体细胞核移植胚胎和皮肤成纤维细胞来源干细胞的相关技术研究
|
摘要
背景及目的人胚胎干细胞(embryonic stem cells, ES cells)是一种具多项分化潜能的细胞,可被诱导分化成各种特化细胞和组织,用来修复或替代人体丧失功能的组织和器官,这就是“干细胞治疗(stem cell therapy)”。干细胞治疗在疾病治疗和损伤修复领域中有潜在的优势,它可治疗包括脑、肾脏、骨髓及组织病变在内的多种疾病。但是,由于每个个体的组织相容性不同,同种异体胚胎干细胞用于临床会引起免疫排斥。解决这一难题的主要途径就是产生患者自己的胚胎干细胞。体细胞核移植法(somatic cell nuclear transfer, SCNT)即治疗性克隆(therapeutic cloning)和诱导产生多能性干细胞这两项技术已成功诱导出组织相容的多能干细胞,并且取得了重大进展。
治疗性克隆是指将体细胞核移入去核的卵母细胞中,在理论上,体细胞的细胞核将被卵母细胞的胞质重编程,沉默表达体细胞基因同时激活干细胞基因。重构胚发育至囊胚阶段后,从囊胚内细胞团(inner cell mass, ICM)中分离出胚胎干细胞。随后胚胎干细胞发育分化产生携带患者自己基因的胚胎干细胞,这些胚胎干细胞及其衍生组织移植后就不会产生免疫排斥。但是,体细胞核移植技术仍不完善,成功的诱导核移植胚胎干细胞系需要依赖多种因素,包括受体细胞的类型、重编程的时间、激活方法、胚胎的培养系统等。此外,损伤小的重构的方法也是关键因素之一。目前,使用该项技术成功获得克隆囊胚的效率仍然很低。
最近,有研究利用异位表达胚胎干细胞标志因子重编程小鼠成纤维细胞,使其产生组织相容胚胎干细胞,为干细胞治疗开辟了新的途径。这类诱导产生的多能干细胞(induced pluripotent stem Cell, iPS)是利用逆转录病毒载体在小鼠成纤维细胞中导入了4个与多能性有关的基因Oct4, Sox2, c-myc和Klf4诱导产生的。得到的iPS细胞在许多方面都与ES细胞十分相似,并且可以产生嵌合体。随后这四个因子又成功诱导人类成纤维细胞重编程为iPS细胞。这一技术的应用对产生组织相容多能干细胞及医学研究有着巨大的价值。但是,c-Myc逆转录病毒的活化增加了嵌合体和子代的致瘤性。此外,诱导产生多能干细胞的效率也不高,阻碍了它的临床应用。
因此,为了提高干细胞治疗的效率,深入研究体细胞重编程的分子机制,本课题从体细胞核移植和诱导产生的多能干细胞两个方面研究了多种因素对其效率产生的影响。
本论文分为6部分:
第1部分不同核移植方法对小鼠体细胞核移植效率的影响
目的研究不同核移植方法对小鼠体细胞核移植效率的影响,建立一种简单有效的核移植方法。
方法以小鼠颗粒细胞作为体细胞核移植的供核细胞,采用四种去核方法(盲吸法、蔗糖辅助去核法、化学去核法、荧光染色去核法)研究卵母细胞去核率的差异,并比较了胞质内注射法和反向核移植法应用于小鼠体细胞核移植的效果。
结果荧光染色法的去核率(88%)显著高于其他试验组(P<0.05);胞质内注射法的囊胚率和囊胚细胞数较反向核移植高(P<0.05)。
结论荧光染色去核法和胞质内注射法构建体细胞核移植胚胎效率较高,可维持胚胎早期发育。
第2部分单一和联合化学激活方法对小鼠体细胞核移植胚胎体外发育的影响
目的探讨单一和联合化学激活方法对小鼠体细胞核移植胚胎体外发育的影响。
方法构建小鼠核移植胚胎。分别使用单一激活剂钙离子载体(A23187)、乙醇(Eth)、氯化锶(SrCl2)及联合蛋白激酶抑制剂6-DMAP、CB激活。
结果10mmol/L SrCl2处理6 d可获得较高卵裂率(68.9%)和囊胚率(7.2%)(P<0.05),也较A23187、Eth组高(P<0.05);联合激活方法中,Eth+6-DMAP+CB组(69.8%和46.05±2.62)、SrCl2 +CB组(71.9%和45.40±2.23)和A23187+6-DMAP+CB组(62%和39.75±1.15)卵裂率和囊胚细胞计数明显高于未联合组(P<0.05)。
结论乙醇联合6-DMAP、CB组合,SrCl2联合CB组合均能较好地激活小鼠体细胞核移植胚胎。
第3部分一步法和二步法培养系统对小鼠体细胞核移植胚胎体外发育的影响
目的比较一步法和二步法两种培养系统对小鼠体细胞核移植胚胎体外发育的影响,确立一种有效的培养系统。
方法构建小鼠核移植胚胎。激活后,随机分为两组。第1组,比较核移植胚胎在KSOMgAA , KSOMgAA / KSOMgAA , cleavage medium and cleavage medium/blastocyst medium中的发育情况;第2组,观察牛黄酸对核移植胚胎发育影响。
结果核移植胚胎发育的囊胚率、孵化率和囊胚细胞计数一步法和两步法培养体系中没有显著差异(P>0.05)。牛磺酸加入KSOMgAA后能有效改善早期核移植胚胎体外发育能力。
结论一步法和两步法培养体系均符合小鼠体细胞核移植胚胎不同发育阶段需求。
第4部分小鼠体细胞核移植囊胚的微卫星DNA鉴定
目的利用微卫星DNA技术进行体细胞核移植囊胚的鉴定。
方法提取近交系小鼠体细胞核移植囊胚、供体BALB/c小鼠、受体C57BL/6小鼠及昆明小鼠的基因组DNA,使用巢式聚合酶链反应扩增4个微卫星位点DNA片段,即D3Mit28, D11Mit258,D12Mit136及D14Mit50。
结果通过微卫星DNA序列的扩增,证明核移植囊胚的微卫星DNA与供体细胞完全相同,而与受体细胞或者对照细胞无亲缘关系。
结论体细胞核移植囊胚基因组来源于供体BALB/c小鼠。
第5部分体细胞核移植胚胎ES细胞样集落分离
目的探讨小鼠体细胞核移植胚胎ES细胞样集落分离和培养方法,比较不同因子对建立核移植胚胎ES细胞样集落效率的影响。
方法构建小鼠体细胞核移植胚胎。当核移植胚胎培养至早期囊胚阶段,移至小鼠胎儿成纤维细胞饲养层上,换成ES细胞培养液继续培养4～5d,分离消化ICM,重新接种。2 d后分离出ES样细胞集落,进行形态学观察。
结果ES样细胞集落具有典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致密,隆起生长,细胞之间界限不清楚;单个细胞体积小、核大。碱性磷酸酶染色呈阳性。体外可分化为拟胚体,体内可分化成上皮样细胞、腺体和肌肉组织。
结论小鼠体细胞核移植胚胎中可以分离出ES细胞样集落。
第6部分小鼠成纤维细胞诱导产生多能干细胞
目的体外诱导小鼠成纤维细胞重编程为多能胚胎干细胞样集落。
方法分别将Pou5f1, Sox2, c-Myc和Klf4真核表达载体转染NIH3T3细胞。分离小鼠成纤维细胞,移至小鼠胎儿成纤维细胞饲养层上,取转染后的上清液培养。3d后,加入G418,筛选克隆2～3w。
结果筛选初期大部分克隆形态学为扁平状,仅4株克隆为ES细胞样生长,后期克隆数增长至11株。ES细胞样克隆具有岛屿状团状隆起结构。
结论四个逆转录因子Pou5f1, Sox2, Klf4和c-Myc可诱导小鼠体细胞ES细胞集落样改变。
Background Human embryonic stem (ES) cells possess the potential to differentiate into all the cell types which serves as a sort of repair system for the body. The remarkable potential of stem cells makes it possible to treat patients by transplanting specialized healthy cells produced from them to repair damaged and diseased body-parts. This concept is known as“stem cell therapy”. Stem cell therapy is now emerging as a potentially revolutionary new way to treat disease and injury. Stem cell therapy has potential applications in treating a wide array of diseases and ailments of the brain, kidney, bone and many other tissues. However, the transplanted cells are grown from stem cells that are not genetically compatible with one patient, their immune system will reject the cells. One approach to overcome transplant rejection of human ES (hES) cells is to derive hES cells from the patient’s own cells. Diverse methods, such as somatic nuclear transfer (also called therapeutic cloning) and the generation of human iPS cells (induced pluripotent stem cells) from somatic cells, have been attempted to produce genetic compatible pluripotent stem cells. These approaches were in progress.
Therapeutic cloning refers to the transfer of the nucleus of a somatic cell into an enucleated donor oocyte. In theory, the oocyte’s cytoplasm would reprogram the transferred nucleus by silencing all the somatic cell genes and activating the embryonic ones. The reconstructed embryos are induced embryonic developments and ES cells would be isolated from the inner cell (ICMs) of the cloned blastocysts. It is proposed that following directed cell differentiation, these cells which carried the nuclear genome of the patient could be transplanted without immune rejection for treatment of degenerative disorders among others. However, Somati cell nuclear transfer(SCNT) is still a developing technique. Success in the production of SCNT-ES cell line is attributed to optimization of several factors including the donor cell type, reprogramming time, activation protocol and use of sequential culture system. Furthermore, use of less-invasive enucleation method is suggested to be one of key factors. Today, the success rate of obtaining cloned blastocysts from this technique remains low.
An approach toward the same end of the generation of“patient-specific”ES cells was recently described, in which murine fibroblasts were reprogrammed by ectopically expressing factors known to be highly expressed in ES cells. Such iPS cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells were indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. While the four transcription factors were used to reprogram human fibroblasts to iPS cells. Application of this approach in human cells would have enormous potential and generate patient-specific pluripotent stem cells to study and potentially ameliorate human diseases. However, reactivation of the c-Myc retrovirus increases tumorigenicity in the chimeras and progeny. Moreover, the efficiency of generating human iPS cells is low, hindering clinical application.
Thus, to improved the efficiency of stem cell therapy and provided necessary theory for the further study of molecular mechenisms of somatic cell reprogram, I have studied some factors on the efficiency of somatic cell nuclear transfer and generating iPS cells from mouse somatic cells.
This thesis divided into six parts as follows:
Part1 Effects of Different Methods of Nuclear Transfer On the Efficiency of Somatic Cell Nuclear Transfer in Mice
Objective To study the effects of different methods of nuclear transfer on the efficiency of somatic cell nuclear transfer in mice, and to establish a simple, efficient method for nuclear transfer.
Methods Granulosa cell was used as a donor of mouse somatic nuclear transfer.Different enucleation methods(blind aspiration,sucrose pretreatment for enucleation, chemical induced enucleation, bisbenzimide stain method) and two methods of enucleation(intracytoplasmic nuclear injection and reverse nuclear transfer)were studied on enucleation rate of the oocyte.
Results The enucleation rate(88 % ) was significantly the higher in bisbenzimide stain method than in other three groups(P<0.05). The intracytoplasmic nuclear injection had a higher rate of blastocyst development and more of total cell numbers per blastocyst than those of reverse nuclear transfer method(P<0.05).
Conclusion Bisbenzimide stain method and intracytoplasmic nuclear injection are efficient procedures in mouse somatic cell nuclear transfer, these procedures have the ability to maintain early nuclear transfer embryos development.
Part2 Effects of Single or Combined chemical activation methods on the Activation of Mouse Somatic Cell Nuclear Transfer Embryos
Objective To establish the optimum procedure of somatic cell nuclear transfer embryos activation in mice, and a comparison has been made of the development of mouse somatic cell nuclear transfer embryos activated by single or combined chemical activation methods.
Methods mouse reconstructed embryos were constructed. The development of reconstructed embryos which have activated in different methods of activation including ethanol(Eth),calcium ionophore(A23187), strontium (SrCl2) and combined these single agent treatment with 6-dimethylaminopurine(6-DMAP), cytochalasin B(CB) were observed.
Results①When NT embryos were treated with 10 mmol/L SrCl2 for 6h, the activation rate (68.9%) and the rate of blastulation (7.2%) were higher than other groups(P <0.05).②The cleavage rate and total cell numbers per blastocyst were significantly higher after treated with Eth + 6-DMAP + CB ( 69.8%;46.05±2.62), SrCl2+ CB(71.9%;45.40±2.23) or A23187+6-DMAP+CB (62%;39.75±1.15) than the control group(P <0.05).
Conclusion The Eth+6-DMAP+CB and the SrCl2+CB combined chemical activation methods could better activate mouse somatic cell nuclear transfer embryos.
Part3 Effect of one-step or two-step culture systems on in vitro development of mouse somatic cell nuclear transferred embryos
Objective To select some culture media profitable for early mouse somatic cell nuclear transfer embryos in vitro development, a comparison has been made of the development of mouse somatic cell nuclear transfer embryos in either one-step or two-step culture systems.
Methods Mouse reconstructed embryos were done. Following activation, NT embryos were allocated randomly to one of two treatment groups. Group 1, NT embryo development was compared in: KSOMgAA, KSOMgAA / KSOMgAA, cleavage medium and cleavage medium/blastocyst medium. Second group, NT embryos were cultured in medium KSOMgAA and KSOMgAA +Taurine. The development of NT embryos in one-step and two-step culture systems was observed, respectively.
Results There were no significantly differences in the proportion of blastocysts, hatching rate, and total cell numbers between one-step and two-step culture systems. Development of early reconstructed and parthenogenetic embryos was significantly increased by adding taurine in KSOMgAA culture medium when compared to KSOMgAA media.
Conclusion Both one-step and two-step culture systems were feasible mouse somatic cell nuclear transffer embryos culture method in vitro.
Part4 Microsatellite DNA analysis of mouse somatic cell nuclear transfer blastocysts
Objective To identify the source of the somatic cell nuclear transfer blastocysts, microsatellite DNA assay was developed.
