RecA-mediated, targeted mutagenesis in zebrafish

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into I-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in I or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems.

Spawning areas and early development of long spiky-head carp (Luciobrama macrocephalus) in the Yangtze River and Pearl River, China

The spawning areas and early development of long spiky-head carp, Luciobrama macrocephalus (Lacepede), an endemic fish species in China, were investigated in the Yangtze River and Pearl River of central and southeastern China between 1961 and 1993. The potamodromous fish migrated upstream to spawn between May and July as the floodwater began to rise. The water-hardened eggs drifted down the river, and the embryos and larvae developed in the course of drifting. The spawning areas of the fish were widely found in the upper and middle main channels and large tributaries. Two large dams (Gezhouba dam and Danjiangkou dam) did not significantly impact on the reproduction of the fish. Fifty stages of the early development from one cell to the juvenile with fully formed fins were observed and characterized pictorially. The larvae of long spiky-head carp could be distinguished from the larvae of other co-occurring species by counting the number of somites and comparing the proportion of sizes of eye to otic capsule.

Comparative ontogenetic behavior and migration of kaluga, Huso dauricus, and Amur sturgeon, Acipenser schrenckii, from the Amur River

We conducted laboratory experiments with kaluga, Huso dauricus, and Amur sturgeon, Acipenser schrenckii, to develop a conceptual model of early behavior. We daily observed embryos (first life phase after hatching) and larvae (period initiating exogenous feeding) to day-30 (late larvae) for preference of bright habitat and cover, swimming distance above the bottom, up- and downstream movement, and diel activity. Day-0 embryos of both species strongly preferred bright, open habitat and initiated a strong, downstream migration that lasted 4 days (3 day peak) for kaluga and 3 days (2 day peak) for Amur sturgeon. Kaluga migrants swam far above the bottom (150 cm) on only 1 day and moved day and night; Amur sturgeon migrants swam far above the bottom (median 130 cm) during 3 days and were more nocturnal than kaluga. Post-migrant embryos of both species moved day and night, but Amur sturgeon used dark, cover habitat and swam closer to the bottom than kaluga. The larva period of both species began on day 7 (cumulative temperature degree-days, 192.0 for kaluga and 171.5 for Amur sturgeon). Larvae of both species preferred open habitat. Kaluga larvae strongly preferred bright habitat, initially swam far above the bottom (median 50-105 cm), and migrated downstream at night during days 10-16 (7-day migration). Amur sturgeon larvae strongly avoided illumination, had a mixed response to white substrate, swam 20-30 cm above the bottom during most days, and during days 12-34 (most of the larva period) moved downstream mostly at night (23-day migration). The embryo-larva migration style of the two species likely shows convergence of non-related species for a common style in response to environmental selection in the Amur River. The embryo-larva migration style of Amur sturgeon is unique among Acipenser yet studied.

Ontogenetic behavior and migration of Chinese sturgeon, Acipenser sinensis

The Chinese sturgeon, Acipenser sinensis, is an anadromous protected species that presently only spawns in the Yangtze River. Using laboratory experiments, we examined the behavioral preference of young Chinese sturgeon to physical habitat (water depth, illumination intensity, substrate color, and cover) and monitored their downstream migration. Hatchling free embryos were photopositive, preferred open habitat, and immediately upon hatching, swam far above the bottom using swim-up and drift. Downstream migration peaked on days 0-1, decreased about 50% or more during days 2-7, and ceased by day 8. Days 0-1 migrants were active both day and night, but days 2-7 migrants were most active during the day. After ceasing migration, days 8-11 embryos were photonegative, preferred dark substrate and sought cover. Free embryos developed into larvae and began feeding on day 12, when another shift in behavior occurred-larvae returned to photopositive behavior and preferred white substrate. The selective factor favoring migration of free embryos upon hatching and swimming far above the bottom may be avoidance of benthic predatory fishes. Free embryos, which must rely on yolk energy for activity and growth, only used 19 cumulative temperature degree-days for peak migration compared to 234 degree-days for growth to first feeding larvae, a 1 : 12 ratio of cumulative temperature units. This ratio suggests that sturgeon species with large migratory embryos, like Chinese sturgeon, which require a high level of energy to swim during migration, may migrate only a short time to conserve most yolk energy for growth.

