通过预先在透明带中制造的孔转移细胞,使单个细胞与卵母细胞接触。然后将胞质体/细胞融合对转移至融合室中的含0.3M甘露醇的200μl蒸馏水中,将融合对在电极之间人工排成一排。施加3秒5V的交流脉冲,然后施加3个80μs 1 25kV/cm的直流脉冲。然后融合对于37℃在含10%FCS的无钙M2中洗涤,在油下,于37℃5%CO2下在同一培养基中培养。在激活前30分钟,将融合对转移至含有5μM nododazole、10%FCS的无钙M2培养基中。在注射hCG后32-34小时时,按照下述方法诱导激活.激活后,于37℃5%CO2下,重组合子在含10%FCS的TC199培养基(Gibco)中再培养3小时.然后将其于37℃在无nocodazole的同一培养基中洗涤3次,每次5分钟,在转移至临时受体母羊之前再培养12-15小时.
(b)“GOAT”(G1/G0激活和转移)
在hCG注射后32-34小时,将单个细胞与去核卵母细胞接触.在将胞质体/细胞融合对转移至融合室(参见下面)中的200μl含0.3M甘露醇、0 1mM MgSO4、0 001mM CaCl2的蒸馏水中.通过施加5秒3V的交流脉冲后,施加3个80μs的1.25kV/cm直流脉冲,诱导融合和激活.然后融合对在含有10%FCS、75μg/ml细胞松弛素B的TC199中洗涤,并于37℃5%CO2下在该培养基中培养1小时.然后融合对在含10%FCS的TC199中洗涤,并于37℃5%CO2下在含10%FCS的TC199中再培养12-15小时.
(c)“通用受体”
去核卵母细胞在hCG注射后32-34小时激活(按下述方法),然后于37℃5%CO2下在含10%FCS的TC199中培养4-6小时。然后将单个细胞与该卵母细胞接触,按下述方法诱导融合.然后将融合对在含有10%FCS、75μg/ml细胞松弛素B的TC199中洗涤,并于37℃5%CO2下在该培养基中培养1小时.然后融合对在含10%FCS的TC199中洗涤并于37℃5%CO2下再培养8-11小时.
34 融合和激活
关于激活,将卵母细胞置于两个平行电极之间的200μl含0 3M甘露醇、0 1mM MgSO4、0 001mM CaCl2的蒸馏水中(Willadsen,Nature 320 63-65(1986))。通过施加80μs 1 25kV/cm的直流脉冲诱导激活。关于融合,操作的胚以相似的方式进行处理,此外将去核卵母细胞和该细胞之间的接触平面与电极平行布置。通过施加5秒3V的交流脉冲,然后施加3个80μs 1 25kV/cm的直流脉冲诱导融合。
35 胚的培养和评价(所有组)
在培养后,将融合的融合对成双地包埋在1%和12%琼脂(DIFCO)的PBS中,将其转移至未同步化母羊的结扎输卵管中.将融合对包埋在琼脂中,以防止或减小受体母羊对该胚的免疫排斥作用,并有助于将融合对保持在一起.6天后,杀死受体母羊,通过用含10%FCS的PBS冲洗输卵管回收胚.用2个针头从琼脂解剖出胚,通过显微镜进行发育评价.尽快将已经发育至桑椹期/胚囊期的所有胚转移至同步化最终受体母羊的子宫角中.
体外技术也适于代替临时受体母羊,使该胚发育至胚囊期.
实施例4:牛的细胞核转移
4.1 体外卵母细胞的成熟
由当地屠宰场获得卵巢,在转移至实验室期间将其保持在28-32℃.用皮下注射针(内径为1.2mm)在直径为3-10mm的滤泡中吸出丘卵卵母细胞复合体(COC’3),将其置于无菌塑料通用容器中.将通用容器置于温室(35℃)中,在倒出四分之三上清液之前,让滤泡物质沉淀10-15分钟.余下的滤泡材料用等体积添加10%牛血清的解剖培养基(具有Earles盐(Gibco)的TCM 199,750mg/l卡那霉素、300mMHepes,pH 74、克分子渗透压浓度为280mOsmols/Kg H2O)稀释,转移至85mm陪替氏培养皿中,在解剖显微镜下寻找COC’s.选择具有至少2-3个致密层的卵丘细胞的复合体,在解剖培养基中洗涤3次,转移至成熟培养基(具有Earles盐(Gibco)的TC培养基199,75mg/l卡那霉素、300mM Hepes、769mM NaHCO3,pH 78、克分子渗透压浓度为280mOsmols/Kg H2O)中,该培养基添加10%牛血清和1×106粒膜细胞/ml,于39℃在含5%CO2的空气中培养在rocking table上。
42 卵母细胞的操作
在成熟开始后18小时,剥下卵丘细胞的成熟卵母细胞。然后将剥下的卵母细胞在含有10%胎牛血清(FCS)的无钙M2培养基中洗涤,并在37℃下将其保持在该培养基中。为了除去染色体(去核),于37℃下将(去核)卵母细胞在含有10%FCS、75μg ml细胞松弛素B(Sigma)和50μg/ml Hoechst 33342(Sigma)的无钙M2中放置20分钟.用20μM玻璃吸管,直接从第一极体下面吸出少量胞质.通过将吸出的胞质部分置于UV光下并检查中期板的存在以证实去核.