Methods DNA isolation from SCNT blastocyst,donor BALB/c mouse, recipient C57BL/6 mouse and KM mouse were tested by nested polymerase-chainreaction(PCR) analysis. Primers designed for four specific microsatellite locus (D3Mit28, D11Mit258, D12Mit136, D14Mit50) were employed.
Results Microsatellite DNA analyses examining four loci confirm that all the SCNT blastocysts were genetically identical to the donor mouse. Additionally, the SCNT embryos were not genetically related to the respective recipient mouse. Furthermore sequences of SCNT blastocysts are diferent from the control KM mouse.
Conclusion The results have proved that the nucleus of somatic cell nuclear transfer blastocysts come from somatic nucleus of donor BALB/c mouse.
Part5 Isolation of Embryonic Stem Cell-like Colonies From Mouse Nuclear Transfer Embryos
Objective The aim of the experiment was to isolate and culture mouse ES cell-like colonies from mouse nuclear transfer embryos, and to compare some factors influencing the efficiency of establishing those stem cell-like colonies.
Methods Mouse NT embryos were done. When NT embryos developed to the stage of early blastocyst,embryos were transferred to the feeder layer of mouse embryo fibroblast with cardiomyocyte media conditioned for 4-5 days,and then ICMs were digested and recultured. ES cell-like colonies were isolated after 2 days,and the morphology of this colonies were observed.
Results Outcome of isolating ES cell-like colonies was good. And the ES cell-like colonies had its typical morphological characteristics such as nest-like colonies,round or elliptic,with clear edge and smooth surface,strong refraction,significant swelling growth, compactness of cell aggregation,and obscure distinction between two edges of neighbour cells within a colony,relatively bigger nucleus compared with little cytoplasm.ES cells showed strong AKP activity. The differentiation of ES cells is outgrowth from embryonic bodies in vitro, while glandular tissues, scalelike cylindrical epithelium and muscular tissue were formed from ES cells in vivo.
Conclusions This result demonstrates that ES cell-like colonies can be isolated from mouse NT embryos.
Part6 Induction of Pluripotent Stem Cells from Mouse Adult Fibroblast
Objective This present study aimed to reprogramm mouse fibroblasts into a pluripotent ES-cell-like state in vitro.
Methods The NIH-3T3 cells were infected with Pou5f1-, Sox2-, c-Myc- and Klf4-expressing retroviral vectors respectly. The mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) were seeded on feeders and incubated in the virus/polybrene-containing supernatants. Three days after infection, we added G418. Clones were selected for 2 to 3 weeks.
Results Most colonies had a flat morphology and 4 colonies were ES-like when selection was applied early, the number of ES-like colonies (11) that increased at later time points. ES-like colonies isolated were with island-like images.
Conclusions This demonstrates that the activity of Pou5f1, Sox2, Klf4 and c-Myc can apparently turn mouse somatic cells into cells that characteristic morphology closely resemble ES cell colony.
治疗性克隆是指将体细胞核移入去核的卵母细胞中,在理论上,体细胞的细胞核将被卵母细胞的胞质重编程,沉默表达体细胞基因同时激活干细胞基因。重构胚发育至囊胚阶段后,从囊胚内细胞团(inner cell mass, ICM)中分离出胚胎干细胞。随后胚胎干细胞发育分化产生携带患者自己基因的胚胎干细胞,这些胚胎干细胞及其衍生组织移植后就不会产生免疫排斥。但是,体细胞核移植技术仍不完善,成功的诱导核移植胚胎干细胞系需要依赖多种因素,包括受体细胞的类型、重编程的时间、激活方法、胚胎的培养系统等。此外,损伤小的重构的方法也是关键因素之一。目前,使用该项技术成功获得克隆囊胚的效率仍然很低。
最近,有研究利用异位表达胚胎干细胞标志因子重编程小鼠成纤维细胞,使其产生组织相容胚胎干细胞,为干细胞治疗开辟了新的途径。这类诱导产生的多能干细胞(induced pluripotent stem Cell, iPS)是利用逆转录病毒载体在小鼠成纤维细胞中导入了4个与多能性有关的基因Oct4, Sox2, c-myc和Klf4诱导产生的。得到的iPS细胞在许多方面都与ES细胞十分相似,并且可以产生嵌合体。随后这四个因子又成功诱导人类成纤维细胞重编程为iPS细胞。这一技术的应用对产生组织相容多能干细胞及医学研究有着巨大的价值。但是,c-Myc逆转录病毒的活化增加了嵌合体和子代的致瘤性。此外,诱导产生多能干细胞的效率也不高,阻碍了它的临床应用。
因此,为了提高干细胞治疗的效率,深入研究体细胞重编程的分子机制,本课题从体细胞核移植和诱导产生的多能干细胞两个方面研究了多种因素对其效率产生的影响。
本论文分为6部分:
第1部分不同核移植方法对小鼠体细胞核移植效率的影响
目的研究不同核移植方法对小鼠体细胞核移植效率的影响,建立一种简单有效的核移植方法。
方法以小鼠颗粒细胞作为体细胞核移植的供核细胞,采用四种去核方法(盲吸法、蔗糖辅助去核法、化学去核法、荧光染色去核法)研究卵母细胞去核率的差异,并比较了胞质内注射法和反向核移植法应用于小鼠体细胞核移植的效果。
结果荧光染色法的去核率(88%)显著高于其他试验组(P<0.05);胞质内注射法的囊胚率和囊胚细胞数较反向核移植高(P<0.05)。
结论荧光染色去核法和胞质内注射法构建体细胞核移植胚胎效率较高,可维持胚胎早期发育。
第2部分单一和联合化学激活方法对小鼠体细胞核移植胚胎体外发育的影响
目的探讨单一和联合化学激活方法对小鼠体细胞核移植胚胎体外发育的影响。
方法构建小鼠核移植胚胎。分别使用单一激活剂钙离子载体(A23187)、乙醇(Eth)、氯化锶(SrCl2)及联合蛋白激酶抑制剂6-DMAP、CB激活。
结果10mmol/L SrCl2处理6 d可获得较高卵裂率(68.9%)和囊胚率(7.2%)(P<0.05),也较A23187、Eth组高(P<0.05);联合激活方法中,Eth+6-DMAP+CB组(69.8%和46.05±2.62)、SrCl2 +CB组(71.9%和45.40±2.23)和A23187+6-DMAP+CB组(62%和39.75±1.15)卵裂率和囊胚细胞计数明显高于未联合组(P<0.05)。
结论乙醇联合6-DMAP、CB组合,SrCl2联合CB组合均能较好地激活小鼠体细胞核移植胚胎。
第3部分一步法和二步法培养系统对小鼠体细胞核移植胚胎体外发育的影响
目的比较一步法和二步法两种培养系统对小鼠体细胞核移植胚胎体外发育的影响,确立一种有效的培养系统。
方法构建小鼠核移植胚胎。激活后,随机分为两组。第1组,比较核移植胚胎在KSOMgAA , KSOMgAA / KSOMgAA , cleavage medium and cleavage medium/blastocyst medium中的发育情况;第2组,观察牛黄酸对核移植胚胎发育影响。
结果核移植胚胎发育的囊胚率、孵化率和囊胚细胞计数一步法和两步法培养体系中没有显著差异(P>0.05)。牛磺酸加入KSOMgAA后能有效改善早期核移植胚胎体外发育能力。
结论一步法和两步法培养体系均符合小鼠体细胞核移植胚胎不同发育阶段需求。
第4部分小鼠体细胞核移植囊胚的微卫星DNA鉴定
目的利用微卫星DNA技术进行体细胞核移植囊胚的鉴定。
方法提取近交系小鼠体细胞核移植囊胚、供体BALB/c小鼠、受体C57BL/6小鼠及昆明小鼠的基因组DNA,使用巢式聚合酶链反应扩增4个微卫星位点DNA片段,即D3Mit28, D11Mit258,D12Mit136及D14Mit50。
结果通过微卫星DNA序列的扩增,证明核移植囊胚的微卫星DNA与供体细胞完全相同,而与受体细胞或者对照细胞无亲缘关系。
结论体细胞核移植囊胚基因组来源于供体BALB/c小鼠。
第5部分体细胞核移植胚胎ES细胞样集落分离
目的探讨小鼠体细胞核移植胚胎ES细胞样集落分离和培养方法,比较不同因子对建立核移植胚胎ES细胞样集落效率的影响。
方法构建小鼠体细胞核移植胚胎。当核移植胚胎培养至早期囊胚阶段,移至小鼠胎儿成纤维细胞饲养层上,换成ES细胞培养液继续培养4～5d,分离消化ICM,重新接种。2 d后分离出ES样细胞集落,进行形态学观察。
结果ES样细胞集落具有典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致密,隆起生长,细胞之间界限不清楚;单个细胞体积小、核大。碱性磷酸酶染色呈阳性。体外可分化为拟胚体,体内可分化成上皮样细胞、腺体和肌肉组织。
结论小鼠体细胞核移植胚胎中可以分离出ES细胞样集落。
第6部分小鼠成纤维细胞诱导产生多能干细胞
目的体外诱导小鼠成纤维细胞重编程为多能胚胎干细胞样集落。
方法分别将Pou5f1, Sox2, c-Myc和Klf4真核表达载体转染NIH3T3细胞。分离小鼠成纤维细胞,移至小鼠胎儿成纤维细胞饲养层上,取转染后的上清液培养。3d后,加入G418,筛选克隆2～3w。
结果筛选初期大部分克隆形态学为扁平状,仅4株克隆为ES细胞样生长,后期克隆数增长至11株。ES细胞样克隆具有岛屿状团状隆起结构。
结论四个逆转录因子Pou5f1, Sox2, Klf4和c-Myc可诱导小鼠体细胞ES细胞集落样改变。
Background Human embryonic stem (ES) cells possess the potential to differentiate into all the cell types which serves as a sort of repair system for the body. The remarkable potential of stem cells makes it possible to treat patients by transplanting specialized healthy cells produced from them to repair damaged and diseased body-parts. This concept is known as“stem cell therapy”. Stem cell therapy is now emerging as a potentially revolutionary new way to treat disease and injury. Stem cell therapy has potential applications in treating a wide array of diseases and ailments of the brain, kidney, bone and many other tissues. However, the transplanted cells are grown from stem cells that are not genetically compatible with one patient, their immune system will reject the cells. One approach to overcome transplant rejection of human ES (hES) cells is to derive hES cells from the patient’s own cells. Diverse methods, such as somatic nuclear transfer (also called therapeutic cloning) and the generation of human iPS cells (induced pluripotent stem cells) from somatic cells, have been attempted to produce genetic compatible pluripotent stem cells. These approaches were in progress.
Therapeutic cloning refers to the transfer of the nucleus of a somatic cell into an enucleated donor oocyte. In theory, the oocyte’s cytoplasm would reprogram the transferred nucleus by silencing all the somatic cell genes and activating the embryonic ones. The reconstructed embryos are induced embryonic developments and ES cells would be isolated from the inner cell (ICMs) of the cloned blastocysts. It is proposed that following directed cell differentiation, these cells which carried the nuclear genome of the patient could be transplanted without immune rejection for treatment of degenerative disorders among others. However, Somati cell nuclear transfer(SCNT) is still a developing technique. Success in the production of SCNT-ES cell line is attributed to optimization of several factors including the donor cell type, reprogramming time, activation protocol and use of sequential culture system. Furthermore, use of less-invasive enucleation method is suggested to be one of key factors. Today, the success rate of obtaining cloned blastocysts from this technique remains low.
An approach toward the same end of the generation of“patient-specific”ES cells was recently described, in which murine fibroblasts were reprogrammed by ectopically expressing factors known to be highly expressed in ES cells. Such iPS cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells were indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. While the four transcription factors were used to reprogram human fibroblasts to iPS cells. Application of this approach in human cells would have enormous potential and generate patient-specific pluripotent stem cells to study and potentially ameliorate human diseases. However, reactivation of the c-Myc retrovirus increases tumorigenicity in the chimeras and progeny. Moreover, the efficiency of generating human iPS cells is low, hindering clinical application.
Thus, to improved the efficiency of stem cell therapy and provided necessary theory for the further study of molecular mechenisms of somatic cell reprogram, I have studied some factors on the efficiency of somatic cell nuclear transfer and generating iPS cells from mouse somatic cells.
This thesis divided into six parts as follows:
Part1 Effects of Different Methods of Nuclear Transfer On the Efficiency of Somatic Cell Nuclear Transfer in Mice
Objective To study the effects of different methods of nuclear transfer on the efficiency of somatic cell nuclear transfer in mice, and to establish a simple, efficient method for nuclear transfer.
Methods Granulosa cell was used as a donor of mouse somatic nuclear transfer.Different enucleation methods(blind aspiration,sucrose pretreatment for enucleation, chemical induced enucleation, bisbenzimide stain method) and two methods of enucleation(intracytoplasmic nuclear injection and reverse nuclear transfer)were studied on enucleation rate of the oocyte.
Results The enucleation rate(88 % ) was significantly the higher in bisbenzimide stain method than in other three groups(P<0.05). The intracytoplasmic nuclear injection had a higher rate of blastocyst development and more of total cell numbers per blastocyst than those of reverse nuclear transfer method(P<0.05).
Conclusion Bisbenzimide stain method and intracytoplasmic nuclear injection are efficient procedures in mouse somatic cell nuclear transfer, these procedures have the ability to maintain early nuclear transfer embryos development.
Part2 Effects of Single or Combined chemical activation methods on the Activation of Mouse Somatic Cell Nuclear Transfer Embryos
Objective To establish the optimum procedure of somatic cell nuclear transfer embryos activation in mice, and a comparison has been made of the development of mouse somatic cell nuclear transfer embryos activated by single or combined chemical activation methods.
Methods mouse reconstructed embryos were constructed. The development of reconstructed embryos which have activated in different methods of activation including ethanol(Eth),calcium ionophore(A23187), strontium (SrCl2) and combined these single agent treatment with 6-dimethylaminopurine(6-DMAP), cytochalasin B(CB) were observed.
