Production of Cloned Goats from Enucleated Oocytes Injected with Cumulus Cell Nuclei or Fused with Cumulus Cells

Zou, Xiangang; Chen, Yong; Wang, Yuge; Luo, Jinping; Zhang, Qingbo; Zhang, Xuchen; Yang, Yaofei; Ju, Huiming; Shen, Yu; Lao, Weide; Xu, Shaofu; Du, Ming

doi:10.1089/152045501300189312

Cited by 54 publications

(27 citation statements)

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“…The morula/blastocyst rate was low in both treatment groups (in 9.5% ionomycin and 5.9% ethanol groups) under our conditions, and the rate was much lower than that (34.4%) previously reported by Zou et al [31] when used in in-vivo matured cytoplasts as recipients and NT embryos cultured in vivo for 6 d. In the case of the ionomycin group, the rate was also much lower than that previously reported by Chesne et al [6], who used in vivo matured oocytes as recipient cytoplasts. This may be due to the in vivo conditions offering a better environment than the in vitro.…”

Section: Discussioncontrasting

confidence: 83%

The Effect of Activation Protocols on the Development of Cloned Goat Embryos

Apimeteetumrong

Thuangsanthia²,

Leingcharoen³

et al. 2004

The Journal of Veterinary Medical Science

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ABSTRACT. This study was conducted to compare the developmental competence of somatic nuclear transfer (NT) embryos, after either ionomycin or ethanol activation, in locally bred goats. Donor cells were prepared from the ear skin fibroblasts of a female goat. Cells, at passage 3-8, starved by culturing in 0.5% FCS for 4-8 d, were used for NT. Immature oocytes were obtained from FSH-stimulated goats and matured for 22 hr before enucleation and NT. After fusion, the reconstructed embryos were activated with either ionomycin or ethanol followed by culturing in 6-dimethylaminopurine (6-DMAP) and cytochalasin B (CB), for 3 hr. In experiment I, the fuse d NT embryos (n=63, ionomycin and n=68, ethanol treatments, respectively) were cultured in B 2 with a Vero co-culture system and their developmental competence was evaluated through to Day 9. In experiment II, the NT embryos at the 2-4 cell stage on Day 2 derived from each treatment (ionomycin n=46, and ethanol n=37), were transferred into 10 synchronous recipients. There were no significant differences between the NT embryos derived from the ionomycin and ethanol groups, in fusion (86.3% versus 82.9%), cleavage (90.5% versus 82.4%) and for morula/blastocyst development rates (9.5% versus 5.9%). Sixty percent (3/5) of the recipients from ionomycin became pregnant by midterm (2.5 mts) while only 20% (1/5) from ethanol treatment was pregnant by Day 45. The results demonstrate that activation with either ionomycin or ethanol in combination with 6-DMAP-CB treatment does not affect the development of cloned goat embryos. KEY WORDS: activation, developmental competence, goat, nuclear transfer.J. Vet. Med. Sci. 66(12): 1529-1534, 2004 Since the cloned sheep named "Dolly", was produced by transferring nuclei from adult cells into enucleated oocytes [27], there have been many studies carried out to produce other cloned mammals such as mice, cattle, goats, pigs and rabbits [1,5,19,25,26]. The nuclear transfer (NT) technique has been efficiently used for producing transgenic, cloned farm animal offspring. These animals are capable of producing valuable proteins, which could have a marked impact on the pharmaceutical industry [22] [1,4,6,11,32]. There are a few reports on the development of goat oocytes after parthenogenetic activation by treatment with ionomycin and ethanol, both followed by exposure to 6-diethylaminopurine (6-DMAP) [17,18]. Nevertheless, comparison of the development of NT goat embryos with either ionomycin or ethanol, as an activating agent, has not been reported. The objective of this study was to compare the developmental competence in vitro and in vivo of NT goat embryos, after activation, by using either ionomycin or ethanol, both followed by incubation in 6-DMAP-cytochalasin B (CB). MATERIALS AND METHODSUnless otherwise indicated, all chemicals used in this study were obtained from Sigma-Aldrich Company (St. Louis, MO) and the media from Gibco Invitrogen Corporation (Grand Island, NY).Preparation of donor cells: Donor cells were prepared from an ear skin f...

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Section: Discussioncontrasting

confidence: 83%

The Effect of Activation Protocols on the Development of Cloned Goat Embryos

Apimeteetumrong

Thuangsanthia²,

Leingcharoen³

et al. 2004

The Journal of Veterinary Medical Science

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“…In addition, and again in contrast to conventional micromanipulation, piezo permits multiple procedures to be performed with a single needle. Thus, although many oocytes (such as those of humans, cattle, pigs and rabbits) can be injected conventionally, the relative speed and ease of piezo means that it is advantageous even in these species 3,[15][16][17][18][19][20] . Ease of pipette fabrication (beveled tips are not required) and speed also make piezo suitable for the injection of blastocysts with ES cells 11 , and the method additionally allows partial ablation of the inner cell mass (i.e., the removal of potentially competing embryonic cells).…”

Section: Introductionmentioning

confidence: 99%

Piezo-actuated mouse intracytoplasmic sperm injection (ICSI)

