CN1202085A - 作为细胞核转移胞质体受体的未激活卵母细胞 - Google Patents

作为细胞核转移胞质体受体的未激活卵母细胞 Download PDF

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CN1202085A
CN1202085A CN96197892A CN96197892A CN1202085A CN 1202085 A CN1202085 A CN 1202085A CN 96197892 A CN96197892 A CN 96197892A CN 96197892 A CN96197892 A CN 96197892A CN 1202085 A CN1202085 A CN 1202085A
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K·H·S·坎贝尔
I·维慕特
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Roslin Institute Edinburgh
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • A01K67/027New breeds of vertebrates
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    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877Techniques for producing new mammalian cloned embryos
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    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877Techniques for producing new mammalian cloned embryos
    • C12N15/8771Bovine embryos

Abstract

重组一个动物胚胎的方法涉及将二倍体核转移至一个停滞在第二成熟分裂中期的卵母细胞中。卵母细胞在转移时是未激活的,因此供体核保持暴露于受体细胞质一定时间。可以由转移时为细胞周期G0或G1期的细胞提供二倍体核。随后,激活重组胚胎。在激活期间,例如通过在诸如nocodazole等的微管抑制剂存在下培养重组胚胎,保持正确的倍性。然后重组胚胎导致一个或多个动物的诞生。本发明可用于转基因动物的生产以及具有高遗传价值的非转基因动物。

Description

作为细胞核转移胞质体受体的未激活卵母细胞
本发明涉及包括(但不限于)遗传选择和/或遗传修饰动物在内的动物的产生,涉及可用于产生这些动物的细胞。
通过将供体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)。
首先计划通过细胞核转移重组胚(Spemann,EmbryonicDevelopment and Induction 210-211 Hofner Publishing Co.,New York(1938)),目的是为了回答核等值或‘核在发育期间改变吗?’的问题。设计这些实验通过转移来自不断发育的胚胎期的核以确定核在什么时候其发育潜能变为受限制。由于技术上的限制以及Spemann的不幸去世,直至1952年才完成这些研究,这时证明在青蛙中,某些核可以指导青蛙发育为性成熟的成体(Briggs和King,Proc.Natl.Acad.Sci.USA38 455-461(1952))。他们的发现产生目前的概念:当来自单一个体的同等全能核转移至去核卵时,产生“遗传上相同”的个体。其真正含义是,由于每个个体中未知的胞质对核转移的影响不同,这些个体不是克隆,并且也必须证明不存在任何染色体重排。
自从证明两栖类动物中的胚克隆,相似的技术已被用于哺乳动物物种中。这些技术分为两类:1)将供体核转移至已经除去染色体DNA的成熟的中期II卵母细胞中,以及2)将供体核转移至一个已除去两个原核的受精细胞合子中。在有蹄类动物中,采用前一方法,因为已经报道不在交换原核时采用后一方法细胞合子不发育。
一般通过诱导细胞融合,将供体核转移到卵母细胞的胞质中。在有蹄类动物中,通过在该对细胞(couplet)的接触/融合平面上施加直流电脉冲诱导融合。诱导细胞融合的同样脉冲也激活受体卵母细胞。胚重组后的进一步发育取决于多种因素,包括该核指导发育的能力-即全能性、受体胞质体的发育感受态(即卵母细胞的成熟)、卵母细胞激活、胚培养(Campbell和Wilmut在Vth World Congress on Genetics asApplied to Livestock 20 180-187(1994)中的综述)。