Results①When NT embryos were treated with 10 mmol/L SrCl2 for 6h, the activation rate (68.9%) and the rate of blastulation (7.2%) were higher than other groups(P <0.05).②The cleavage rate and total cell numbers per blastocyst were significantly higher after treated with Eth + 6-DMAP + CB ( 69.8%;46.05±2.62), SrCl2+ CB(71.9%;45.40±2.23) or A23187+6-DMAP+CB (62%;39.75±1.15) than the control group(P <0.05).
Conclusion The Eth+6-DMAP+CB and the SrCl2+CB combined chemical activation methods could better activate mouse somatic cell nuclear transfer embryos.
Part3 Effect of one-step or two-step culture systems on in vitro development of mouse somatic cell nuclear transferred embryos
Objective To select some culture media profitable for early mouse somatic cell nuclear transfer embryos in vitro development, a comparison has been made of the development of mouse somatic cell nuclear transfer embryos in either one-step or two-step culture systems.
Methods Mouse reconstructed embryos were done. Following activation, NT embryos were allocated randomly to one of two treatment groups. Group 1, NT embryo development was compared in: KSOMgAA, KSOMgAA / KSOMgAA, cleavage medium and cleavage medium/blastocyst medium. Second group, NT embryos were cultured in medium KSOMgAA and KSOMgAA +Taurine. The development of NT embryos in one-step and two-step culture systems was observed, respectively.
Results There were no significantly differences in the proportion of blastocysts, hatching rate, and total cell numbers between one-step and two-step culture systems. Development of early reconstructed and parthenogenetic embryos was significantly increased by adding taurine in KSOMgAA culture medium when compared to KSOMgAA media.
Conclusion Both one-step and two-step culture systems were feasible mouse somatic cell nuclear transffer embryos culture method in vitro.
Part4 Microsatellite DNA analysis of mouse somatic cell nuclear transfer blastocysts
Objective To identify the source of the somatic cell nuclear transfer blastocysts, microsatellite DNA assay was developed.
Methods DNA isolation from SCNT blastocyst,donor BALB/c mouse, recipient C57BL/6 mouse and KM mouse were tested by nested polymerase-chainreaction(PCR) analysis. Primers designed for four specific microsatellite locus (D3Mit28, D11Mit258, D12Mit136, D14Mit50) were employed.
Results Microsatellite DNA analyses examining four loci confirm that all the SCNT blastocysts were genetically identical to the donor mouse. Additionally, the SCNT embryos were not genetically related to the respective recipient mouse. Furthermore sequences of SCNT blastocysts are diferent from the control KM mouse.
Conclusion The results have proved that the nucleus of somatic cell nuclear transfer blastocysts come from somatic nucleus of donor BALB/c mouse.
Part5 Isolation of Embryonic Stem Cell-like Colonies From Mouse Nuclear Transfer Embryos
Objective The aim of the experiment was to isolate and culture mouse ES cell-like colonies from mouse nuclear transfer embryos, and to compare some factors influencing the efficiency of establishing those stem cell-like colonies.
Methods Mouse NT embryos were done. When NT embryos developed to the stage of early blastocyst,embryos were transferred to the feeder layer of mouse embryo fibroblast with cardiomyocyte media conditioned for 4-5 days,and then ICMs were digested and recultured. ES cell-like colonies were isolated after 2 days,and the morphology of this colonies were observed.
Results Outcome of isolating ES cell-like colonies was good. And the ES cell-like colonies had its typical morphological characteristics such as nest-like colonies,round or elliptic,with clear edge and smooth surface,strong refraction,significant swelling growth, compactness of cell aggregation,and obscure distinction between two edges of neighbour cells within a colony,relatively bigger nucleus compared with little cytoplasm.ES cells showed strong AKP activity. The differentiation of ES cells is outgrowth from embryonic bodies in vitro, while glandular tissues, scalelike cylindrical epithelium and muscular tissue were formed from ES cells in vivo.
Conclusions This result demonstrates that ES cell-like colonies can be isolated from mouse NT embryos.
Part6 Induction of Pluripotent Stem Cells from Mouse Adult Fibroblast
Objective This present study aimed to reprogramm mouse fibroblasts into a pluripotent ES-cell-like state in vitro.
Methods The NIH-3T3 cells were infected with Pou5f1-, Sox2-, c-Myc- and Klf4-expressing retroviral vectors respectly. The mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) were seeded on feeders and incubated in the virus/polybrene-containing supernatants. Three days after infection, we added G418. Clones were selected for 2 to 3 weeks.
Results Most colonies had a flat morphology and 4 colonies were ES-like when selection was applied early, the number of ES-like colonies (11) that increased at later time points. ES-like colonies isolated were with island-like images.
Conclusions This demonstrates that the activity of Pou5f1, Sox2, Klf4 and c-Myc can apparently turn mouse somatic cells into cells that characteristic morphology closely resemble ES cell colony.
引文
1 Ueno M, Matsumura M, Watanabe K,et al.Neural conversion of ES cells by an inductive activity on human amniotic membrane matrix.Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9554-9.
2 Norstr?m A, Akesson K, Hardarson T,et al.Molecular and pharmacological properties of human embryonic stem cell-derived cardiomyocytes.Exp Biol Med (Maywood). 2006 Dec;231(11):1753-62.
3 Hwang NS, Varghese S, Zhang Z, et al.Chondrogenic differentiation of human embryonic stem cell-derived cells in arginine-glycine-aspartate-modified hydrogels.Tissue Eng. 2006 Sep;12(9):2695-706.
4 D'Amour KA, Bang AG, Eliazer S,et al.Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.Nat Biotechnol. 2006 Nov;24(11):1392-401.
5 Cummings BJ, Uchida N, Tamaki SJ,et al.Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice.Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):14069-74.
6 Yoon BS, Yoo SJ, Lee JE, et al.Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.Differentiation. 2006 Apr;74(4):149-59.
7 Lanza RP, Cibelli JB, West MD.Prospects for the use of nuclear transfer inhuman transplantation. Nat Biotechnol. 1999 Dec;17(12):1171-4.
8 Hochedlinger K, Jaenisch R. Nuclear transplantation,embryonic stem cells, and the potential for cell therapy. N Eng J Med.2003, 349, 275-286.
9 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126(4): 663?676.
1 Hochedlinger K, Jaenisch R. Nuclear reprogramming and pluripotency. Nature, 2006, 441(7097): 1061-1067.
2 Rodolfa K T, Eggan K. A transcriptional logic for nuclear reprogramming. Cell, 2006, 126(4): 652—655.
3 Cervera RP, Stojkovic M.Commentary: somatic cell nuclear transfer-progress and promise.Stem Cells,2008,26(2):494-495.
4 Wakayama T, Perry ACF, Zueeotti M. et al. Full-term development of mice from enucleated oocytes infected with cumulus cell nuclei. Nature, 1998, 394:369-374.
5 Zhou QI, Jean-Paul Renard, Gaelle LE Friec, et al. Generation of fertile cloned rats by regulating oocyte activation. Science, 2003, 302:1179.
6 Shin T, Kraemer D, Pryor J, et al. A cat cloned by nuclear transplantation. Nature. 2002, 415:859.
7 Chesne P, Adenot PCB Viglietta C, et al. Cloned rabbits produced by nuclear transfer from adult somatic cells[J]. Nat Biotechnol, 2002, 20(4): 366-369.
8 Lee BC, Kim MK, Jang G,et al. Dogs cloned from adult somatic cells.Nature. 2005 , 436(7051):641.
9 Baguisi A, Behboodi E, Melican DT, et al.Production of goats by somatic cell nuclear transfer. Nat Biotechnol,1999,17(5):456-461.
10郭继彤.成年体细胞克隆山羊研究. [学位论文],西北农林科技大学,中国,西安,2000.
11 Wilmut I, Schnieke AE, Mcwhir J, et al. Viable offspring derived from fetal and adult mammalian cells. Nature. 1997, 385: 810-813.
12 Woods GL, White KL, Vanderwall DK, et al. A mule cloned from fetal cellsby nuclear transfer. Science. 2003 ,301(5636):1063.
13 Kato Y, Tani T, Sotomaru Y, et al. Eight calves cloned from somatic cells of single adult.Science, 1998,282: 2095-2098.
14陈大元,李劲松,韩之明,等.体细胞克隆牛:供体细胞和受体的影响.科学通报.2003,48:768-773.
15 Galli C, Lagutina I, Crotti G, et al.Pregnancy: a cloned horse born to its dam twin.Nature,2003,424(6949):635.
16 Polejaeva IA, Chen SH, Vaught TD, et al. Cloned pigs produced by nuclear transfer adult somatic cells. Nature, 2000, 407: 86-90.
17陆凤花,石德顺,韦英明,等.不同融合条件对水牛体细胞核移植效果的影响.广西农业生物科学,2005,24 (1): 1-5.
18 Kato Y, Tani T, Tsunoda Y Cloning of calves from various somatic cell types of male and female adult newborn and fetal cows. Reprod Fertil, 2000,120: 231-237.
19 Meissner A, Jaenisch R.Mammalian nuclear transfer.Dev Dyn,2006, 235(9):2460-2469.
20 Reik W, Dean W, Walter J.Epigenetic reprogramming in mammalian development.Science, 2001,293(5532):1089-1093.
21 Wrenxyckic, Wellsd, et al. Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts . Biol Reprod, 2001 ,65:309-317.
22 Rideout WM 3rd, Eggan K, Jaenisch R.Nuclear cloing and epigenetic regprogramming of the genome. Science. 2001 ,293(5532):1093-1098.
23 Wolffe AP, Matzke MA. Epigenetics: regulation through regression . Science. 1999 Oct 15;286(5439):481-486.
24 Smith LC, Wilmut I, Hunter RHF. Influence of cell cycle stage at nuclear transplantation on the development of mouse embryos. Reprod. Fertil. 1988,84: 619-624.
25 Smith S D,Soloy E,Avery B,et al. Nucleus remodeling in reconstructed cattle embryos.Theriogenology.1994,41:298-304.
26 Kikyo N, Wolffe AP.Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons.J Cell Sci, 2000 ,113 ( Pt 1):11-20.
27 Steinbach OC, Wolffe AP, Rupp RA.Somatic linker histones cause loss of mesodermal competence in Xenopus.Nature. 1997,389(6649):395-399.
28 Ogura A, Inoue K, Ogonuki N, et al. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol Reprod, 2000a,62(6): 1579-1584.
29 Butler D.French clone provides support for Dolly. Nature, 1998 ,12;392(6672):113.
30 Kubota C, Yamakuchi H, Todoroki J, et al. Six cloned calves produced from adult fibroblast cells after long–term cultire . Proc Natl Acad Sci USA, 2000, 97: 990-995.
31 Ogura A, Inoue K, Takano K, et al. Birth of mice after nuclear transfer by electrofusion using tail tip cells. Mol Reprod Dev,2000,57(1): 55-59.
32 Inoue K,Ogonuki N,Mochida K,et al.Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning. Biol.Reprod, 2003,69:1394-1400.
33 Campbell KHS. Nuclear equivalence, nuclear transfer and the cell cycle. Cloning,1999, 1(1): 3-15.
34 Wilmut I, Campbell KH. Quiescence in nuclear transfer. Science 1998;281:1611.
35 Campbell KHS, McWhir J, Ritchi WA, et al. Sheep cloned by nuclear transfer from a cultured cell line . Nature,1996, 380: 64-66.
36 Wells DN, Misica PM, Tervit HR. Production of cloned calves following Nuclear Transfer with Cultured Adult Mural Granulosa cells. J Biol Reprod. 1999, 60: 996-1005.
37 Zakhartchenko V, Alberio R, Stojkovic M, et al. Adult cloning in cattle: potential of nuclei from permanent cell line and from primary cultures. Mol Reprod Dev. 1999, 54: 264-272.
38 Cibelli JB, Stice SL, Golueke PJ,et al. Cloned transgenic calves produced from nonquiescent fetal fibroblast. Science,1998, 280(5367):1256-1258.
39 Zakhartchenko V, Durcova-Hills G, Stojkovic M,et al. Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts. J Reprod Fertil. 1999 ,115(2):325-331.
40 Peura TT, Lewis IM, Trounson AO. The effect of recipient oocyte volume on nuclear transfer in cattle. Mol Reprod Dev. 1998, 50 (2): 185-191.
41 Westhusin ME, Collas P, Marek D, et al. Reducing the amount of cytoplasm available for early embryonic development decreases the quality but quantity of embryos produced by in vitro fertilization and nuclear transplantation. Theriogenology. 1996, 46: 243-252.
42 McGrath J, Solter D.Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro.Science,1984,14;226(4680): 1317-1319.
43 Robl JM, Prather R, Barnes F, et al. Nuclear transplantation in bovine embryos. Anim Sci,1987, 64: 642-647.
44 Prather RS, Barnes FL, Sims MM, et al. Nuclear transplantation in the bovine embryos: assessment of donor nuclei and recipient oocyte. J Biol Reprod,1987, 37: 859-866.
45 Takano H, Koyama K, Kozzi C, et al. Effects of aging of recepient oocytes in the development of bovine nuclear transfer embryos in vitro. Theriogenology. 1993, 39: 909-917.
46 Yang X, Jiang S, Farrell D, et al. Nuclear transfer in cattle: effect of nuclear donor cells cytoplast age co-culture and embryo transfer. Mol Reprod Dev. 1993, 35(1): 29-36.
47 Stice SL, Robl JM. Nuclear reprogramming in nuclear transplant rabbit embryos. Biol Reprod. 1988, 39: 657-664.
48 Prather RS,Sims MM, First NL. Nuclear transplantation in early pig embryos. J Biol Reprod,1989, 44: 414-418.
49 Westhusin ME,.Levanduski MJ, Scarborough R, et al. Viable embryos and normal calves after nuclear transfer into Hoechst staind enucleated demi-oocytes of cows. J Reprod Fertil. 1992. 95(2): 475-480.
50 Stice SL, Keefer CL. Multiple generational bovine embryo cloning. Biol Reprod, 1993, 48: 715-719.
51 Bordignon V, Smith LC. Telophase enucleated: an improved method to prepare recipient cytoplasts for use in bovine nuclear transfer. Mol Reprod Dev,1998, 49(1): 29-36.