Nuclear transplantation in different strains of zebrafish

Single later blastula nuclei from AB strain of zebrafish (Danio rerio) were transplanted into enucleated unfertilized eggs of Long fin strain. Of 1119 cloning embryos, 14 reconstructed embryos developed into fry. DNA fingerprinting systems of the cloned fish were similar to those of the nuclear donor fish, but were distinctly different from those of the unclear recipient fish. It confirmed that the genetic material originated from nuclear donor cell other than from nuclear recipient egg. The research suggested that the basic technique for nuclear transplantation performed with different strains of zebrafish has made a breakthrough. It should be helpful for the study of some important developmental problems such as gene function, the regulation of gene expression during animal development, the developmental potential of a nucleus and the interactions between the donor nucleus and the recipient cytoplasm, etc.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach, Misguruns mizolepis Gunthe

Microcystin-LR, a specific and potent hepatotoxin, was tested for its effects oil loach embryo-larval and juvenile development, The results of this study showed that loach embryos were more sensitive when exposed to microcystin-LR at a later than at an earlier stage of development, Juveniles were far less sensitive to MC-LR than were embryos and larvae. Mortality and developmental abnormality were proven to be dose-dependent and to be stage-specific sensitive. Among the abnormal changes noted were: pericardial edema and tubular heart, bradycardia, homeostasis, poor yolk resumption. small head, curved body and tail, and abnormal hatching, Liver and heart were the main targets of microcystin-LR toxicity. Ultrastructural analysis documented a complex set of sublethal effects of microcystin-LR on loach hepatocytes, chiefly including morphological alteration in nuclear and RER of loach liver cells. fit addition, microcystin-LR was lethal to loach juvenile in the subacute (7 days) exposure (LC50) = 593.3 mug/l). (C) 2002 Elsevier Science Ltd. All rights reserved.

In vitro culture of embryonic hearts from guppy fish (Poecilia reticulata)

Forty embryonic hearts were taken out by anatomical needle from denuded embryos of the ovoviviparity guppy fish that were dechorioned by mechanic method or by trypsin digestion, and were in vitro cultured. In the cultured hearts, 80% have maintained beating in vitro for 4 weeks, and the longest record for beating was 142 d. Owing to fish embryo transparency, beating frequency and blood color changes are easily viewed from the embryonic hearts under a dissecting microscope. The current study established the in vitro culture method of embryonic hearts in guppy fish, which can be used as a model for the study of heart and cardiovascular system in vertebrates.

Margulis' theory on division of labour in cells revisited

Division of labour is a marked feature of multicellular organisms. Margulis proposed that the ancestors of metazoans had only one microtubule organizing center (MTOC), so they could not move and divide simultaneously. Selection for simultaneous movement and cell division had driven the division of labour between cells. However, no evidence or explanation for this assumption was provided. Why could the unicellular ancetors not have multiple MTOCs? The gain and loss of three possible strategies are discussed. It was found that the advantage of one or two MTOC per cell is environment-dependent. Unicellular organisms with only one MTOC per cell are favored only in resource-limited environments without strong predatory pressure. If division of labour occurring in a bicellular organism just makes simultaneous movement and cell division possible, the possibility of its fixation by natural selection is very low because a somatic cell performing the function of an MTOC is obviously wasting resources. Evolutionary biologists should search for other selective forces for division of labour in cells.

Embryonic and genetic manipulation in fish

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An "all-fish" gene construct CAgcGH has been made by splicing the common carp beta-actin gene (CA) promoter onto the grass carp growth hormone gene (gcGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21(st) century.

长俊木瓜体细胞胚胎诱导的初步研究

以长俊木瓜为材料,研究了其体细胞胚胎诱导过程中各个环节的影响因素。结果表明:愈伤组织诱导的适宜外植体是叶片,培养基为MS+6-BA1.0 mg.L-1+2,4-D0.2 mg.L-1,黑暗培养;非胚性愈伤组织向胚性愈伤组织转化的适宜培养基是MS+6-BA1.0 mg.L-1+NAA1.0 mg.L-1;胚性愈伤组织的保持与增殖应在黑暗条件下进行,培养基为MS+6-BA1.0 mg.L-1+2,4-D0.2 mg.L-1;长俊木瓜叶片体胚的发生以加入ABA2.0 mg.L-1的MS培养基最为有利。