除上述因素外,我们已经表明在重组胚的第一细胞周期维持正确的倍性是至关重要的(Campbell等,Biol.Reprod. 49 933-942(1993);Campbell等,Biol.Reprod.50 1385-1393(1994))。在单个细胞周期中,在有丝分裂前所有基因组DNA必须复制一次并且只复制一次。如果任何DNA或者不能复制,或者复制多于一次,那么在有丝分裂时该核的倍性将不正确。在每个细胞周期中复制限于一个循环的机制尚不清楚,然而,几种证据的迹象表明,完整的核膜对于这一控制是决定性的。已经在包括小鼠(Czolowiska等,J.Cell Sci.69 19-34(1984))、兔(Collas和Robl,Biol.Reprod. 45 455-465(1991))、猪(Prather等,J.Exp.Zool.225 355-358(1990))、母牛(Kanka等,Mol.Reprod. Dev.29 110-116(1991))在内的多个物种中研究了在供体核转移到去核中期II卵母细胞后发生在供体核中的形态学现象。恰好在融合时,供体核膜破裂(NEBD),染色体早熟浓缩(PCC)。这些作用是由称为成熟/有丝分裂/减数分裂启动因子(MPF)的胞质活性催化。在所有有丝分裂和减数分裂的细胞中都发现了该活性,在中期达到最大活性。成熟的哺乳动物卵母细胞停顿在第二成熟分裂的中期(中期II)并具有高MPF活性。受精时或激活时,MPF活性下降,完成第二成熟分裂并挤出第二极体,然后染色质去浓缩,发生原核形成。在核转移重组胚中,当MPF水平高时,发生NEBD和PCC;当MPF活性下降时,在这些现象发生后,染色质去浓缩,再形成核,随后进行DNA复制。在重组胚中,可以用两种方法中的一种维持正确的倍性;第一种方法是在激活时,将限定的细胞周期阶段的核(例如G1期细胞的二倍体核)转移到中期II卵母细胞中;或第二种方法是激活受体卵母细胞,在MPF活性消失后转移供体核。在羊中,采用16个细胞胚的分裂球作为核供体时,这后一方法已经将发育至胚囊期的频率由重组胚的21%提高至55%(Campbell等,Biol.Reprod. 50 1385-1393(1994))。
这些重组胚发育频率的提高尚未回答核重编程序的问题。在发育期间,某些基因变为“刻上记号”,即发生改变,使得他们不再转录。对印痕的研究已经表明,在生殖细胞形成期间除去该“印痕”(即重编程序)。一个可能性是,这种重编程序受染色质暴露于经历减数分裂细胞中存在的胞质因子的影响。这产生一个问题:在通过核转移重组胚期间,我们如何模拟这一情形以便重编供体核发育钟的程序。
现已发现,如果维持正常倍性(即二倍性)并且该胚在核转移时不激活,那么将核转移到停顿在中期II的卵母细胞中可以产生能活胚。激活的延迟使得该核一直暴露于受体胞质中。
按照本发明的第一个方面,提供一个重组动物胚的方法,该方法包括将二倍体核转移到停顿在第二成熟分裂中期的卵母细胞中,同时不激活卵母细胞,保持该核暴露于受体胞质中,暴露时间足以使重组胚变得能够产生活体分娩,随后激活重组胚,同时维持正确的倍性。在该阶段,重组胚为单细胞。
原则上,本发明可应用于所有动物,包括诸如家禽之类的鸟、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)可以采用克隆的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,在通过胚囊注射包含修饰的干细胞种群后,
   通过原核注射或嵌合现象生产转基因动物,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,
   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,对供体核进行遗传修饰。供体核可以含有一个或多个转基因,可以在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,二倍体核由供体转移至去核受体卵母细胞中。供体在转移时必需为二倍体,以便维持重组胚的正确倍性;因此,供体可以或者处于G1期,或者最好为我们今天申请的共同未决的PCT专利申请PCT/GB96/02099(由GB 9517780.4要求优先权)的对象-处于细胞周期的G0期。
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。同G1细胞的核一样,静止G0细胞的核具有二倍体DNA含量;这两种二倍体核都可以用于本发明中。
根据上述描述,我们相信对于可以用于核供体的细胞没有明显的限制:如细胞可以体外培养或体外(ex vivo)提炼一样,可以使用完分化或部分分化的细胞或未分化细胞。唯一的限制是,供体细胞具有正常的DNA含量并且核型正常。在我们共同未决的专利申请PCT专利申请PCT/GB95/02095(以WO 96/07732公开)中公开了推荐的细胞源。我们相信所有这类正常细胞都含有产生成年动物所需的所有遗传信息。本发明通过改变染色质结构,允许为发育中的胚提供该信息,使得遗传物质可以重新指导发育。