52 Monamed Nour MS, Takahashi Y. Preparation of young preactivated oocyte with high enucleation efficiency for bovine nuclear transfer. Theriogenology. 1999, 51(3): 661-666.
53王敏康,刘冀珑,李光鹏,等.蔗糖预处理去核:一种可靠的无损害的小鼠卵母细胞MII期核去除法.生殖医学杂志, 2001, 10(4): 227-232.
54 Willadsen SM. Nuclear transplantation in sheep embryos. Nature, 1986, 320:63-65.
55 Booth PJ, Tan SJ, Reipurth R,et a1.Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique.Cloning Stem Cells,2001,3(3):139-150.
56 Oldenbourg R.A new view on polarization microscopy. Nature, 1996, 381(6585):811-812.
57 Liu L, Oldenbourg R, Trimarchi JR, et al. A reliable, noninvasive technique for spindle imaging and enucleation of mammalian ooctes. Nature,2000, 18: 223-225.
58 Tatham BG, Dowsing AT, Tyounson AO. Enucleated by centrifugation of in vitro~matured bovine oocytes for use in nuclear transfer. Biol. Reprod. 1995,53(5): 1088-1094.
59 Tsunoda Y, Shioda Y, Onodera M, et a1.Differential sensitivity of mouse pronuclei and zygote cytoplasm to Hoechst staining and ultraviolet irradiation.J.Reprod.Fertil,l988, 82:173-178.
60 Fulka JR, Moor RM. Noninvasive chemical enucleation of mouse oocytes. Mol Repord Dev. 1993, 34: 427-430.
61 Gasparrini B, Gao S, Ainslie A, et al.Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation. Biol Reprod, 2003, 68: 1259-1266.
62 Gasparrini B, Dinneyes A, Ainslie A. Attempted chemical enucleation of in vitro matured oocytes after activation and their use in nuclear transfer. Theriogenology, 2002, 414.
63 Yin XJ, Tani T, Yonemura I, et al.Productionn of cloned pigs from adult somatic cells by chemical assisted removal of maternal chromosomes. Biol Reprod, 2002, 67:442-446.
64 Kawakami M, Tani T, Yabuuchi A, et al.Effect of demecolcine and nocodazole on the efficiency of chemically assisted removal of chromosomes and the developmental potential of nuclear transferred porcine oocytes. Cloning Stem Cells. 2003;5(4):379-387.
65 Ficsher D, Ibanez E, Albertini DF, Activated bovine cytoplasts produced by induced enucleation support development of bovine nuclear transfer embryos in vitro. Bicsl Reprod, 2002, 66, 238:346.
66 Yin XJ, Kato Y, Tsunoda Y.Effect of delayed enucleation on the developmental potential of nuclear-transferred oocytes receiving adult and fetal fibroblast cells.Zygote. 2002,10(3):217-222.
67 Illmensee K, Hoppe PC. Nuclear transplantation in Mus musculus: developmental potential of nuclei from preimplantation embryos. Cells. 1981, 23: 9-18.
68 Meng L, Ely JJ, Stouffer RL, et al. Rhesus monkeys produced by nuclear transfer. Biol Reprod. 1997, 57(2): 454-459.
69 Kono T, Kwon OY, Watanabe T, et al. Development of mouse enucleated oocytes receiving a nucleus from different stages of the second cell cycle. J Reprod Fertil. 1992, 94: 481-487.
70 Kono T , Tsunoda Y. Development of Single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryos. Gamete Res. 1989, 2: 427-434.
71张涌,王建辰,钱菊汾,等.山羊卵核移植的研究.中国农业科学,1991,24(5): 1-6.
72 Goto K, A Kinoshita, TY Takuma, et al. Fertilization by sperm injection in cattle.Theriogenology,1990,33:238
73 GoTo K, Tanaka M, Ookutsu S, et al. Production of a Nuclear Transferred Calf by the Intracytoplasmic Injection of Donor Cells. Reprod. Dev. 1997; 43: 3.
74 Wakayama T , Yanagimachi R. Cloning of male mice from adult tail-trip cells. Nature Genetics,1999,22: 127-128.
75 Onishi A, Iwamoto M, Akita T, et al. Pig cloning by microinjection of fetal fibroblast nuclei. Science,2000, 289: 1188-1190.
76 Machaty Z, Prather RS. Strategies for activating nuclear transfer oocytes. J Reprod Fertil Dev.1998, 10(7-8): 599-613.
77 Marcus GJ.Activation of cumulus-free mouse oocytes.Mol.Reprod.Dev., 1990,26:159-162.
78 Chang MC.Development of parthenogenetic rabbit blastocysts induced by low temperature storage of unfertilized ova.J.Exp.Zoo1.,1954, 125:127-149.
79 Keefer CL, Stice SL, Matthews DL. Bovine inner cell mass cells as donor nuclei in the production of nuclear transfer embryos and calves.Bio1. Reprod.,1994,50:935-939.
80 Fissore RA, Robl JM.Intracellular Ca2+ response of rabbit oocytes to electrical stimulation. Mol Reprod Dev, 1992,32(1):9-16.
81 Robl JM.Development and application of technology for large scale cloning of cattle.Theriogenology,1999,51(2):499-508.
82 Miyazaki S, Shirakawa H, Nakada K, Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs.Dev Biol. 1993,158(1):62-78.
83 Fissore RA, Gordo AC ,Wu H.Activation of development in mammalians:is there a role for a sperm cytosolic factor?Theriogenology,1998,49:43-52.
84 Bos-Mikich A, Swann K, Whittingham DG.Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes.Mol Reprod Dev,1995,41(1):84-90.
85 Tateno H, Kamiguchi Y.Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation.Mol Reprod Dev. 1997 May;47(1):72-78.
86 Liu L, Ju JC ,Yang X.Parthenogenetic development and protein patterns of newly matured bovine oocytes after chemical activation. Mol.Reprod.Dev.1998,49:298-307.
87 Liu L, Yang X.Interplay of maturation-promoting factor and mitogen-activated protein kinase inactivation during metaphase-to-interphase transition of activated bovine oocytes. Biol. Reprod.1999,61:1-7.
88 Shiga K, Fujita T, Hirose K, et al. Production of calves by transfer of nuclei from cultured somatic cells obtained from Japanese black bulls.Theriogenology. 1999,52(3):527-535.
89 Hoth M, R Penner. Depletion of intracellular calcium stores activates a calcium current in mast cells.Nature,355, 353-356.
90 Susko-Parrish JL, Leibfried-Rutledge ML, Northey DL, et al. Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion. Dev Biol. 1994,166(2):729-739.
91 Mitalipov SM, White KL,Farrar VR,et a1.Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol1,4,5-trisphosphate.Biol. Reprod.1999,60:821-827.
92 Sato A, Hattori Y, Fukao M, et al. A role of myofilament Ca2+ sensitivity in enhanced vascular reactivity in cardiomyopathic hamsters.Eur J Pharmacol. 1998 Jul 24;353(2-3):247-256.
93 Navara CS, First NL, Schatten G.Microtubule organization in the cow during fertilization,polyspermy,parthenogenesis,and nuclear transferahe role of sperm aster.De v.Bio1.,1994,162:29-40.
94 Cuthbertson KSR, Whittingham DG and Cobbold PH. Free calcium increases in exponential phases during mouse oocyte activation. Nature, 1981,294:754-757.
95 Cuthbertson KSR. Parthenogenetic activation of mouse oocytes in vitro with ethanol and benzyl alcohol.J.Exp.Zoo1.,1983,226:311-314.
96 Mueller S, Prelle K, Rieger N, et a1.Chimeric pigs following blastocyst injection of transgenic porcine primordial germ cells.Mo1. Reprod.Dev.,1999,54:244-254.
97 Loi P, Ledda S, Fulka J,et a1.Development of parthenogenetic and cloned ovine embryos:effect of activation protocol.Bio1. Reprod., 1998,58:1177-1187.
98 Didion BA, Martin MJ and Markert CL. Parthenogenetic activation of mouse and pig oocytes matured in vitro.Theriogenology,1990,33:1165-1175.
99 Boediono A, Saha S, Sumantri C, et a1.Development in vitro and in vivo of aggregated parthenogenetic bovine embryos.Reprod. Fertil.Dev., 1995, 7:1073-1079.
100 Ledda S, Loi P, Bogliolo L, et al. The effect of 6-dimethylaminopurine (6-DMAP) on DNA synthesis in activated mammalian oocytes.Zygote,1996,4(1):7-9.
101 Fraser LR. Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs.Gamete Res,1987,18:363-374.
102 Mau JC, Grupen CG, Verma PJ, et al. MPF activity of porcine oocytes following electrical activation with and without 6-dimethylaminopurin. Theriogenology, 2001,55(1):457.
103 Bavister BD, Arlotto T. Influence of single amino acids on the development of hamster one-cell embryos in vitro. Mol Reprod Dev,1990,25(1):45-51.
104 Barmat L I, K C Worrilow, B V Paynton. Growth factor expression by human oviduct and buffalo rat liver coculture cells. Fertil Steril,1997, 67(4):775-779.
105 Wiemer KE, Cohen J, Tucker MJ,et al.The application of co-culture in assisted reproduction: 10 years of experience with human embryos.Hum Reprod. 1998,13 Suppl 4:226-238.
106 Gardner DK, Lane M.Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod, 1998 ,13 Suppl 3:148-159.
107 Lane M, Hooper K, Gardner DK. Effect of essential amino acids on mouse embryo viability and ammonium production. J Assist Reprod Genet. 2001, 18(9):519-525.
108 Lane M, Gardner DK. Differential regulation of mouse embryo development and viability by amino acids. J Reprod Fertil, 1997, 109(1):153-164.
109 Gardner DK. Lane M. The 2-cell block in CF1 mouse embryos is associated with an increase in glycolysis and a decrease in tricarboxylic acid (TCA) cycle activity: alleviation of the 2-cell block is associated with the restoration of invivo metabolic pathway activities. Biol. Reprod, 1993,49:52.
110 Gardner DK, Lane M.Alleviation of the '2-cell block' and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum Reprod, 1996,11(12):2703-2712.
111杨素芳,石德顺,等.激活方法对水牛卵母细胞孤雌发育的影响.中国兽医学报,2002,22(4):325-327.
112 Munsie M J,Michalska A E,O'Brien C M,et o1.Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.Curr Biol,2000,10(16):989-992.
113 Chen Y,He Z X,Liu A,et o1.Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes . Cell Research ,2003,13(4):25 1-263.
114 Wakayama T,Tabar V,Rodriguez I,el o1.Diferentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer.Science,2001,292(5517):740-743.
115 Hochedlinger K, Jaenisch R.Monoclonal mice generated by nuclear transfer from mature B and T donor cells.Nature,2002,415(6875): 1035-1038.
116 Rideout W M,Hochedlinger Kyba M,et a1.Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell, 2002,109(1):17-27.
117 Barberi T, Klivenyi P, Calingasan NY, et al.Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice.Nat Biotechnol,2003,21(10):1200-1207.
118 Wakayama T.Cloned mice and embryonic stem cell lines generated fromadult somatic cells by nuclear transfer.Oncol Res,2003,13(6-10):309-314.
119 Lee SH,Lumelsky N,Studer L,et a1.Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells.Nat Biotechnol,2000,18(6):675-679.
120 Tauer CA . International policy failures : cloning and stem cell research.Lancet,2004,364(9429):209-214.
121 Brustle O.From ES cells to functional neurons and glia.J Neurochem, 2005,94(1):55-55.
122 Vats A,Bielby RC,Tolley NS,et a1.Stem cells.Lancet,2005,366(9485):592-602.
123 Nishimura F,Yoshikawa M ,Kanda S,et a1.Potential use of embryonic stem cells for the treatment of mouse Parkinsonian models: Improved behavior by transplantation of in vitro differentiated dopaminergic neurons from embryonic stem cells.Stern Cells,2003,21(2):171-180.
124 Carpenter MK, Inokuma MS, Denham J, et al. Enrichment of neurons and neural precursors from human embryonic stem cells.Exp Neuro,l. 2001,172(2):383-397.
125 Zhang SC.Wemig M,Duncan ID,et al.In vitro diferentiation of transplantable neural precursors from human emb ryonic stem cells.Nat Biotechnol,2001,19(12):l129-l133.
126 Ishizaka S,Shiroi A,Kanda S,et a1.Development ofhepatocytes from ES cells after transfection with the HNF-3beta gene.Faseb J,2002,16(11):1444-1446.
127 Chinzei R, Tanaka Y, Shimizu-Saito K, et al. Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functionalhepatocytes.Hepatology, 2002 ,36(1):22-29.
128 Rambhatla L, Chiu CP, Kundu P, et a1.Generation of hepatocyte-like cells from human embryonic stem cells.Cell Transplant. 2003;12(1):1-11.
129 Kehat I, Kenyagin-Karsenti D, Snir M,et al.Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes.J Clin Invest. 2001,108(3):407-414.
130 Xu C, Police S, Rao N, Carpenter MK.Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res. 2002,91(6):501-508.
131 Min JY,Yang Y, L Liu, et a1.Improvement of heart function in postinfarcted rats by transplantation of embryonic stem cells.Circulation,2000,102(18):35-35.
132 Wang JF,Yang Y, Wang G,et a1.Embryonic stem cells attenuate viral myocarditis in murine model.Cell Transplant,2002,11(8):753-758.
133 Shiroi A, Yoshikawa M, Yokota H, et al.Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.Stem Cells. 2002;20(4):284-292.
134 Blyszczuk P,Czyz J,Kania G,et a1.Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive Progenitor and insulin-producing cells.P Natl Acad Sci USA,2003,100(3):998-1003.
135 Assady S, Maor G, Amit M, Itskovitz-Eldor J,et al.Insulin production by human embryonic stem cells.Diabetes,2001,50(8):1691-1697.