非洲爪蛙分泌型叶酸结合蛋白及NKX6.3在早期胚胎神经发育中的功能研究

叶酸是B族维生素的一员，参与体内一系列重要的生命过程包括DNA，氨基酸的合成，调控细胞周期，参与一碳单位供体循环，调节DNA,蛋白质甲基化等。叶酸的许多功能都和叶酸结合蛋白有关，体内有多种跨膜形式的叶酸结合蛋白，比如Folbp1，RFC，HCP等。以前的研究表明这些不同的叶酸结合蛋白具有不同的功能。分泌型叶酸结合蛋白是另外一类叶酸结合蛋白，在人类，小鼠，猪中都有序列报道，但是其功能却知之甚少。 我们在非洲爪蛙中鉴定出一个全新的分泌型叶酸结合蛋白并命名为Secreted Folate Binding Protein(sFBP)。在胚胎和转染细胞系中我们都证明该蛋白是分泌性的，表面等离子共振实验发现sFBP能够结合叶酸。在胚胎早期这个基因表达于粘液腺和神经板区域，神经管闭合后在神经管、粘液腺、眼睛，头部以及鳃弓都有表达。特异morpholino 阻断sFBP翻译后发现粘液腺发育异常，神经管闭合缺陷，前后体轴聚集延伸运动受到抑制，尾芽期胚胎表现出体轴缩短，无眼，小头或无头的表型。进一步研究发现显微注射sFBP morpholino 的胚胎神经板区域细胞发生凋亡，中胚层和神经外胚层的一系列粘附分子表达异常，神经细胞的正常分化也受到抑制。通过显微移植实验我们还发现抑制sFBP的翻译后，神经嵴细胞的正常分化和迁移都受到抑制。但是，显微注射叶酸及其类似物或者显微注射甲基供体S-腺苷甲硫氨酸或者亮氨酸甲基转移酶都不能挽救阻断sFBP造成的表形，由此提示sFBP可能不是通过叶酸传统的参与营养合成或者甲基化的途径发挥作用。我们发现注射sFBP morpholino可以抑制Islet-1mRNA和蛋白质的表达，Islet-1的表达区域与sFBP类似。共同注射Islet-1 mRNA和sFBP morpholino可以极大的挽救sFBP morpholino的表型。最后通过morpholino特异阻断Islet-1的表达后，我们发现其表现出与sFBP morpholino类似的粘液腺发育缺陷，神经板细胞凋亡，小头无眼的表形。由此叶酸是B族维生素的一员，参与体内一系列重要的生命过程包括DNA，氨基酸的合成，调控细胞周期，参与一碳单位供体循环，调节DNA,蛋白质甲基化等。叶酸的许多功能都和叶酸结合蛋白有关，体内有多种跨膜形式的叶酸结合蛋白，比如Folbp1，RFC，HCP等。以前的研究表明这些不同的叶酸结合蛋白具有不同的功能。分泌型叶酸结合蛋白是另外一类叶酸结合蛋白，在人类，小鼠，猪中都有序列报道，但是其功能却知之甚少。 我们在非洲爪蛙中鉴定出一个全新的分泌型叶酸结合蛋白并命名为Secreted Folate Binding Protein(sFBP)。在胚胎和转染细胞系中我们都证明该蛋白是分泌性的，表面等离子共振实验发现sFBP能够结合叶酸。在胚胎早期这个基因表达于粘液腺和神经板区域，神经管闭合后在神经管、粘液腺、眼睛，头部以及鳃弓都有表达。特异morpholino 阻断sFBP翻译后发现粘液腺发育异常，神经管闭合缺陷，前后体轴聚集延伸运动受到抑制，尾芽期胚胎表现出体轴缩短，无眼，小头或无头的表型。进一步研究发现显微注射sFBP morpholino 的胚胎神经板区域细胞发生凋亡，中胚层和神经外胚层的一系列粘附分子表达异常，神经细胞的正常分化也受到抑制。通过显微移植实验我们还发现抑制sFBP的翻译后，神经嵴细胞的正常分化和迁移都受到抑制。但是，显微注射叶酸及其类似物或者显微注射甲基供体S-腺苷甲硫氨酸或者亮氨酸甲基转移酶都不能挽救阻断sFBP造成的表形，由此提示sFBP可能不是通过叶酸传统的参与营养合成或者甲基化的途径发挥作用。我们发现注射sFBP morpholino可以抑制Islet-1mRNA和蛋白质的表达，Islet-1的表达区域与sFBP类似。共同注射Islet-1 mRNA和sFBP morpholino可以极大的挽救sFBP morpholino的表型。最后通过morpholino特异阻断Islet-1的表达后，我们发现其表现出与sFBP morpholino类似的粘液腺发育缺陷，神经板细胞凋亡，小头无眼的表形。由此我们认为sFBP结合叶酸后可能通过细胞膜上的受体传递信号，并且Islet-1可能在sFBP的下游发挥作用。 神经嵴是脊椎动物特有的一群多潜能干细胞，产生于表皮和神经板的边界，在原肠运动之后这群细胞通过表皮间充值转换从神经管背侧迁移到不同的区域，分化成不同的细胞类型，包括外周神经系统，色素细胞，软骨等。神经嵴的发生是一个多步骤多基因参与的精细调控过程。目前理论认为最初由一些分泌性信号分子又叫形态生成素比如BMP,Wnt，FGF，Notch等通过不同浓度梯度的相互作用调节一组在表皮和神经板边界的转录因子（Msx、Pax3/7、Zic1、Dlx3/5等）的表达，即边界决定。这些边界决定因子进一步在预定形成神经嵴的区域激活神经嵴特化基因比如Slug/Snail、FoxD3、Twist、Sox9/10的表达完成神经嵴的特化（Specification）。 Nkx6.3是Nkx6家族的一个转录因子，RT-PCR显示其呈现母源性表达。特异抗体显示Nkx6.3蛋白第9期在整个胚胎都表达，大部分蛋白集中在细胞核，有少部分蛋白定位于细胞膜上；神经板时期主要定位于神经嵴区域的细胞膜上。过表达Nkx6.3会影响细胞粘连分子的表达，由此干扰正常的胚胎原肠运动和Activin诱导的动物帽聚集延伸运动。显微注射Nkx6.3特异morpholino阻断其蛋白表达会抑制神经嵴的marker基因Wnt8，Fgf8，Pax3，Msx1，Zic1，FoxD3，Slug的转录，阻碍神经嵴的发育。在动物帽中单独注射Nkx6.3可以在mRNA水平上诱导Wnt8、Fgf8另一方面抑制BMP4的表达进而诱导神经嵴基因Pax3，Zic1，Slug的表达。报告基因实验也显示Nkx6.3能够激活Wnt信号而在动物帽中抑制BMP信号。Nkx6.3蛋白功能域分析发现其EH1结构域（domain）参与对Wnt8信号的激活，而EH1结构域和HD结构域之间的连接区域（linker domain）参与对FGF的激活和对BMP的抑制。进一步在动物帽和胚胎中分析发现Nkx6.3对Wnt8的激活依赖于FGF家族受体信号但是不依赖于Fgf8。有趣的是4细胞时期过表达Nkx6.3促进Fgf8和Wnt8 mRNA表达，但是抑制边界决定基因Msx1、Pax3和神经嵴特化基因Slug的转录。在32细胞时期显微注射Nkx6.3可以在内源神经嵴发生区域抑制Slug的表达，而异位却诱导Slug的mRNA。我们发现与动物帽中对BMP的调节不同，在胚胎中，过表达Nkx6.3会强烈的激活Smad1蛋白在细胞核中的表达即BMP信号被激活，高的BMP信号会抑制神经嵴的发生。另外我们发现过表达Nkx6.3在胚胎中抑制Dlx5而在动物帽中却不影响Dlx5的表达水平，Morpholino阻断Dlx5会抑制Msx1、Pax3和Slug的表达。BMP信号和Dlx5在动物帽和在整体胚胎中对Nkx6.3的不同响应可以一定程度上解释过表达Nkx6.3在2个系统中对神经嵴基因Slug相反的影响结果。