43 胚重组
按照下面详细描述的方法,将去核卵母细胞用于以下三个重组方法(a)、(b)和(c)中的每个方法.
(a)“MAGIC”(中期停顿的G1/G0接纳胞质体Metaphase Arrested G1/G0 AcceptIng Cytoplast)
去核后,将去核卵母细胞保持在39℃含10%FCS的无钙M2中,采用玻璃吸管通过预先在透明带中制造的孔转移细胞,将单个细胞与卵母细胞接触.然后将胞质体/细胞融合对转移至融合室中的含03M甘露醇的200μl蒸馏水中.将融合对在电极之间人工排成一排.施加5秒3V的交流脉冲,然后施加3次80μs 1 25kV/cm的直流脉冲.然后融合对于37℃在含10%FCS的无钙M2中洗涤,在油下,于37℃5%CO2下在同一培养基中培养.在激活前30分钟,将融合对转移至含有5μM nododazole、10%FCS的无钙M2培养基中.在按照下述方法诱导激活,然后于37℃5%CO2下,重组合子在含10%FCS的TC199培养基中再培养3小时.然后将其于37℃在无nocodazole的同一培养基中洗涤3次,每次5分钟,在转移至临时受体母羊之前(母羊作为重组胚的临时受体,为较为便宜的选择)再培养12-15小时。
(b)“GOAT”(G1/G0激活和转移)
将去核卵母细胞转移到成熟培养基上。在成熟开始后30-42小时,将单个细胞与去核卵母细胞接触.将融合对转移至融合室(参见下面)中的200μl含03M甘露醇、0 1mM MgSO4、0 001mM CaCl2的蒸馏水中.通过施加5秒3V的交流脉冲后,施加3次80μs的125kV/cm直流脉冲,诱导融合和激活.然后融合对在含有10%FCS的TC199中洗涤,并于37℃5%CO2下培养15-20小时(30hpm组)或4-8小时(42hpm组)[缩写“hpm”表示“成熟后小时数”].
(c)“通用受体”
在成熟开始后30或42小时时激活去核卵母细胞(按下述方法),然后于37℃5%CO2下含10%FCS的TC199中培养8-10小时(30hpm组)或4-6小时(42hpm组).然后将单个细胞与该卵母细胞接触,按下述方法诱导融合.然后融合对于37℃5%CO2下在含有10%FCS的TC199中再培养12-16小时(30hpm组)或4-6小时(42hpm组).
44 融合和激活
关于激活,将卵母细胞置于两个平行电极之间的200μl含0.3M甘露醇、0 1mM MgSO4、 0 001mM CaCl2的蒸馏水中(Willadsen,Nature 320 63-65(1986)).通过施加80μs 1 25kV/cm的1次直流脉冲诱导激活.关于融合,以相似的方式进行处理操作的胚,此外将去核卵母细胞和该细胞之间的接触平面与电极平行布置.通过施加5秒3V的交流脉冲,然后施加3次80μs 1 25kV/cm的直流脉冲诱导融合。
45 胚的培养和评价(所有组)
在培养后,将融合的融合对成双地包埋在1%和12%琼脂,转移至未同步化母羊的结扎输卵管中(DIFCO)的PBS中(母羊作为重组胚的临时受体为较便宜的选择)。将融合对包埋在琼脂中,以防止或减小受体母羊对该胚的免疫排斥作用,并有助于将融合对保持在一起.6天后,杀死母羊受体,用含10%FCS的PBS冲洗输卵管回收胚.用2个针头从琼脂块中解剖出胚,通过显微镜进行发育评价。
体外技术也可能适于代替临时受体,使该胚发育至胚囊期。
实施例3(羊细胞)和实施例4(牛细胞)的结果
本技术已经用于羊胚和牛胚两者的重组.目前,在牛中已经获得胚囊期胚;然而,这些胚尚未转移至最终受体中.在羊中,7只受体母羊受孕,导致5只活羔羊出生(其中2只出生后不久死亡)。这些实验的结果总结于表1-3中.