136 Soria B, Roche E, BernáG,et al.Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.Diabetes,2000 ,49(2):157-162.
137 Levenberg S,Colt JS,Amit M,et a1.Endothelial cells derived from human embryonic stem cels.Proc Natl AcadSci USA, 2002,99:439l-4396.
138 Kaufman DS, Lewis RL, Hanson ET,et al.Functional endothelial cells derived from rhesus monkey embryonic stem cells.Blood, 2004 , 103(4):1325-1332.
139 Amit M, Carpenter MK, Inokuma MS,et al.Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol,2000,15;227(2):271-278.
140 Stephereon J.Green light for federally funded research on elllbryonic stem cell.JAMA,2000, 284(14)1773
141 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell, 2006, 126(4): 663-676.
142 Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells.Nature,2007,448: 313-317.
143 Wernig M, Meissner A, Foreman R, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature, 2007,448(7151): 318-324.
144 Aoi T, Yae K, Nakagawa M, et al. Generation of pluripotent stem cells from adult mouse liver and stomach cells.Science,2008 ,321 (5889):699-702.
145 Yamanaka S.Strategies and new developments in the generation of patient-specific pluripotent stem cells.Cell Stem Cell. 2007 ,1(1):39-49.
146 Yu J, Vodyanik MA, Smuga-Otto K, et al.Induced pluripotent stem cell lines derived fromhuman somatic cells. Science,2007,318: 1917-1920.
147 Park IH, Zhao R, West JA, et al. Reprogramming of human somatic cells topluripotency with defined factors. Nature,2008,451: 141-146.
148 Hanna J, Wernig M, Markoulaki S, et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 2007, 318(5858): 1920-1923.
149 Higgs DR.A new dawn for stem-cell therapy.N Engl J Med. 2008, 28;358(9):964-966.
150 Qin D, Li W, Zhang J, et al. Direct generation of ES-like cells from unmodified mouse embryonic fibroblasts by Oct4/Sox2/Myc/Klf4. Cell Res, 2007, 17(11): 959-962.
151 Liao J, Wu Z, Wang Y, Cheng L,et a1.Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors.Cell Res. 2008,18(5):600-603.
152 Takahashi K, Okita K, Nakagawa M, et al. Generation of high quality iPS cells. Neurosci Res, 2007, 58(Suppl1): S19
153 Nakagawa M, Koyanagi M, Tanabe K, et a1.Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts.Nat Biotechnol,2008 ,26(1):101-106.
154 Wernig M, Meissner A, Cassady JP,et al.c-Myc is dispensable for direct reprogramming of mouse fibroblasts.Cell Stem Cell. 2008,2(1):10-12.
155 Kanatsu-Shinohara M,Inoue K,Lee J,et a1.Generation of Pluripotent Stem Cells from Neonatal Mouse Testis.Cell, 2004, 119: 1001.
156 Rodda DJ, Chew JL, Lim LH,et al. Transcriptional regulation of nanog by OCT4 and SOX2.J Biol Chem. 2005,280(26):24731-24737.
157 Chambers I, Colby D, Robertson M, Nichols J, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stemcells.Cell,2003,30;113(5):643-55.
158常灏,郭彤,彦萌,等.体细胞重编程为多能干细胞的研究进展.细胞生物学杂志,2008, 30: 545-552.
159 Masui S, Nakatake Y, Toyooka Y,et al.Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells.Nat Cell Biol,2007,9(6):625-635.
160 Boyer LA, Lee TI, Cole MF, Johnstone SE,et a1.Core transcriptional regulatory circuitry in human embryonic stem cells. Cell, 2005, 23;122(6):947-956.
161 Maherali N, Sridharan R, Xie W, et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell, 2007, 1(1): 55-70.
162 Meissner A, Wernig M, Jaenisch R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol, 2007, 25(10): 1177-1181.
163 Chen H, Faller JW, Crabtree RH, et al. Dimer-of-dimers model for the oxygen-evolving complex of photosystem II. Synthesis and properties of [MnIV4O5(terpy)4(H2O)2](ClO4)6. J Am Chem Soc, 2004 , 16;126 (23): 7345-7349.
164 Huangfu D, Maehr R, Guo W, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.Nat Biotechnol, 2008 ,26(7):795-797.
[1] Wilmut I, Schnieke AE, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells[J]. Nature,1997,385(6619):810-813.
[2] Fulka J Jr, Loi P, Fulka H, et al. Nucleus transfer in mammals: noninvasive approaches for the preparation of cytoplasts[J]. Trends Biotechnol,2004,22(6):279-283.
[3] Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy[J]. N Engl J Med,2003,349(3):275-286.
[4] Hua S, Zhang Z, Zhang C, et al. An improved enucleation method of bovine somatic cell nuclear transfer[J]. J Genet Genomics, 2007,34(6):491-496.
[5] Elsheikh AS, Takahashi Y, Katagiri S, et al. Sucrose-exposed chemically enucleated mouse oocytes support blastocyst development of reconstituted embryos[J]. Reprod Fertil Dev,2006,18(6):697-701.
[6] Fulka J Jr, Moor RM. Noninvasive chemical enucleation of mouse oocytes[J]. Mol Reprod Dev,1993,34(4):427-430.
[7]韦精卫,石德顺,韦英明,等.荧光染色及紫外照射(UV)对黄牛孤雌和克隆胚胎早期发育的影响[J].广西农业生物科学, 2005,24(3):196-199,219.
[8] Munsie M, O'brien C, Mountford P. Transgenic strategy for demonstrating nuclear reprogramming in the mouse[J]. Cloning Stem Cells,2002,4(2):121-130.
[9] Li GP, White KL, Bunch TD. Review of enucleation methods and procedures used in animal cloning: state of the art[J]. Cloning Stem Cells, 2004,6(1):5-13.
1 Jones KT.Mammalian egg activation: from Ca21 spiking to cell cycle progression. Reproduction 2005;130:813-823
2 Tamada H, Kikyo N. Nuclear reprogramming in mammalian somatic cell nuclear cloning. Cytogenet Genome Res 2004;105 :285-291
3杨素芳,石德顺,韦精卫,等.激活方法对水牛卵母细胞孤雌发育的影响[J].中国兽医学报,2002,22(4)325-327
4 Vajta G.Somatic cell nuclear transfer in its first and second decades: successes, setbacks,paradoxes and perspectives. Reprod Biomed Online 2007;15(5):582-590 5 Méo SC, Leal CL, Garcia JM. Activation and early parthenogenesis of bovine oocytes treated with ethanol and strontium. Anim Reprod Sci 2004;81(1-2):35-46
6 Jean L,Orly LK. The employment of strontium to activate mouse oocytes: effects on spermatid-injection outcome.Reproduction 2006;131:259–267
7 Kawasumi M, Anzai M, Takehara T,et al. Abnormal distribution of chromosomes in the first division of nuclear transferred mouse embryos. J Reprod Dev 2007;53: 615–622
8 Wani NA.Chemical activation of in vitro matured dromedary camel(Camelus dromedarius) oocytes: Optimization of protocols. Theriogenology2008;69:591–602
9李雪峰,安志兴,郭继彤,等.几种不同化学激活方法对牛卵母细胞孤雌激活的影响[J].西北农林科技大学学报,2002,30(2):43-47
10 SedmíkováM, BurdováJ, Petr J, et al. Induction and activation of meiosis and subsequent parthenogenetic development of growing pig oocytes using calcium ionophore A23187. Theriogenology 2003;60(9):1609-1620
11 Che L,Lalonde A,Bordignon V.Chemical activation of parthenogenetic and nuclear transfer porcine oocytes using ionomycin and strontium chloride.Theriogenology 2007;67:1297–1304
12 Lan GC, Ma SF, Wang ZY, et al.Effects of post-treatment with 6-dimethylaminopurine (6-DMAP) on ethanol activation of mouse oocytes at different ages. J Exp Zoolog A Comp Exp Biol 2004;301(10):837-843
13 Wakayama T, Yanagimachi R. Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei. Reproduction 2001;122(1):49-60
14 Revazova ES,Turovets OD,Kochetkova LS,et al.HLA homozygous stem cell lines derived from human parthenogenetic blastocysts.Cloning Stem Cells 2008;10(1):11-24
15 Yvan H.Nuclear transfer: a new tool for reproductive biotechnology in cattle.Reprod Nutr Dev 2005;45:353–361
16 Ma SF, Liu XY, Miao DQ, et al. Parthenogenetic activation of mouse oocytes by strontium chloride: a search for the best conditions. Theriogenology 2005;15;64(5):1142-1157
17 Tian JH,Wu ZH,Liu L,et al.Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from invitro-matured oocytes and intracytoplasmic sperm injection.Theriogenology 2006;66:439–448
18 Alexander M,Rudolf J.Mammalian Nuclear Transfer.Dev Dyn 2006;235:2460–2469
1 Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N Engl J Med,2003,349(3):275-286.
2 Gardner DK.Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology, 1998,49(1):83-102.
3 Chatot CL,Ziomek CA,Bavister BD. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J-Reprod-Fertil,1989,86(2): 679-688.
4 LAWITTS JA,BIGGERS JD. Culture and preimplantation embryos. Guide to techniques in mouse development,1993,225 (26):153-164.
5 Aoki VW, Wilcox AL, Peterson CM, et al.Comparison of four media types during 3-day human IVF embryo culture . Reprod Biomed Online, 2005 ,10(5):600-606.
6 Biggers JD. Reflections on the culture of the preimplantation embryo. Int J Dev Biol, 1998;42(7):879-884.
7 Van Winkle LJ. Amino acid transport regulation and early embryo development. Biol Reprod, 2001 ,64(1):1-12.
8 Devreker F, Hardy K. Effects of glutamine and taurine on preimplantation development and cleavage of mouse embryos in vitro. Biol Reprod, 1997 ,57(4):921-928.
9 Heindryckx B, Rybouchkin A, Van Der Elst J, et al. Effect of culture media on in vitro development of cloned mouse embryos. Cloning, 2001;3(2):41-50.
10 Summers MC, McGinnis LK, Lawitts JA, et al.Mouse embryo development following IVF in media containing either L-glutamine or glycyl-L-glutamine.Hum Reprod, 2005,20(5):1364-1371.
11莫曾南,覃敏,何敏,等.近交系小鼠体细胞核移植胚胎的构建和鉴定.中国组织工程研究与临床康复,2008,12(31):6001-6006.
12 Lane M, Gardner DK. Nonessential amino acids and glutamine decrease the time of the first three cleavage divisions and increase compaction of mouse zygotes in vitro. J Assist Reprod Genet, 1997,14(7):398-403.
13 Biggers JD, McGinnis LK, Lawitts JA. One-step versus two-step culture of mouse preimplantation embryos: is there a difference? Hum Reprod, 2005 ,20(12):3376-3384.
14 Zander DL, Thompson JG, Lane M. Perturbations in mouse embryo development and viability caused by ammonium are more severe after exposure at the cleavage stages. Biol Reprod, 2006,74(2):288-294.
15 Martin PM, Sutherland AE, Van Winkle LJ. Amino acid transport regulates blastocyst implantation. Biol Reprod, 2003 ,69(4):1101-1108.
16 Choi YH,Seng S,Toyoda Y. Effect of taurine on in vitro fertilization and embryo development of BALB/c mouse strain. Journal of Reproduction and Development,1998,44(1):29-34.
17 Liu Z, Foote RH. Development of bovine embryos in KSOM with added superoxide dismutase and taurine and with five and twenty percent O2. Biol Reprod, 1995,53(4):786-790
18 Devreker F, Van den Bergh M, Biramane J, et al.Effects of taurine on human embryo development in vitro. Hum Reprod,1999,14(9):2350-2356.
19 Van Winkle LJ, Dickinson HR. Differences in amino acid content of preimplantation mouse embryos that develop in vitro versus in vivo: in vitro effects of five amino acids that are abundant in oviductal secretions. BiolReprod, 1995 ,52(1):96-104.
20 Guérin P, El Mouatassim S, Ménézo Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update,2001,7(2):175-189.
1 Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy[J]. N Engl J Med, 2003,349(3):275-286.
2 Ribas R, Oback B, Ritchie W, et al. Development of a zona-free method of nuclear transfer in the mouse[J]. Cloning Stem Cells, 2005,7(2):126-138.
3 Tomii R, Kurome M, Ochiai T, et al. Production of cloned pigs by nuclear transfer of preadipocytes established from adult mature adipocytes[J]. Cloning Stem Cells, 2005,7(4):279-288.
4 Li Shangang, Chen Xuejin, Fang Zhenfu, et al. Rabbits generated from fibroblasts through nuclear transfer[J]. Reproduction, 2006 ,131(6):1085-1090.
5 Andrew J, Catharine A, Linda S, et al. Development of Human Cloned Blastocysts Following Somatic Cell Nuclear Transfer with Adult Fibroblasts[J]. Stem Cells, 2008,26(2):485 -493.
6 Tamada H, Kikyo N. Nuclear reprogramming in mammalian somatic cell nuclear cloning[J]. Cytogenet Genome Res, 2004,105(2-4):285-291.
7 Saliba-Colombani V, Causse M, Gervais L, et al. Efficiency of RFLP, RAPD, and AFLP markers for the construction of an intraspecific map of the tomato genome[J]. Genome, 2000,43(1):29-40.
8王芳,孙以方,段天林,等.(GTG)5探针产生的DNA纹图在近交系小鼠遗传检测中的应用[J].中国实验动物学报, 2007,15(5):361-364.
9 Sakai T, Kikkawa Y, Miura I, et al. Origins of mouse inbred strains deduced from whole-genome scanning by polymorphic microsatellite loci[J]. Mamm Genome, 2005 ,16(1):11-19.
10陈振文,欧阳兆,董罡,等.用微卫星标记技术对国内BALB/c小鼠遗传质量的分析[J].遗传, 2004,26(6):845-848.
11 Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for sonic hedgehog mutation causing familial holoprosencephaly[J]. N Engl J Med, 2003,348(15):1449-1454.