可用于本发明的受体细胞为停顿在第二成熟分裂中期的去核卵母细胞。在大多数脊椎动物中,卵母细胞的成熟在体内进行至这一卵成熟过程的相当晚的时期,然后停顿。排卵时,停顿的卵母细胞从卵巢中排出(如果发生受精,那么自然刺激卵母细胞完成减数分裂)。在本发明的实施中,卵细胞可以或者体外成熟,或者体内成熟,分别在出现第一极体时,或在排卵后尽可能快地收集卵母细胞。
受体最好去核。尽管一般假定在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,并且胞质体积减小将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相邻的胞质除去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,用该方法去核可以保证最小限度地减少胞质体积(Tsunoda等,J.Rprod.Fertil.82173(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.41125(1990));Westhusin等,Biol.Reprod.(Suppl.)42 176(1990))。
在哺乳动物中很少有非侵入性去核方法,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.101 299-311(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。
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。
去核后,或者在不诱导卵母细胞激活的条件下与供体细胞融合,或者通过在非激活条件下进行注射导入供体核。为维持重组胚的正确倍性,供体核融合时必须为二倍体(即为细胞周期的G0或G1期)。
一旦已经制备出适当的供体细胞和受体细胞,那么前者的核必需转移到后者中。更好是通过融合进行核转移。不应该在融合时进行激活。
已经建立的用来诱导融合的三个方法为:
(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.Monopr.919(1969)),其另一实验优点是不诱导激活。在有蹄类动物中,通常使用与诱导单性生殖激活相同的电刺激达到融合(Willadsen Nature 320(6)63-65(1986);Prather等,Biol.Rprod.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,但不能完全用于常规应用(Willadsen Nature 320(6)63-65(1986))。
尽管细胞-细胞融合为进行核转移的推荐方法,但它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,J.Reproduction and Fertility Abstract Series No.15,第60页)。
在本发明的推荐实施方案中,在缺乏激活的情况下,通过在0.3M甘露醇溶液或0.27M蔗糖溶液中进行电脉冲处理,完成卵母细胞细胞核对(couplet)的融合;另一方法是,可以通过在无钙培养基中进行注射导入该核。融合/注射时卵母细胞的年龄和融合/注射介质缺乏钙离子,防止受体卵母细胞激活。
实际上,最好在卵母细胞达到中期II后尽可能快地去核和进行转移。该时间是在成熟开始(体内)或激素处理(体外)后,这取决于物种。对于牛或羊,核转移应该最好在24小时内进行;对于猪,应该在48小时内进行;对于小鼠,在12小时内进行;而对于兔,最好在20-24小时内进行。尽管可以较迟进行转移,但随着卵母细胞年龄变得越来越难以达到转移。需要高MPF活性。
随后,一般回到成熟培养基的融合重组胚维持不被激活,使得供体核暴露于受体胞质中,其时间足以使重组胚变得最终能够产生活体分娩(最好为可育后代)。
激活前的最适时间随物种而变化,这可容易地通过实验确定。对于牛,6-20小时的时间是合适的。该时间可能至少不低于允许染色体形成的时间,但该时间也不能长到融合对同时激活或者在极端情况下重组胚死亡。
当应该激活时,可以使用任何常规的或其它适当的激活方案。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(OzilDevelopment 109 117-127(1990))。在受精期间,胞内钙浓度重复地瞬时提高(Cutbertson & Cobbold Nature 316 541-542(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 109 117-127(1990)),在小鼠中,为10-20分钟(Cutbertson & Cobbold Nature 316 541-542(1985)),而在母牛中的初步观察为20-30分钟(Robl等,Symposium on Cloning Mammals byNuclear 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,通过暴露于几个脉冲,将增加发育的重组胚比例(Collas & RoblBiol.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。