表1表明采用静止TNT4细胞群体和3种不同的胞质体受体重组的羊胚发育至胚囊期的结果.重组胚在临时受体母羊的结扎输卵管中培养至重组后第7天.结果以相对于回收胚总数的桑椹期/胚囊期胚的百分比表示。
表1
表2表明将所有桑椹期/胚囊期重组胚转移至同步化最终受体黑脸母羊的子宫角后诱导妊娠的结果。该表表明每个转移组的胚总数和以母羊和胚为单位的妊娠频率(在大多数情况下,每只母羊转移2个胚)采用“MAGIC”胞质体建立了单次双妊娠。
表2
传代数 | “MAGIC” | “GOAT” | “UNIVERSAL” |
P6 | 4 | 6 | 0 |
P7 | 1 | 1 | 0 |
P11 | 2 | 9 | 0 |
P12 | 0 | 0 | 6 |
P13 | 3 | 0 | 2 |
MOR/L总数 | 10 | 16 | 8 |
母羊总数 | 6 | 9 | 4 |
受孕母羊% | 1(16.7) | 5(55.5) | 1(25.0) |
胎儿/转移总数(%) | 2/10(20.0) | 5/16(31.25) | 1/8(12.5) |
表3表明桑椹期胚/胚囊期胚转移至最终受体母羊后产生的妊娠结果.
表3
母羊 | 方法 | 传代 | 结果 |
4E468 | GOAT | 6 | 活羔羊 |
4E302 | GOAT | 7 | 胎儿死亡(大约130天) |
4E210 | GOAT | 11 | 活羔羊 |
4E286 | GOAT | 11 | 活羔羊(出生后不久死亡) |
4E453 | GOAT | 11 | 胎儿死亡(大约80天) |
4E294 | UNIVERSAL | 11 | 活羔羊 |
4E272 | MAGIC | 13 | 活羔羊(出生后不久死亡) |
实施例5.采用OME、BLWF1和SEC1细胞进行羊的细胞核转移和
胚重组
已经采用命名为OME、BLWF1和SEC1的三个新细胞类型进行了细胞核转移.OME(羊乳腺上皮)细胞为按照Finch等的方法(Biochem Soc Trans 24 369S(1996),由成年6岁的Fin-Dorset母羊的乳腺通过活组织检查建立的上皮细胞系.BLWF1(黑威尔士成纤维细胞)细胞为在黑威尔士母羊与黑威尔士公羊自然交配后获得的26天大黑威尔士胎儿解剖和培养获得的成纤维细胞细胞系.分离初级胎儿成纤维细胞的方法为按照Robertson,E J在Teratocarcinomas andembryonic stem cell A practical approach,71-112,IRL Press Oxford(1987)中描述的方法.SEC1(羊胚性细胞)为由超数排卵和与Pol-Dorset公羊交配的Pol-Dorset母羊获得的9天大的胚得到的上皮样细胞系.由于以下原因,SEC1细胞不同于共同未决的PCT申请PCT/GB95/02095(以WO 96/07732公布)中描述的TNT细胞.第一,两个细胞系的细胞形态学完全不同,第二,用来分离细胞系的方法不同.SEC1细胞系由单个胚建立,而TNT细胞系得自多组细胞.
所有细胞系都进行核型分析,表明典型的染色体数为54(2n).在用作胚重组的细胞核供体之前,按前述方法监测在将血清水平降至0 5%后静止的诱导(Campbell等,Nature 380 64-66(1996)).按先前实施例中描述的方法制备重组胚.