1 Solter D , Gearhart J . Putting stem cells to work. Science,1999 , 283 : 1468 - 1470.
2 Lanza RP , Cibelli JB , West MD. Human therapeutic cloning. Nature Med. 1999 , 5 : 975 - 977.
3 Colman A, Kind A. Therapeutic cloning : concepts and practicalities. Trends Biotechnol,2000 , 18 :192 - 196.
4 Cibelli JB,Stice SL,Golueke PJ,et a1.Transgenic bovine chimeric ofspring produced from somatic cell-derived stem-like cel1.Nat Biotechnol,1998,16(7):642-664.
5 Munsie MJ,Michalska AE,O'Brien CM,et a1.Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.Curr Biol,2000,10(16):989-992.
6 Chen Y,He ZX,Liu A,et a1.Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes.Cell Research,2003,13(4):251-263.
7 Simerly C ,Cominko T , Navara C , et al. Molecular corre2lates of primate nuclear transfer failures. Science , 2003 ,300 : 297
8 Dunglison GF,Barlow DH,Sargent IL. Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium. Hum Roped,1996,11:191-196.
9 Xu C,Inokuma MS,Denham J,Golds K,et al,Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol , 2001 ,19:971-974.
10曹鸿国,张涌.小鼠体细胞核移植及ES细胞样集落分离.自然科学进展,2005,15(4):481-485.
11刘登华,肖文伍,陈红.小鼠胚胎干细胞体外分离培养影响因素的实验研究.华中科技大学学报(医学版),2004,33(6);734-736.
12 Gardener RL,Papaioannou VE.Differentiation in the trophectoderm and inner cell mass. Cambridge University Press,1975,12(1):107-132.
13 Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Gene Development, 2006, 19: 1129-1155.
14 Solter D. From teratocarcinomas to embryonic stem cells and beyond: a history of embryonic stem cell research. Nat. Rev. Genet., 2006, 7(4): 319-327.
1 Takahashi K, S Yamanaka. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined FactorsCell, 2006, 126(4): 663-676
2 Müller FJ, Laurent LC, Kostka D, et al. Regulatory networks define phenotypic classes of human stem cell lines. Nature, 2008, 451(7175): 141-146
3 Fujimori T, Kurotaki Y, Miyazaki J, et al. Analysis of cell lineage in two- and four-cell mouse embryos. Development, 2003, 130(21):5113—5122
4 Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature, 2007, 448(7151): 313-317
5 Wernig M, Meissner A, Foreman R, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007, 19;448(7151):318-324
6 Hanna J, Wernig M, Markoulaki S, et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 2007, 318(5858): 1920-1923
7 Wernig M, Zhao JP, Pruszak J, et al. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci USA, 2008, 105: 5856-5861
2 Norstr?m A, Akesson K, Hardarson T,et al.Molecular and pharmacological properties of human embryonic stem cell-derived cardiomyocytes.Exp Biol Med (Maywood). 2006 Dec;231(11):1753-62.
3 Hwang NS, Varghese S, Zhang Z, et al.Chondrogenic differentiation of human embryonic stem cell-derived cells in arginine-glycine-aspartate-modified hydrogels.Tissue Eng. 2006 Sep;12(9):2695-706.
4 D'Amour KA, Bang AG, Eliazer S,et al.Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.Nat Biotechnol. 2006 Nov;24(11):1392-401.
5 Cummings BJ, Uchida N, Tamaki SJ,et al.Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice.Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):14069-74.
6 Yoon BS, Yoo SJ, Lee JE, et al.Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.Differentiation. 2006 Apr;74(4):149-59.
7 Lanza RP, Cibelli JB, West MD.Prospects for the use of nuclear transfer inhuman transplantation. Nat Biotechnol. 1999 Dec;17(12):1171-4.
8 Hochedlinger K, Jaenisch R. Nuclear transplantation,embryonic stem cells, and the potential for cell therapy. N Eng J Med.2003, 349, 275-286.
9 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126(4): 663?676.
1 Hochedlinger K, Jaenisch R. Nuclear reprogramming and pluripotency. Nature, 2006, 441(7097): 1061-1067.
2 Rodolfa K T, Eggan K. A transcriptional logic for nuclear reprogramming. Cell, 2006, 126(4): 652—655.
3 Cervera RP, Stojkovic M.Commentary: somatic cell nuclear transfer-progress and promise.Stem Cells,2008,26(2):494-495.
4 Wakayama T, Perry ACF, Zueeotti M. et al. Full-term development of mice from enucleated oocytes infected with cumulus cell nuclei. Nature, 1998, 394:369-374.
5 Zhou QI, Jean-Paul Renard, Gaelle LE Friec, et al. Generation of fertile cloned rats by regulating oocyte activation. Science, 2003, 302:1179.
6 Shin T, Kraemer D, Pryor J, et al. A cat cloned by nuclear transplantation. Nature. 2002, 415:859.
7 Chesne P, Adenot PCB Viglietta C, et al. Cloned rabbits produced by nuclear transfer from adult somatic cells[J]. Nat Biotechnol, 2002, 20(4): 366-369.
8 Lee BC, Kim MK, Jang G,et al. Dogs cloned from adult somatic cells.Nature. 2005 , 436(7051):641.
9 Baguisi A, Behboodi E, Melican DT, et al.Production of goats by somatic cell nuclear transfer. Nat Biotechnol,1999,17(5):456-461.
10郭继彤.成年体细胞克隆山羊研究. [学位论文],西北农林科技大学,中国,西安,2000.
11 Wilmut I, Schnieke AE, Mcwhir J, et al. Viable offspring derived from fetal and adult mammalian cells. Nature. 1997, 385: 810-813.
12 Woods GL, White KL, Vanderwall DK, et al. A mule cloned from fetal cellsby nuclear transfer. Science. 2003 ,301(5636):1063.
13 Kato Y, Tani T, Sotomaru Y, et al. Eight calves cloned from somatic cells of single adult.Science, 1998,282: 2095-2098.
14陈大元,李劲松,韩之明,等.体细胞克隆牛:供体细胞和受体的影响.科学通报.2003,48:768-773.
15 Galli C, Lagutina I, Crotti G, et al.Pregnancy: a cloned horse born to its dam twin.Nature,2003,424(6949):635.
16 Polejaeva IA, Chen SH, Vaught TD, et al. Cloned pigs produced by nuclear transfer adult somatic cells. Nature, 2000, 407: 86-90.
17陆凤花,石德顺,韦英明,等.不同融合条件对水牛体细胞核移植效果的影响.广西农业生物科学,2005,24 (1): 1-5.
18 Kato Y, Tani T, Tsunoda Y Cloning of calves from various somatic cell types of male and female adult newborn and fetal cows. Reprod Fertil, 2000,120: 231-237.
19 Meissner A, Jaenisch R.Mammalian nuclear transfer.Dev Dyn,2006, 235(9):2460-2469.
20 Reik W, Dean W, Walter J.Epigenetic reprogramming in mammalian development.Science, 2001,293(5532):1089-1093.
21 Wrenxyckic, Wellsd, et al. Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts . Biol Reprod, 2001 ,65:309-317.
22 Rideout WM 3rd, Eggan K, Jaenisch R.Nuclear cloing and epigenetic regprogramming of the genome. Science. 2001 ,293(5532):1093-1098.
23 Wolffe AP, Matzke MA. Epigenetics: regulation through regression . Science. 1999 Oct 15;286(5439):481-486.
24 Smith LC, Wilmut I, Hunter RHF. Influence of cell cycle stage at nuclear transplantation on the development of mouse embryos. Reprod. Fertil. 1988,84: 619-624.
25 Smith S D,Soloy E,Avery B,et al. Nucleus remodeling in reconstructed cattle embryos.Theriogenology.1994,41:298-304.
26 Kikyo N, Wolffe AP.Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons.J Cell Sci, 2000 ,113 ( Pt 1):11-20.
27 Steinbach OC, Wolffe AP, Rupp RA.Somatic linker histones cause loss of mesodermal competence in Xenopus.Nature. 1997,389(6649):395-399.
28 Ogura A, Inoue K, Ogonuki N, et al. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol Reprod, 2000a,62(6): 1579-1584.
29 Butler D.French clone provides support for Dolly. Nature, 1998 ,12;392(6672):113.
30 Kubota C, Yamakuchi H, Todoroki J, et al. Six cloned calves produced from adult fibroblast cells after long–term cultire . Proc Natl Acad Sci USA, 2000, 97: 990-995.
31 Ogura A, Inoue K, Takano K, et al. Birth of mice after nuclear transfer by electrofusion using tail tip cells. Mol Reprod Dev,2000,57(1): 55-59.
32 Inoue K,Ogonuki N,Mochida K,et al.Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning. Biol.Reprod, 2003,69:1394-1400.
33 Campbell KHS. Nuclear equivalence, nuclear transfer and the cell cycle. Cloning,1999, 1(1): 3-15.
34 Wilmut I, Campbell KH. Quiescence in nuclear transfer. Science 1998;281:1611.
35 Campbell KHS, McWhir J, Ritchi WA, et al. Sheep cloned by nuclear transfer from a cultured cell line . Nature,1996, 380: 64-66.
36 Wells DN, Misica PM, Tervit HR. Production of cloned calves following Nuclear Transfer with Cultured Adult Mural Granulosa cells. J Biol Reprod. 1999, 60: 996-1005.
37 Zakhartchenko V, Alberio R, Stojkovic M, et al. Adult cloning in cattle: potential of nuclei from permanent cell line and from primary cultures. Mol Reprod Dev. 1999, 54: 264-272.
38 Cibelli JB, Stice SL, Golueke PJ,et al. Cloned transgenic calves produced from nonquiescent fetal fibroblast. Science,1998, 280(5367):1256-1258.
39 Zakhartchenko V, Durcova-Hills G, Stojkovic M,et al. Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts. J Reprod Fertil. 1999 ,115(2):325-331.
40 Peura TT, Lewis IM, Trounson AO. The effect of recipient oocyte volume on nuclear transfer in cattle. Mol Reprod Dev. 1998, 50 (2): 185-191.
41 Westhusin ME, Collas P, Marek D, et al. Reducing the amount of cytoplasm available for early embryonic development decreases the quality but quantity of embryos produced by in vitro fertilization and nuclear transplantation. Theriogenology. 1996, 46: 243-252.
42 McGrath J, Solter D.Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro.Science,1984,14;226(4680): 1317-1319.
43 Robl JM, Prather R, Barnes F, et al. Nuclear transplantation in bovine embryos. Anim Sci,1987, 64: 642-647.
44 Prather RS, Barnes FL, Sims MM, et al. Nuclear transplantation in the bovine embryos: assessment of donor nuclei and recipient oocyte. J Biol Reprod,1987, 37: 859-866.
45 Takano H, Koyama K, Kozzi C, et al. Effects of aging of recepient oocytes in the development of bovine nuclear transfer embryos in vitro. Theriogenology. 1993, 39: 909-917.
46 Yang X, Jiang S, Farrell D, et al. Nuclear transfer in cattle: effect of nuclear donor cells cytoplast age co-culture and embryo transfer. Mol Reprod Dev. 1993, 35(1): 29-36.
47 Stice SL, Robl JM. Nuclear reprogramming in nuclear transplant rabbit embryos. Biol Reprod. 1988, 39: 657-664.
48 Prather RS,Sims MM, First NL. Nuclear transplantation in early pig embryos. J Biol Reprod,1989, 44: 414-418.
49 Westhusin ME,.Levanduski MJ, Scarborough R, et al. Viable embryos and normal calves after nuclear transfer into Hoechst staind enucleated demi-oocytes of cows. J Reprod Fertil. 1992. 95(2): 475-480.
50 Stice SL, Keefer CL. Multiple generational bovine embryo cloning. Biol Reprod, 1993, 48: 715-719.
51 Bordignon V, Smith LC. Telophase enucleated: an improved method to prepare recipient cytoplasts for use in bovine nuclear transfer. Mol Reprod Dev,1998, 49(1): 29-36.
52 Monamed Nour MS, Takahashi Y. Preparation of young preactivated oocyte with high enucleation efficiency for bovine nuclear transfer. Theriogenology. 1999, 51(3): 661-666.
53王敏康,刘冀珑,李光鹏,等.蔗糖预处理去核:一种可靠的无损害的小鼠卵母细胞MII期核去除法.生殖医学杂志, 2001, 10(4): 227-232.
54 Willadsen SM. Nuclear transplantation in sheep embryos. Nature, 1986, 320:63-65.
55 Booth PJ, Tan SJ, Reipurth R,et a1.Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique.Cloning Stem Cells,2001,3(3):139-150.
56 Oldenbourg R.A new view on polarization microscopy. Nature, 1996, 381(6585):811-812.
57 Liu L, Oldenbourg R, Trimarchi JR, et al. A reliable, noninvasive technique for spindle imaging and enucleation of mammalian ooctes. Nature,2000, 18: 223-225.
58 Tatham BG, Dowsing AT, Tyounson AO. Enucleated by centrifugation of in vitro~matured bovine oocytes for use in nuclear transfer. Biol. Reprod. 1995,53(5): 1088-1094.
59 Tsunoda Y, Shioda Y, Onodera M, et a1.Differential sensitivity of mouse pronuclei and zygote cytoplasm to Hoechst staining and ultraviolet irradiation.J.Reprod.Fertil,l988, 82:173-178.
60 Fulka JR, Moor RM. Noninvasive chemical enucleation of mouse oocytes. Mol Repord Dev. 1993, 34: 427-430.
61 Gasparrini B, Gao S, Ainslie A, et al.Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation. Biol Reprod, 2003, 68: 1259-1266.
62 Gasparrini B, Dinneyes A, Ainslie A. Attempted chemical enucleation of in vitro matured oocytes after activation and their use in nuclear transfer. Theriogenology, 2002, 414.
63 Yin XJ, Tani T, Yonemura I, et al.Productionn of cloned pigs from adult somatic cells by chemical assisted removal of maternal chromosomes. Biol Reprod, 2002, 67:442-446.
64 Kawakami M, Tani T, Yabuuchi A, et al.Effect of demecolcine and nocodazole on the efficiency of chemically assisted removal of chromosomes and the developmental potential of nuclear transferred porcine oocytes. Cloning Stem Cells. 2003;5(4):379-387.