在本发明的实施中,在激活期间必须维持正确的倍性。需要抑制或稳定微管聚合,以便防止产生多个原核,由此保持正确的倍性。通过应用有效浓度(诸如大约5μg/ml)的微管抑制剂(诸如nocodazole)可以做到这一点。另一方法是,可以使用微管稳定剂,诸如taxol。
微管的分子组分(微管蛋白)处于聚合和非聚合状态之间的动态平衡。微管抑制剂(诸如nocodazole)防止微管蛋白分子加入微管中,由此打破平衡并导致微管解聚和纺锤体分解。最好在激活前加入微管抑制剂并留有足够的时间,以确保完成或几乎完成微管的解聚。在大多数情况下20-30分钟可能是足够的。诸如taxol的微管抑制剂防止纺锤体分解,因此也可以防止产生多个原核。最好在与使用微管抑制剂的相似条件下使用微管稳定剂。
在激活后应该保持微管抑制剂或稳定剂的存在,直至形成原核。此后以及在发生第一分裂前的任何活动中应将其除去。
在本发明的推荐实施方案中,成熟开始后30-42小时(对于牛和羊,即核转移后6-18小时)时,将重组卵母细胞置于含有nocodazole(5μg/ml)的培养基中,采用常规方法激活。在激活刺激后,可以在nocodazole中继续培养4-6小时(取决于物种和卵母细胞的年龄)。
按照本发明的第二个方面,提供用前述方法制备的活重组动物胚。
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(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。每个能自身发育的胚在32-64细胞阶段可以用作核供体;另一方法是,可以使用胚囊期的内细胞群细胞。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核事实上发生重编程序(看上去可能是),那么这样每个发育中的胚的繁殖有赖于核转移方法的效率。可能得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原则,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。
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 Egg Transfer(Adams,E.E.,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核生产的动物共享相同的核,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 StateUniversity 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 Cloning Mammals byNuclear Transplantation(Seidel,G.E.Jr,ed.)17-20,Colorado StateUniversity,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)分离二倍体供体细胞;
(2)可选地,例如通过转染适当的具有或不具有选择标记的构建物进行转基因;
(2a)可选地筛选和选择稳定的整合体(微注射越过此步骤);
(3)通过核转移进行胚重组;
(4)体内或体外培养至胚囊;
(4a)可选地筛选和选择稳定的整合体(如果在2a进行筛选,则省略此步骤)或其它所需的特性;
(5)必要时转移至最终受体中。
与先前建立的核转移方法相比,该方案具有多个优点:
1)供体核的染色质可以在缺乏激活的受体卵母细胞的减数分裂胞质中暴露适当的时间。这可以通过改变染色质的结构增加供体核的“重编程序”。
2)当转移G0/G1核时,维持重组胚的正确倍性。
3)现有研究已经表明,牛/羊卵母细胞的激活反应性随年龄而提高。先前观察到的一个问题是,在未去核的老化卵母细胞中发生减数分裂纺锤体极体的复制,并观察到多极纺锤体。然而我们报道,在重组并维持该MPF水平的胚中,尽管核膜破裂并且发生染色质浓缩,但没有观察到形成的纺锤体。早熟浓缩的染色质保持紧密集拢,因此我们可以利用老化过程,提高重组胚的激活反应,而对重组胚的倍性不产生消极影响。
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。在以下描述中参考附图,其中:
图1表明牛卵母细胞的体外成熟速率。
实施例1:采用钠卵母细胞的“MAGIC”方法
该实验方法的对象-受体卵母细胞命名为MAGIC(中期停顿的G1/G0接纳胞质体Metaphase Arrested G1/G0 AcceptIng Cytoplast)受体。
研究了体外卵母细胞成熟期间核和胞质的活动。此外,也研究了融合和激活在不同年龄重组的胚中的作用。研究已经表明,卵母细胞的成熟是不同步的;然而,可以从形态学上选择18小时的成熟卵母细胞群体(图1)。卵母细胞的形态学选择