Yoshida¹,

Perry²

2007

Nat Protoc

156

135

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The mouse is a genetically tractable model organism widely used to study mammalian development and disease. However, mouse metaphase II (mII) oocytes are exquisitely sensitive and intracytoplasmic sperm injection (ICSI) with conventional pipettes generally kills them. This problem can be solved with piezo-actuated micromanipulation, in which the piezo-electric effect (crystal deformation in response to an externally applied voltage) propels a microinjection needle tip forward in a precise and rapid movement. Piezoactuated micromanipulation enhances the penetration of membranes and matrices, and mouse ICSI is a major application. Here we describe a comprehensive, step-by-step mouse piezo ICSI protocol for non-specialists that can be completed in 2-4 h. The protocol is a basic prelude to multiple applications, including nuclear transfer cloning, spermatid injection, blastocyst injection, mII transgenesis, and streamlining micromanipulation in primates and livestock. Moreover, piezo ICSI can be used to obtain offspring from 'dead' (non-motile) sperm, enabling trivial sperm freezing protocols for mouse strain storage and shipment. INTRODUCTIONPiezo-actuated micromanipulation harnesses the piezo-electric effect to transmit a small crystal lattice distortion to the tip of a pipette, driving it forward in a precise and controlled manner. Piezo-actuated micromanipulation has multiple applications in the study and engineering of gametes and embryos. It enabled the first intracytoplasmic sperm injection (ICSI) to produce mice 1 , the first nuclear transfer cloning of mice 2 and pigs 3 , the first productive frozen 4 and freeze-dried sperm injections 5 , and the first production of nuclear transfer embryonic stem (ES) cells 6 . Piezo was utilized to generate the first transgenic offspring by injecting unfertilized oocytes in metaphase II (mII) transgenesis 7 and has been extended to the delivery of artificial chromosome transgenes 8,9 . Piezo has been employed for RNA interference (RNAi) in mII oocytes 10 ; it enhances blastocyst injection with ES cells 11 and the manipulation of gamete precursors 12 and facilitates the renaissance of stem cell biology 13 .Piezo-actuated micromanipulation was developed by Atsushi Mimatsu and colleagues, and its application as a biological research tool was demonstrated in the mouse by Dr. Yasuyuki Kimura, who used it to generate the first mouse offspring by ICSI 1 . Piezo was necessary because in the mouse, oocyte plasma membranes are exquisitely sensitive and survival rates following ICSI with conventional (i.e., manual, non-piezo) microinjection rarely exceed 50% (ref. 14). Contrastingly, piezo ICSI can achieve mouse oocyte survival rates of 100%, with 90% development to morula/blastocyst stages in vitro. The efficacy of piezo is highly desirable, not only in ICSI but also where development following microinjection is less efficient, such as in nuclear transfer. Most mouse nuclear transfer

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“…Neste sentido, vários estudos já foram desenvolvidos buscando avaliar o estádio nuclear adequado deste citoplasto receptor em programas de transferência nuclear. Contudo, diferentes estudos têm demonstrado que oócitos em metáfase II (MII) são mais adequados para suportar a reprogramação nuclear São comparados aos demais estádios de maturação nuclear (Zou et al, 2001;Lee et al, 2007). Por esta razão, a maioria das pesquisas relata o uso de oócitos em MII maturados in vivo (Kato et al, 2000) ou in vitro (Zhou et al, 2007).…”

Section: Seleção De Oócitos Receptoresunclassified

Clonagem de embriões bovinos a partir de células somáticas

2014

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Titulo en español: Clonación de embriones bovinos a partir de células somáticastitulo en ingles: Cloning bovine embryos from somatic cellsResumo: No presente artigo revisa-se dedicadamente a literatura do tema da clonagem tentando levar o leitor paulatinamente através dos avanços da técnica até alcançar as mais novas alternativas propostas pelos cientistas, e incluindo os erros mais comumente acontecidos nelas. A finalidade é mostrar o amplio panorama que tem aberto esta biotecnologia para a humanidade toda.Palavras chave: Transferência nuclear, fissão, paraclonagem, clonagem verdadeira, genoma mitocondrial.Resumen: En el presente artículo se revisa detenidamente la literatura en el tema de clonación, intentando llevar al lector paulatinamente a través de los avances de la técnica hasta alcanzar las más nuevas alternativas propuestas por los investigadores, incluyendo los errores más comúnmente cometidos. La finalidad es mostrar el amplio panorama que a abierto esta biotecnología para la humanidad.Palabras clave: Transferência nuclear, fissão, paraclonagem, clonagem verdadeira, genoma mitocondrial.Abstract: The present article provides a review of the pertinent literature on the topic of cloning. It aims to take the reader gradually through the advances made so far regarding the technique, to the newest alternatives proposed by researchers, including the most commonly committed errors, to unveil the broad panorama this biotechnology has opened up for humankind.Key words: Nuclear transfer, fission, paraclone colony, true cloning, mitochondrial genome.

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Production of Cloned Goats from Enucleated Oocytes Injected with Cumulus Cell Nuclei or Fused with Cumulus Cells

Cited by 54 publications

References 27 publications

The Effect of Activation Protocols on the Development of Cloned Goat Embryos

The Effect of Activation Protocols on the Development of Cloned Goat Embryos

Piezo-actuated mouse intracytoplasmic sperm injection (ICSI)

Clonagem de embriões bovinos a partir de células somáticas

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