65 Ficsher D, Ibanez E, Albertini DF, Activated bovine cytoplasts produced by induced enucleation support development of bovine nuclear transfer embryos in vitro. Bicsl Reprod, 2002, 66, 238:346.
66 Yin XJ, Kato Y, Tsunoda Y.Effect of delayed enucleation on the developmental potential of nuclear-transferred oocytes receiving adult and fetal fibroblast cells.Zygote. 2002,10(3):217-222.
67 Illmensee K, Hoppe PC. Nuclear transplantation in Mus musculus: developmental potential of nuclei from preimplantation embryos. Cells. 1981, 23: 9-18.
68 Meng L, Ely JJ, Stouffer RL, et al. Rhesus monkeys produced by nuclear transfer. Biol Reprod. 1997, 57(2): 454-459.
69 Kono T, Kwon OY, Watanabe T, et al. Development of mouse enucleated oocytes receiving a nucleus from different stages of the second cell cycle. J Reprod Fertil. 1992, 94: 481-487.
70 Kono T , Tsunoda Y. Development of Single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryos. Gamete Res. 1989, 2: 427-434.
71张涌,王建辰,钱菊汾,等.山羊卵核移植的研究.中国农业科学,1991,24(5): 1-6.
72 Goto K, A Kinoshita, TY Takuma, et al. Fertilization by sperm injection in cattle.Theriogenology,1990,33:238
73 GoTo K, Tanaka M, Ookutsu S, et al. Production of a Nuclear Transferred Calf by the Intracytoplasmic Injection of Donor Cells. Reprod. Dev. 1997; 43: 3.
74 Wakayama T , Yanagimachi R. Cloning of male mice from adult tail-trip cells. Nature Genetics,1999,22: 127-128.
75 Onishi A, Iwamoto M, Akita T, et al. Pig cloning by microinjection of fetal fibroblast nuclei. Science,2000, 289: 1188-1190.
76 Machaty Z, Prather RS. Strategies for activating nuclear transfer oocytes. J Reprod Fertil Dev.1998, 10(7-8): 599-613.
77 Marcus GJ.Activation of cumulus-free mouse oocytes.Mol.Reprod.Dev., 1990,26:159-162.
78 Chang MC.Development of parthenogenetic rabbit blastocysts induced by low temperature storage of unfertilized ova.J.Exp.Zoo1.,1954, 125:127-149.
79 Keefer CL, Stice SL, Matthews DL. Bovine inner cell mass cells as donor nuclei in the production of nuclear transfer embryos and calves.Bio1. Reprod.,1994,50:935-939.
80 Fissore RA, Robl JM.Intracellular Ca2+ response of rabbit oocytes to electrical stimulation. Mol Reprod Dev, 1992,32(1):9-16.
81 Robl JM.Development and application of technology for large scale cloning of cattle.Theriogenology,1999,51(2):499-508.
82 Miyazaki S, Shirakawa H, Nakada K, Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs.Dev Biol. 1993,158(1):62-78.
83 Fissore RA, Gordo AC ,Wu H.Activation of development in mammalians:is there a role for a sperm cytosolic factor?Theriogenology,1998,49:43-52.
84 Bos-Mikich A, Swann K, Whittingham DG.Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes.Mol Reprod Dev,1995,41(1):84-90.
85 Tateno H, Kamiguchi Y.Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation.Mol Reprod Dev. 1997 May;47(1):72-78.
86 Liu L, Ju JC ,Yang X.Parthenogenetic development and protein patterns of newly matured bovine oocytes after chemical activation. Mol.Reprod.Dev.1998,49:298-307.
87 Liu L, Yang X.Interplay of maturation-promoting factor and mitogen-activated protein kinase inactivation during metaphase-to-interphase transition of activated bovine oocytes. Biol. Reprod.1999,61:1-7.
88 Shiga K, Fujita T, Hirose K, et al. Production of calves by transfer of nuclei from cultured somatic cells obtained from Japanese black bulls.Theriogenology. 1999,52(3):527-535.
89 Hoth M, R Penner. Depletion of intracellular calcium stores activates a calcium current in mast cells.Nature,355, 353-356.
90 Susko-Parrish JL, Leibfried-Rutledge ML, Northey DL, et al. Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion. Dev Biol. 1994,166(2):729-739.
91 Mitalipov SM, White KL,Farrar VR,et a1.Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol1,4,5-trisphosphate.Biol. Reprod.1999,60:821-827.
92 Sato A, Hattori Y, Fukao M, et al. A role of myofilament Ca2+ sensitivity in enhanced vascular reactivity in cardiomyopathic hamsters.Eur J Pharmacol. 1998 Jul 24;353(2-3):247-256.
93 Navara CS, First NL, Schatten G.Microtubule organization in the cow during fertilization,polyspermy,parthenogenesis,and nuclear transferahe role of sperm aster.De v.Bio1.,1994,162:29-40.
94 Cuthbertson KSR, Whittingham DG and Cobbold PH. Free calcium increases in exponential phases during mouse oocyte activation. Nature, 1981,294:754-757.
95 Cuthbertson KSR. Parthenogenetic activation of mouse oocytes in vitro with ethanol and benzyl alcohol.J.Exp.Zoo1.,1983,226:311-314.
96 Mueller S, Prelle K, Rieger N, et a1.Chimeric pigs following blastocyst injection of transgenic porcine primordial germ cells.Mo1. Reprod.Dev.,1999,54:244-254.
97 Loi P, Ledda S, Fulka J,et a1.Development of parthenogenetic and cloned ovine embryos:effect of activation protocol.Bio1. Reprod., 1998,58:1177-1187.
98 Didion BA, Martin MJ and Markert CL. Parthenogenetic activation of mouse and pig oocytes matured in vitro.Theriogenology,1990,33:1165-1175.
99 Boediono A, Saha S, Sumantri C, et a1.Development in vitro and in vivo of aggregated parthenogenetic bovine embryos.Reprod. Fertil.Dev., 1995, 7:1073-1079.
100 Ledda S, Loi P, Bogliolo L, et al. The effect of 6-dimethylaminopurine (6-DMAP) on DNA synthesis in activated mammalian oocytes.Zygote,1996,4(1):7-9.
101 Fraser LR. Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs.Gamete Res,1987,18:363-374.
102 Mau JC, Grupen CG, Verma PJ, et al. MPF activity of porcine oocytes following electrical activation with and without 6-dimethylaminopurin. Theriogenology, 2001,55(1):457.
103 Bavister BD, Arlotto T. Influence of single amino acids on the development of hamster one-cell embryos in vitro. Mol Reprod Dev,1990,25(1):45-51.
104 Barmat L I, K C Worrilow, B V Paynton. Growth factor expression by human oviduct and buffalo rat liver coculture cells. Fertil Steril,1997, 67(4):775-779.
105 Wiemer KE, Cohen J, Tucker MJ,et al.The application of co-culture in assisted reproduction: 10 years of experience with human embryos.Hum Reprod. 1998,13 Suppl 4:226-238.
106 Gardner DK, Lane M.Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod, 1998 ,13 Suppl 3:148-159.
107 Lane M, Hooper K, Gardner DK. Effect of essential amino acids on mouse embryo viability and ammonium production. J Assist Reprod Genet. 2001, 18(9):519-525.
108 Lane M, Gardner DK. Differential regulation of mouse embryo development and viability by amino acids. J Reprod Fertil, 1997, 109(1):153-164.
109 Gardner DK. Lane M. The 2-cell block in CF1 mouse embryos is associated with an increase in glycolysis and a decrease in tricarboxylic acid (TCA) cycle activity: alleviation of the 2-cell block is associated with the restoration of invivo metabolic pathway activities. Biol. Reprod, 1993,49:52.
110 Gardner DK, Lane M.Alleviation of the '2-cell block' and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum Reprod, 1996,11(12):2703-2712.
111杨素芳,石德顺,等.激活方法对水牛卵母细胞孤雌发育的影响.中国兽医学报,2002,22(4):325-327.
112 Munsie M J,Michalska A E,O'Brien C M,et o1.Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.Curr Biol,2000,10(16):989-992.
113 Chen Y,He Z X,Liu A,et o1.Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes . Cell Research ,2003,13(4):25 1-263.
114 Wakayama T,Tabar V,Rodriguez I,el o1.Diferentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer.Science,2001,292(5517):740-743.
115 Hochedlinger K, Jaenisch R.Monoclonal mice generated by nuclear transfer from mature B and T donor cells.Nature,2002,415(6875): 1035-1038.
116 Rideout W M,Hochedlinger Kyba M,et a1.Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell, 2002,109(1):17-27.
117 Barberi T, Klivenyi P, Calingasan NY, et al.Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice.Nat Biotechnol,2003,21(10):1200-1207.
118 Wakayama T.Cloned mice and embryonic stem cell lines generated fromadult somatic cells by nuclear transfer.Oncol Res,2003,13(6-10):309-314.
119 Lee SH,Lumelsky N,Studer L,et a1.Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells.Nat Biotechnol,2000,18(6):675-679.
120 Tauer CA . International policy failures : cloning and stem cell research.Lancet,2004,364(9429):209-214.
121 Brustle O.From ES cells to functional neurons and glia.J Neurochem, 2005,94(1):55-55.
122 Vats A,Bielby RC,Tolley NS,et a1.Stem cells.Lancet,2005,366(9485):592-602.
123 Nishimura F,Yoshikawa M ,Kanda S,et a1.Potential use of embryonic stem cells for the treatment of mouse Parkinsonian models: Improved behavior by transplantation of in vitro differentiated dopaminergic neurons from embryonic stem cells.Stern Cells,2003,21(2):171-180.
124 Carpenter MK, Inokuma MS, Denham J, et al. Enrichment of neurons and neural precursors from human embryonic stem cells.Exp Neuro,l. 2001,172(2):383-397.
125 Zhang SC.Wemig M,Duncan ID,et al.In vitro diferentiation of transplantable neural precursors from human emb ryonic stem cells.Nat Biotechnol,2001,19(12):l129-l133.
126 Ishizaka S,Shiroi A,Kanda S,et a1.Development ofhepatocytes from ES cells after transfection with the HNF-3beta gene.Faseb J,2002,16(11):1444-1446.
127 Chinzei R, Tanaka Y, Shimizu-Saito K, et al. Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functionalhepatocytes.Hepatology, 2002 ,36(1):22-29.
128 Rambhatla L, Chiu CP, Kundu P, et a1.Generation of hepatocyte-like cells from human embryonic stem cells.Cell Transplant. 2003;12(1):1-11.
129 Kehat I, Kenyagin-Karsenti D, Snir M,et al.Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes.J Clin Invest. 2001,108(3):407-414.
130 Xu C, Police S, Rao N, Carpenter MK.Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res. 2002,91(6):501-508.
131 Min JY,Yang Y, L Liu, et a1.Improvement of heart function in postinfarcted rats by transplantation of embryonic stem cells.Circulation,2000,102(18):35-35.
132 Wang JF,Yang Y, Wang G,et a1.Embryonic stem cells attenuate viral myocarditis in murine model.Cell Transplant,2002,11(8):753-758.
133 Shiroi A, Yoshikawa M, Yokota H, et al.Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.Stem Cells. 2002;20(4):284-292.
134 Blyszczuk P,Czyz J,Kania G,et a1.Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive Progenitor and insulin-producing cells.P Natl Acad Sci USA,2003,100(3):998-1003.
135 Assady S, Maor G, Amit M, Itskovitz-Eldor J,et al.Insulin production by human embryonic stem cells.Diabetes,2001,50(8):1691-1697.
136 Soria B, Roche E, BernáG,et al.Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.Diabetes,2000 ,49(2):157-162.
137 Levenberg S,Colt JS,Amit M,et a1.Endothelial cells derived from human embryonic stem cels.Proc Natl AcadSci USA, 2002,99:439l-4396.
138 Kaufman DS, Lewis RL, Hanson ET,et al.Functional endothelial cells derived from rhesus monkey embryonic stem cells.Blood, 2004 , 103(4):1325-1332.
139 Amit M, Carpenter MK, Inokuma MS,et al.Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol,2000,15;227(2):271-278.
140 Stephereon J.Green light for federally funded research on elllbryonic stem cell.JAMA,2000, 284(14)1773
141 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell, 2006, 126(4): 663-676.
142 Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells.Nature,2007,448: 313-317.
143 Wernig M, Meissner A, Foreman R, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature, 2007,448(7151): 318-324.
144 Aoi T, Yae K, Nakagawa M, et al. Generation of pluripotent stem cells from adult mouse liver and stomach cells.Science,2008 ,321 (5889):699-702.
145 Yamanaka S.Strategies and new developments in the generation of patient-specific pluripotent stem cells.Cell Stem Cell. 2007 ,1(1):39-49.
146 Yu J, Vodyanik MA, Smuga-Otto K, et al.Induced pluripotent stem cell lines derived fromhuman somatic cells. Science,2007,318: 1917-1920.
147 Park IH, Zhao R, West JA, et al. Reprogramming of human somatic cells topluripotency with defined factors. Nature,2008,451: 141-146.
148 Hanna J, Wernig M, Markoulaki S, et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 2007, 318(5858): 1920-1923.
149 Higgs DR.A new dawn for stem-cell therapy.N Engl J Med. 2008, 28;358(9):964-966.
150 Qin D, Li W, Zhang J, et al. Direct generation of ES-like cells from unmodified mouse embryonic fibroblasts by Oct4/Sox2/Myc/Klf4. Cell Res, 2007, 17(11): 959-962.
151 Liao J, Wu Z, Wang Y, Cheng L,et a1.Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors.Cell Res. 2008,18(5):600-603.
152 Takahashi K, Okita K, Nakagawa M, et al. Generation of high quality iPS cells. Neurosci Res, 2007, 58(Suppl1): S19
153 Nakagawa M, Koyanagi M, Tanabe K, et a1.Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts.Nat Biotechnol,2008 ,26(1):101-106.
154 Wernig M, Meissner A, Cassady JP,et al.c-Myc is dispensable for direct reprogramming of mouse fibroblasts.Cell Stem Cell. 2008,2(1):10-12.