在图1中,由当地屠宰场获得卵巢,在运输至实验室期间维持28-32℃。用皮下注射针(内径为1.2mm)在直径为3-10mm的卵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,让卵泡物质沉淀10-15分钟。余下的卵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 199,75.0mg/l卡那霉素、30.0mMHepes,pH 7.4、克分子渗透压浓度为280 mOsmols/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、30.0mM Hepes、7.69mM NaHCO3,pH7.8、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℃在空气中5%CO2的氛围中培养在摇床(rocking table)上。从成熟培养皿中取出卵母细胞,在乙醇清洗的玻片上湿装片,在盖玻片下用5%石油胶冻和95%蜡的混合物将其粘附。然后装片的胚在新鲜制备的甲醇∶冰醋酸(3∶1)中固定24小时,用45%的乙酸地衣红(Sigma)染色,用Nikon Microphot-SA相差DIC显微镜检查,图1中的图表示MII卵母细胞的百分比以及具有可见极体的卵母细胞的百分比。牛卵泡卵母细胞的激活
如果继续成熟直至24小时,那么这些卵母细胞在含钙甘露醇中的激活比率非常低(24%)(表1a)。然而,从电脉冲介质中除去钙和镁将防止激活。
表1a表示在体外不同时间成熟的牛卵泡卵母细胞的激活。从成熟培养基中取出卵母细胞,在激活培养基中洗涤1次,将其置于激活室中,给予80μs的电脉冲1.25 kV/cm。
                              表1a
    卵母细胞数(N)     成熟开始后的小时数(hpm)[年龄(hrs)]     原核形成(激活%)
    73     24     24.6
    99     30     84.8
    55     45     92.7*
*许多2个或多个原核假去核牛卵母细胞的激活反应
表1b表示体外成熟的在成熟开始后大约22小时(hpm)时假去核的牛卵母细胞的激活反应。卵母细胞完全按去核的方法进行处理,吸出不含中期板的小体积胞质。操作后,给予卵母细胞1.25KV/cm的单个直流脉冲,放回成熟培养基中,在30hpm和42hpm时将卵母细胞组装片、固定和用乙酸地衣红染色。结果表明5个单独实验每个时间点时的卵母细胞数,按相对于细胞总数具有原核的细胞数计。
                              表1b
  实验     具有原核的细胞数/细胞总数30hpm     具有原核的细胞数/细胞总数42hpm
    1     1/8     -
    2     0/24     0/30
    3     0/21     0/22
    4     0/27     0/25
    5     0/19     0/1
hpm=开始成熟后的小时数在去核牛卵母细胞中形成原核
表2表示在与初级牛成纤维细胞融合(24hpm)并随后激活(42hpm)的去核卵母细胞中原核的形成。结果代表5个实验。将卵母细胞分为两组,A组在激活前在nocodazole中温育1小时,激活后在nocodazle中温育6小时。B组不用nocodazole处理。在激活后12小时固定激活的卵母细胞,并用乙酸地衣红染色。然后在相差下记录每个parthenote中的原核(PN)数。结果以含有1个或多个原核的激活卵母细胞百分比表示。
                            表2
  总数    1PN   2PN   3PN   4PN   >4PN
  A组     52     100    0    0     0     0
  B组     33     45.2    25.8    16.1     3.2     9.7
缺乏形成的纺锤体和缺乏极体表示,为了维持重组胚的正确倍性,那么应该只将二倍体(即G0/G1)核转移至该胞质环境中。激活的卵母细胞在激活刺激前后,在微管抑制剂nocodazole中温育5小时、1小时,防止形成小核(表2),因此当供体核处于细胞周期的G0/G1期时,维持重组胚的正确倍性。结果
这些结果表明:
i)这些卵母细胞可以在成熟开始后18小时去核(图1);
ii)去核卵母细胞可以在0.3M甘露醇中或者在0.27M蔗糖中与供体分裂球/供体细胞融合,也可以在缺乏任何激活反应的情况下,在无钙介质中注射供体细胞或供体核;
iii)重组胚或脉冲处理的去核卵母细胞可以在成熟培养基中培养,不进行同时激活;
iv)观察转移的核经历核膜破裂(NEBD)和染色体浓缩。无论在转移核的任何细胞周期阶段,都没有观察到形成的减数分裂/有丝分裂纺锤体;
v)这类操作的融合对在30小时和42小时时激活,激活频率等于未操作的对照卵母细胞;
vi)无论在转移核的任何细胞周期阶段,在随后激活后都没有观察到极体;
viii)随后激活时,每个重组合子形成1-5个小核(表2)。采用“MAGIC”方法重建牛胚