哺乳动物体细胞核移植影响因素的研究

题。 在已有的基础上，建立了基于单个植入前胚胎的逆转录-聚合酶链反应（RT-PCR）技术，成功实现了针对单个猕猴植入前胚胎进行可重复的，多个特定基因的表达检测。在此基础上，对一些重要的早期胚胎发育相关基因在猕猴体细胞核移植胚胎和体外受精胚胎中的表达进行了比较研究。这其中包括核仁相关蛋白基因nucleolin，nucleophosmin，fibrillarin，PAF53，UBF和mRNA早期装配相关蛋白snrpn，这些基因被认为与植入前胚胎的再程序化有着非常密切的关系。我们的研究结果发现，相对于体外受精胚胎，被检测基因在核移植胚胎中的转录出现了不同程度的异常，表现为表达丰度相对较低，这意味着体细胞核移植植入前胚胎的再程序化，或者说供体核的再程序化可能出现了异常。通过进一步对fibrillarin（胚胎合子基因组启动的标志基因）的表达研究，比较了合子基因组启动这一再程序化分子事件在核移植胚胎和体外受精胚胎中的发生时序。研究结果证实，体细胞核移植胚胎的合子基因组启动出现了明显的滞后。我们还发现这与核移植胚胎的异常卵裂速度有着一定的关联，同时说明某些源自核移植操作技术和方法的未知因素可能是造成核移植胚胎再程序化异常的主要原因。为了验证合子基因组滞后对核移植胚胎后续发育的影响，本论文研究分析了着床相关基因Mamu-AG在猕猴核移植胚胎中的表达。研究首次发现Mamu-AG mRNA只在囊胚期开始出现，这与在人类中的情况有所不同（始于8细胞时期）。在正常体外受精囊胚中，不论发育时间如何，第6和第7天的囊胚均正常表达Mamu-AG mRNA。而在核移植囊胚中，仅有第7天的囊胚检测到该mRNA，第6天的囊胚中则没有，这进一步说明了胚胎合子基因组启动的延迟是影响核移植胚胎发育的重要环节。 本研究从分子水平上对猕猴植入前胚胎基因转录和表达进行了分析研究，首次发现了在猕猴体细胞核移植胚胎中发育相关基因表达异常，以及合子基因组启动滞后的分子证据，这为我们改进现有的猕猴核移植操作程序提供了新的思路，同时也为进一步研究猕猴植入前胚胎发育的再程序化奠定了基础。