155 Kanatsu-Shinohara M,Inoue K,Lee J,et a1.Generation of Pluripotent Stem Cells from Neonatal Mouse Testis.Cell, 2004, 119: 1001.
156 Rodda DJ, Chew JL, Lim LH,et al. Transcriptional regulation of nanog by OCT4 and SOX2.J Biol Chem. 2005,280(26):24731-24737.
157 Chambers I, Colby D, Robertson M, Nichols J, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stemcells.Cell,2003,30;113(5):643-55.
158常灏,郭彤,彦萌,等.体细胞重编程为多能干细胞的研究进展.细胞生物学杂志,2008, 30: 545-552.
159 Masui S, Nakatake Y, Toyooka Y,et al.Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells.Nat Cell Biol,2007,9(6):625-635.
160 Boyer LA, Lee TI, Cole MF, Johnstone SE,et a1.Core transcriptional regulatory circuitry in human embryonic stem cells. Cell, 2005, 23;122(6):947-956.
161 Maherali N, Sridharan R, Xie W, et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell, 2007, 1(1): 55-70.
162 Meissner A, Wernig M, Jaenisch R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol, 2007, 25(10): 1177-1181.
163 Chen H, Faller JW, Crabtree RH, et al. Dimer-of-dimers model for the oxygen-evolving complex of photosystem II. Synthesis and properties of [MnIV4O5(terpy)4(H2O)2](ClO4)6. J Am Chem Soc, 2004 , 16;126 (23): 7345-7349.
164 Huangfu D, Maehr R, Guo W, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.Nat Biotechnol, 2008 ,26(7):795-797.
[1] Wilmut I, Schnieke AE, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells[J]. Nature,1997,385(6619):810-813.
[2] Fulka J Jr, Loi P, Fulka H, et al. Nucleus transfer in mammals: noninvasive approaches for the preparation of cytoplasts[J]. Trends Biotechnol,2004,22(6):279-283.
[3] Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy[J]. N Engl J Med,2003,349(3):275-286.
[4] Hua S, Zhang Z, Zhang C, et al. An improved enucleation method of bovine somatic cell nuclear transfer[J]. J Genet Genomics, 2007,34(6):491-496.
[5] Elsheikh AS, Takahashi Y, Katagiri S, et al. Sucrose-exposed chemically enucleated mouse oocytes support blastocyst development of reconstituted embryos[J]. Reprod Fertil Dev,2006,18(6):697-701.
[6] Fulka J Jr, Moor RM. Noninvasive chemical enucleation of mouse oocytes[J]. Mol Reprod Dev,1993,34(4):427-430.
[7]韦精卫,石德顺,韦英明,等.荧光染色及紫外照射(UV)对黄牛孤雌和克隆胚胎早期发育的影响[J].广西农业生物科学, 2005,24(3):196-199,219.
[8] Munsie M, O'brien C, Mountford P. Transgenic strategy for demonstrating nuclear reprogramming in the mouse[J]. Cloning Stem Cells,2002,4(2):121-130.
[9] Li GP, White KL, Bunch TD. Review of enucleation methods and procedures used in animal cloning: state of the art[J]. Cloning Stem Cells, 2004,6(1):5-13.
1 Jones KT.Mammalian egg activation: from Ca21 spiking to cell cycle progression. Reproduction 2005;130:813-823
2 Tamada H, Kikyo N. Nuclear reprogramming in mammalian somatic cell nuclear cloning. Cytogenet Genome Res 2004;105 :285-291
3杨素芳,石德顺,韦精卫,等.激活方法对水牛卵母细胞孤雌发育的影响[J].中国兽医学报,2002,22(4)325-327
4 Vajta G.Somatic cell nuclear transfer in its first and second decades: successes, setbacks,paradoxes and perspectives. Reprod Biomed Online 2007;15(5):582-590 5 Méo SC, Leal CL, Garcia JM. Activation and early parthenogenesis of bovine oocytes treated with ethanol and strontium. Anim Reprod Sci 2004;81(1-2):35-46
6 Jean L,Orly LK. The employment of strontium to activate mouse oocytes: effects on spermatid-injection outcome.Reproduction 2006;131:259–267
7 Kawasumi M, Anzai M, Takehara T,et al. Abnormal distribution of chromosomes in the first division of nuclear transferred mouse embryos. J Reprod Dev 2007;53: 615–622
8 Wani NA.Chemical activation of in vitro matured dromedary camel(Camelus dromedarius) oocytes: Optimization of protocols. Theriogenology2008;69:591–602
9李雪峰,安志兴,郭继彤,等.几种不同化学激活方法对牛卵母细胞孤雌激活的影响[J].西北农林科技大学学报,2002,30(2):43-47
10 SedmíkováM, BurdováJ, Petr J, et al. Induction and activation of meiosis and subsequent parthenogenetic development of growing pig oocytes using calcium ionophore A23187. Theriogenology 2003;60(9):1609-1620
11 Che L,Lalonde A,Bordignon V.Chemical activation of parthenogenetic and nuclear transfer porcine oocytes using ionomycin and strontium chloride.Theriogenology 2007;67:1297–1304
12 Lan GC, Ma SF, Wang ZY, et al.Effects of post-treatment with 6-dimethylaminopurine (6-DMAP) on ethanol activation of mouse oocytes at different ages. J Exp Zoolog A Comp Exp Biol 2004;301(10):837-843
13 Wakayama T, Yanagimachi R. Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei. Reproduction 2001;122(1):49-60
14 Revazova ES,Turovets OD,Kochetkova LS,et al.HLA homozygous stem cell lines derived from human parthenogenetic blastocysts.Cloning Stem Cells 2008;10(1):11-24
15 Yvan H.Nuclear transfer: a new tool for reproductive biotechnology in cattle.Reprod Nutr Dev 2005;45:353–361
16 Ma SF, Liu XY, Miao DQ, et al. Parthenogenetic activation of mouse oocytes by strontium chloride: a search for the best conditions. Theriogenology 2005;15;64(5):1142-1157
17 Tian JH,Wu ZH,Liu L,et al.Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from invitro-matured oocytes and intracytoplasmic sperm injection.Theriogenology 2006;66:439–448
18 Alexander M,Rudolf J.Mammalian Nuclear Transfer.Dev Dyn 2006;235:2460–2469
1 Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N Engl J Med,2003,349(3):275-286.
2 Gardner DK.Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology, 1998,49(1):83-102.
3 Chatot CL,Ziomek CA,Bavister BD. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J-Reprod-Fertil,1989,86(2): 679-688.
4 LAWITTS JA,BIGGERS JD. Culture and preimplantation embryos. Guide to techniques in mouse development,1993,225 (26):153-164.
5 Aoki VW, Wilcox AL, Peterson CM, et al.Comparison of four media types during 3-day human IVF embryo culture . Reprod Biomed Online, 2005 ,10(5):600-606.
6 Biggers JD. Reflections on the culture of the preimplantation embryo. Int J Dev Biol, 1998;42(7):879-884.
7 Van Winkle LJ. Amino acid transport regulation and early embryo development. Biol Reprod, 2001 ,64(1):1-12.
8 Devreker F, Hardy K. Effects of glutamine and taurine on preimplantation development and cleavage of mouse embryos in vitro. Biol Reprod, 1997 ,57(4):921-928.
9 Heindryckx B, Rybouchkin A, Van Der Elst J, et al. Effect of culture media on in vitro development of cloned mouse embryos. Cloning, 2001;3(2):41-50.
10 Summers MC, McGinnis LK, Lawitts JA, et al.Mouse embryo development following IVF in media containing either L-glutamine or glycyl-L-glutamine.Hum Reprod, 2005,20(5):1364-1371.
11莫曾南,覃敏,何敏,等.近交系小鼠体细胞核移植胚胎的构建和鉴定.中国组织工程研究与临床康复,2008,12(31):6001-6006.
12 Lane M, Gardner DK. Nonessential amino acids and glutamine decrease the time of the first three cleavage divisions and increase compaction of mouse zygotes in vitro. J Assist Reprod Genet, 1997,14(7):398-403.
13 Biggers JD, McGinnis LK, Lawitts JA. One-step versus two-step culture of mouse preimplantation embryos: is there a difference? Hum Reprod, 2005 ,20(12):3376-3384.
14 Zander DL, Thompson JG, Lane M. Perturbations in mouse embryo development and viability caused by ammonium are more severe after exposure at the cleavage stages. Biol Reprod, 2006,74(2):288-294.
15 Martin PM, Sutherland AE, Van Winkle LJ. Amino acid transport regulates blastocyst implantation. Biol Reprod, 2003 ,69(4):1101-1108.
16 Choi YH,Seng S,Toyoda Y. Effect of taurine on in vitro fertilization and embryo development of BALB/c mouse strain. Journal of Reproduction and Development,1998,44(1):29-34.
17 Liu Z, Foote RH. Development of bovine embryos in KSOM with added superoxide dismutase and taurine and with five and twenty percent O2. Biol Reprod, 1995,53(4):786-790
18 Devreker F, Van den Bergh M, Biramane J, et al.Effects of taurine on human embryo development in vitro. Hum Reprod,1999,14(9):2350-2356.
19 Van Winkle LJ, Dickinson HR. Differences in amino acid content of preimplantation mouse embryos that develop in vitro versus in vivo: in vitro effects of five amino acids that are abundant in oviductal secretions. BiolReprod, 1995 ,52(1):96-104.
20 Guérin P, El Mouatassim S, Ménézo Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update,2001,7(2):175-189.
1 Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy[J]. N Engl J Med, 2003,349(3):275-286.
2 Ribas R, Oback B, Ritchie W, et al. Development of a zona-free method of nuclear transfer in the mouse[J]. Cloning Stem Cells, 2005,7(2):126-138.
3 Tomii R, Kurome M, Ochiai T, et al. Production of cloned pigs by nuclear transfer of preadipocytes established from adult mature adipocytes[J]. Cloning Stem Cells, 2005,7(4):279-288.
4 Li Shangang, Chen Xuejin, Fang Zhenfu, et al. Rabbits generated from fibroblasts through nuclear transfer[J]. Reproduction, 2006 ,131(6):1085-1090.
5 Andrew J, Catharine A, Linda S, et al. Development of Human Cloned Blastocysts Following Somatic Cell Nuclear Transfer with Adult Fibroblasts[J]. Stem Cells, 2008,26(2):485 -493.
6 Tamada H, Kikyo N. Nuclear reprogramming in mammalian somatic cell nuclear cloning[J]. Cytogenet Genome Res, 2004,105(2-4):285-291.
7 Saliba-Colombani V, Causse M, Gervais L, et al. Efficiency of RFLP, RAPD, and AFLP markers for the construction of an intraspecific map of the tomato genome[J]. Genome, 2000,43(1):29-40.
8王芳,孙以方,段天林,等.(GTG)5探针产生的DNA纹图在近交系小鼠遗传检测中的应用[J].中国实验动物学报, 2007,15(5):361-364.
9 Sakai T, Kikkawa Y, Miura I, et al. Origins of mouse inbred strains deduced from whole-genome scanning by polymorphic microsatellite loci[J]. Mamm Genome, 2005 ,16(1):11-19.
10陈振文,欧阳兆,董罡,等.用微卫星标记技术对国内BALB/c小鼠遗传质量的分析[J].遗传, 2004,26(6):845-848.
11 Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for sonic hedgehog mutation causing familial holoprosencephaly[J]. N Engl J Med, 2003,348(15):1449-1454.
1 Solter D , Gearhart J . Putting stem cells to work. Science,1999 , 283 : 1468 - 1470.
2 Lanza RP , Cibelli JB , West MD. Human therapeutic cloning. Nature Med. 1999 , 5 : 975 - 977.
3 Colman A, Kind A. Therapeutic cloning : concepts and practicalities. Trends Biotechnol,2000 , 18 :192 - 196.
4 Cibelli JB,Stice SL,Golueke PJ,et a1.Transgenic bovine chimeric ofspring produced from somatic cell-derived stem-like cel1.Nat Biotechnol,1998,16(7):642-664.
5 Munsie MJ,Michalska AE,O'Brien CM,et a1.Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.Curr Biol,2000,10(16):989-992.
6 Chen Y,He ZX,Liu A,et a1.Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes.Cell Research,2003,13(4):251-263.
7 Simerly C ,Cominko T , Navara C , et al. Molecular corre2lates of primate nuclear transfer failures. Science , 2003 ,300 : 297
8 Dunglison GF,Barlow DH,Sargent IL. Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium. Hum Roped,1996,11:191-196.
9 Xu C,Inokuma MS,Denham J,Golds K,et al,Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol , 2001 ,19:971-974.
10曹鸿国,张涌.小鼠体细胞核移植及ES细胞样集落分离.自然科学进展,2005,15(4):481-485.
11刘登华,肖文伍,陈红.小鼠胚胎干细胞体外分离培养影响因素的实验研究.华中科技大学学报(医学版),2004,33(6);734-736.
12 Gardener RL,Papaioannou VE.Differentiation in the trophectoderm and inner cell mass. Cambridge University Press,1975,12(1):107-132.
13 Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Gene Development, 2006, 19: 1129-1155.
14 Solter D. From teratocarcinomas to embryonic stem cells and beyond: a history of embryonic stem cell research. Nat. Rev. Genet., 2006, 7(4): 319-327.
1 Takahashi K, S Yamanaka. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined FactorsCell, 2006, 126(4): 663-676
2 Müller FJ, Laurent LC, Kostka D, et al. Regulatory networks define phenotypic classes of human stem cell lines. Nature, 2008, 451(7175): 141-146
3 Fujimori T, Kurotaki Y, Miyazaki J, et al. Analysis of cell lineage in two- and four-cell mouse embryos. Development, 2003, 130(21):5113—5122
4 Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature, 2007, 448(7151): 313-317
5 Wernig M, Meissner A, Foreman R, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007, 19;448(7151):318-324
6 Hanna J, Wernig M, Markoulaki S, et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 2007, 318(5858): 1920-1923
7 Wernig M, Zhao JP, Pruszak J, et al. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci USA, 2008, 105: 5856-5861