在初步实验中,使用通过血清饥饿5天在细胞周期G0期同步的初级成纤维细胞,已经将该技术应用于牛胚的重组。结果总结于表3中。
表3表明通过将核由血清饥饿的(G0)牛初级成纤维细胞转移至去核未激活MII卵母细胞中重组的牛胚的发育。在24hpm时重组胚,而在42hpm时该融合对激活。在激活前,融合对在含nocodazole(5μg/ml)的M2培养基中培养1小时,在激活后培养5小时。用80μs的单个直流脉冲1.25 KV/cm激活融合对。
                                表3
   实验数  胚囊数/融合对总数    胚囊%
    12     1/304/31     3.312.9
实施例2:采用羊卵母细胞的“MAGIC”方法
我们也已经在在体内已成熟的羊卵母细胞中进行了与实施例1相似的观察。可以通过在前列腺素处理后24小时,冲洗超刺激母羊的输卵管,取出新排卵的卵母细胞。使用无钙镁的PBS/1.0%FCS作为冲洗介质,防止卵母细胞激活。卵母细胞可以在无钙介质中去核,在缺乏激活的情况下按上述方法导入供体细胞。没有观察到形成的纺锤体,在随后激活时形成多个核,这可以通过nocodazole处理抑制。结果
在羊的初步实验中,单次妊娠已经导致一个活羔羊出生。结果总结于表4和表5中。
表4表示通过将得自建立的细胞系的胚转移至未激活的体内成熟的去核羊卵母细胞中重组的羊胚的发育。由超刺激的苏格兰黑脸母羊获得卵母细胞,由得自威尔士mountain ewe获得的9天大的胚的胚盘建立细胞系。重组胚在临时受体母羊的结扎输卵管中培养6天,然后取出,分析发育。
                                表4
  核转移的日期    继代数 桑椹胚、胚囊数/总数
    17.1.95     6     4/28
    19.1.95     7     1/10
    31.1.95     13     0/2
    2.2.95     13     0/14
    7.2.95     11     1/9
    9.2.95     11     1/2
    14.2.95     12
    16.2.95     13     3/13
    总数     10/78(12.8%)
表5表示将所有桑椹胚期/胚囊期重组的胚转移至同步的最后受体黑脸母羊的子宫角后,诱导妊娠。该表表明,来自妊娠频率的每个转移组的胚总数,以母羊和胚计,在大多数情况下,每只母羊转移2个胚。建立了导致单个活羔羊出生的单次双妊娠。
                            表5
       继代数     “MAGIC”
    P6     4
    P7     1
    P11     2
    P12     0
    P13     3
    总MOR/BL     10
    母羊总数     6
    妊娠母羊%     1(16.7)
    胎儿/总转移数(%)     2/10(20.0)

Claims (18)

1.一个重组动物胚的方法,该方法包括将二倍体核转移到停顿在第二成熟分裂中期的卵母细胞中,同时不激活卵母细胞,保持该核暴露于受体胞质中,暴露时间足以使该胚变得能够产生活体分娩,随后激活重组胚,同时维持正确的倍性。
2.权利要求1中要求保护的一个方法,其中该动物为有蹄类动物。
3.权利要求2中要求保护的一个方法,其中该动物为母牛或公牛、猪、山羊、羊、骆驼或水牛。
4.权利要求1-3的任一项中要求保护的一个方法,其中对供体核进行遗传修饰。
5.权利要求1-4的任一项中要求保护的一个方法,其中由静止期细胞贡献二倍体核。
6.权利要求1-5的任一项中要求保护的一个方法,其中将受体卵母细胞去核。
7.权利要求1-6的任一项中要求保护的一个方法,其中通过细胞融合达到核转移。
8.权利要求1-7的任一项中要求保护的一个方法,其中该动物为母牛或公牛,而在激活前保持供体核暴露于受体胞质6-20小时。
9.权利要求1-8的任一项中要求保护的一个方法,其中在激活期间,通过微管抑制维持正确的倍性。
10.权利要求9中要求保护的一个方法,其中通过应用nocodazole达到微管抑制。
11.权利要求1-8的任一项中要求保护的一个方法,其中在激活期间,通过微管稳定维持正确的倍性。
12.权利要求11中要求保护的一个方法,其中通过应用taxol达到微管稳定。
13.制备动物的方法,该方法包括:
(a)按任一前述权利要求要求保护的方法重组动物胚;
(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育种。
14.权利要求13中要求保护的一个方法,其中该动物胚在完全发育之前进行进一步操作。
15.权利要求14中要求保护的一个方法,其中由该胚得到不止1个动物。
16.重组动物胚,它能够产生活体分娩,并用权利要求1-12的任一项中要求保护的方法制备。
17.用权利要求13-15的任一项中要求保护的方法制备的动物。
18.由权利要求16中要求保护的胚发育的动物。
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