体细胞核移植影响因素的研究

尽管体细胞核移植(somatic cell nuclear transfer, SCNT)技术仍然处于 起步发展阶段，但是随着体细胞核移植技术在近些年来的飞速发展，人们已经得 到了多种哺乳动物体细胞核移植的存活后代。体细胞核移植技术在生物医药、农 业及其它领域的应用显示了这项技术的巨大发展前景。另一方面，人们发现体细 胞核移植效率低下并且体细胞克隆动物存在许多缺陷，这些主要是由于核移植技 术，供体细胞选择，体外培养系统以及卵母细胞的状态等的差异导致的。本研究 主要围绕着这些因素在体细胞核移植过程中对于克隆胚胎植入前后发育的影响 而开展，目的在于提高体细胞核移植的效率。 实验一，研究了几个因素对克隆胚胎发育和克隆效率的影响，为提高体细胞 核移植效率提供一些依据。这部分研究主要包括四倍体半克隆小鼠研究和蛋白酶 体抑制剂在克隆胚胎和孤雌激活胚胎发育中的影响。四倍体体细胞半克隆 （tetraploid semi-cloned , TSC）胚胎的体外发育结果表明，虽然TSC 胚胎可 能避免二倍体半克隆胚胎发育过程中的非整倍体现象，但是仍然不能形成胎儿。 另外，利用蛋白酶体抑制剂MG132 处理克隆胚胎的结果表明，虽然MG132 通过抑 制成熟促进因子（maturation promoting factors，MPF）活性的降低可以提高 克隆胚胎的体外发育率，但是没有改变克隆胚胎的质量。 实验二，研究了克隆技术在克隆胚胎构建和发育上的影响。首先，我们研究 了完整颗粒细胞注射到卵母细胞中所引起的变化。我们发现完整的颗粒细胞注入 到卵母细胞中之后很快引起细胞膜裂解和细胞核碎裂，从而导致激活后重构胚胎 的碎裂。这一实验表明完整供体细胞核注入的方法不适用于小鼠体细胞克隆。接 下来我们研究了不同的核移植技术――利用piezo 的直接注射法（piezoelectric microinjection，PEM）和电融合法（Electrofusion，EF）――对植入前后的克隆胚 胎以及出生后的克隆动物的影响。研究的结果发现，在PEM 法中，提高piezo 脉 冲的强度会导致供体细胞核DNA 断裂增加，使植入前克隆胚胎的细胞数目减少， 凋亡增加，从而降低克隆胚胎的质量。相反，在用EF 法产生的胚胎中细胞核DNA 的断裂很少，克隆胚胎质量相对较好。实验的结果表明，由于两种克隆技术利用 的原理不同，会对克隆胚胎的发育产生一定的影响，从而改变克隆的效率。 实验三，研究了猕猴体细胞克隆胚胎早期发育过程中细胞核内有丝分裂器蛋 白[Nuclear Mitotic Apparatus Protein，NuMA]的表达和分布。尽管在有些猕猴体 细胞核移植胚胎中没有检测到NuMA 的正确表达和分布并且有些胚胎出现了微 管组装异常，但是大多数猕猴体细胞核移植胚胎具有正常的NuMA 蛋白表达和 正常的核型。通过改进操作技术我们得到了可以发育到正常囊胚的猕猴体细胞核 移植胚胎。这些结果提示猕猴SCNT 胚胎发育失败的原因不是NuMA 等重要蛋 白的缺失。