表4表明由不同细胞类型重组的细胞核转移胚发育的总结.该表表明三种细胞类型中的每种发育至胚囊期的重组胚数和受孕数.所有细胞系在用于胚重组前都进行核型分析.这些细胞系的典型染色体数为54.每只同步化最终受体母羊转移1-3个胚囊期胚.将体外培养的重组胚置于含有10%人血清的10μl(4个胚)的SOFM(合成输卵管流体培养基)小滴中,于39℃5%O2、5%CO2和90%N2的潮湿氛围下培养.每2天将培养的胚转移至新鲜的培养基中。按照Gardner等,Biologyof Reproduction 50 3 90-400(1994)和Thompson等,Biology ofReproduction 53 1385-1391(1995)的方法制备SOFM培养基。
表5表明于1994年6月24日保持受孕的受体母羊的鉴定、用于胚重组的细胞类型和预期的小羊出生日期.通过将1-3个桑椹期/胚囊期胚(重组后第7天)转移至同步化的最终受体母羊中产生妊娠。重组数的细节示于表4中。缩写为:PD=Pol-Dorset,BW=黑威尔士,FD=Fin-DOrset,*=体外培养至胚囊期的胚.
Claims (10)
1. 一种克隆非人哺乳动物的方法,包括:
(i)将静止二倍体供体细胞与去核的卵母细胞融合,从而获得重建细胞;
(ii)融合之前、之中或之后激活所述卵母细胞;
(iii)培养所述重建细胞形成胚胎;
(iv)将所述胚胎转移到非人哺乳动物宿主,使胚胎发育至足月。
2. 一种克隆非人哺乳动物的方法,包括:
(i)将静止二倍体供体细胞与去核的卵母细胞融合,从而获得重建细胞;
(ii)融合之前、之中或之后激活所述卵母细胞;
(iii)培养所述重建细胞形成胚胎;
(iv)用步骤(iii)的胚胎的细胞作为供体细胞重复步骤(i)至(iii)从而获得胚胎;
(v)将步骤(iv)的胚胎转移到非人哺乳动物宿主,使胚胎发育至足月。
3. 一种克隆非人哺乳动物的方法,包括:
(i)将静止二倍体供体细胞显微注射入去核的卵母细胞,从而获得重建细胞;
(ii)显微注射之前、之中或之后激活所述卵母细胞;
(iii)培养所述重建细胞形成胚胎;
(iv)将所述胚胎转移到非人哺乳动物宿主,使胚胎发育至足月.
4. 一种克隆非人哺乳动物的方法,包括:
(i)将静止二倍体供体细胞显微注射入去核的卵母细胞,从而获得重建细胞;
(ii)显微注射之前、之中或之后激活所述卵母细胞;
(iii)培养所述重建细胞形成胚胎;
(iv)用步骤(iii)的胚胎的细胞作为供体细胞重复步骤(i)至(iii)从而获得胚胎;
(v)将步骤(iv)的胚胎转移到非人哺乳动物宿主,使胚胎发育至足月。
5. 权利要求1-4任一项要求保护的方法,其中所述非人哺乳动物选自牛、猪、绵羊、山羊、马、大鼠、小鼠和兔。
6. 一种重建非人哺乳动物胚胎的方法,包括:
(i)将静止二倍体供体细胞与去核的卵母细胞融合以重建胚胎;
(ii)融合之前、之中或之后激活所述卵母细胞;
(iii)培养所述重建胚胎从而使重建胚胎发育。
7. 一种重建非人哺乳动物胚胎的方法,包括:
(i)将静止二倍体供体细胞显微注射入去核的卵母细胞融合以重建胚胎;
(ii)显微注射之前、之中或之后激活所述卵母细胞;
(iii)培养所述重建胚胎从而使重建胚胎发育.
8. 一种重建非人哺乳动物胚胎的方法,包括:
(i)将静止二倍体供体细胞与去核的卵母细胞融合以重建胚胎;
(ii)融合之前、之中或之后激活所述卵母细胞;
(iii)培养所述重建胚胎从而使重建胚胎发育;
(iv)使用步骤(iii)的胚胎的细胞作为供体细胞重复步骤(i)至(iii)获得进一步的胚胎。
9. 一种重建非人哺乳动物胚胎的方法,包括:
(i)将静止二倍体供体细胞显微注射入去核的卵母细胞以重建胚胎;
(ii)显微注射之前、之中或之后激活所述卵母细胞;
(iii)培养所述重建胚胎从而使重建胚胎发育;
(iV)使用步骤(iii)的胚胎的细胞作为供体细胞重复步骤(i)至(iii)获得进一步的胚胎。
10. 权利要求6-9任一项要求保护的方法,其中所述非人哺乳动物胚胎选自牛、猪、绵羊、山羊、马、大鼠、小鼠和兔.
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