Production of Cloned Goats after Nuclear Transfer Using Adult Somatic Cells1

Keefer, Carol L.; Keyston, Rebecca; Lazaris, Anthoula; Bhatia, B.; Begin, I.; Bilodeau, A.S.; Zhou, Fanny; Kafidi, N.; Wang, B.; Baldassarre, Hernán; Karatzas, C.N.

doi:10.1095/biolreprod66.1.199

Cited by 141 publications

(76 citation statements)

References 30 publications

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“…This pregnancy rate, based on the number of pregnant recipients, per total number of recipients, was slightly higher than that previously reported by others [10,22]. Similar to the findings of earlier studies [10,11], prenatal loss after Day 30 ultrasound scan was not observed in the ionomycin treatment group in the present study. A 20% (1/5) pregnancy rate at Day 30 was achieved after embryo transfer of NT embryos, derived from the ethanol treatment, which was lower than that reported (55.5%) by Baguisi et al [1] but those authors lost all thereafter.…”

Section: Discussionsupporting

confidence: 86%

“…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].…”

mentioning

confidence: 99%

“…Activation is an important step in the NT procedure and ethanol and ionomycin have been widely used as activating agents in several species, such as cattle [15,20,21], mice [8], sheep [16], and goats [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].…”

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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: Discussionsupporting

confidence: 86%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…The maturation medium was composed of: TCM199 (Sigma M5017-1L, St. Louis, MO, USA) supplemented with 10% FBS, 0.02U/ml bFSH (Sioux Biochemicals 715, IA, USA), 0.02U/ml bLH (Sioux Biochemicals 725, IA, USA), 1 µg/ml estradiol-17β, 0.2 mM sodium pyruvate, 50 µg/ml kanamycin, 100 µM cysteamine [32]. To assess maturation, the oocytes were stained with a supra vital concentration of Hoechst (Sigma B2261, St. Louis, MO, USA), 20-22 h after in vitro maturation.…”

Section: In Vitro Maturationmentioning

confidence: 99%

In vitro maturation, apoptotic gene expression and incidence of numerical chromosomal abnormalities following cryotop vitrification of sheep cumulus-oocyte complexes

Ebrahimi

Valojerdi

Eftekhari‐Yazdi

et al. 2010

J Assist Reprod Genet

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Purpose The purpose of this study was to evaluate the effects of cryotop vitrification of sheep cumulus-oocyte complexes (COCs) on oocyte maturation, apoptotic gene expression and incidence of chromosomal abnormalities. Methods Freshly isolated (control group) and vitrifiedwarmed COCs (cryotop group) were matured in vitro. The expression of pro-and anti-apoptotic genes was investigated by real-time PCR. The incidence of numerical chromosomal abnormalities was evaluated by cytogenetic analysis. Results The mean percentage of oocytes in the cryotop and control groups that reached metaphase II was 49.25±3.01% and 51.94±2.7% respectively. The expression rates of proand anti-apoptotic genes were similar in both groups, whereas the incidence of numerical chromosomal abnormalities was higher in the cryotop group compared to the control group (42.5% vs. 20%, p<0.05). Conclusion Although cryotop vitrification of COCs did not affect the incidence of oocyte maturation or apoptotic gene expression, significant deficiencies in the maintenance of oocyte chromosomal organization were seen.

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“…Sequential incubations with the calcium ionophore, ionomycin, and the protein kinase inhibitor, 6-DMAP, is another activation treatment that has been used to produce cloned sheep [130], cattle [177][178][179], pigs [180,181] and goats [182]. Incubation with cycloheximide following exposure to ionomycin or electric pulses has also induced cattle [172,183,184], rhesus monkey [185] and mouflon [186] NT embryos to develop to term.…”

Section: Activation Of Nuclear Transfer Embryosmentioning

confidence: 99%

Calcium Release at Fertilization: Artificially Mimicking the Oocyte's Response to Sperm.

Grupen¹,

Nottle²,

Nagashima

2002

Journal of Reproduction and Development

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Abstract. The mechanism of sperm-induced calcium release has been the subject of many studies since the development in the late 1950s of in vitro culture systems that support mammalian fertilization. Despite efforts to elucidate the nature of the signal from the sperm that triggers both the early and late events of oocyte activation, the precise mechanism remains unresolved. Now, with the advent of somatic nuclear transfer technologies, the need to better understand this unique process has been recognised. Nuclear transfer embryos must be induced to commence development artificially because the activating signal from the sperm is absent. The primary activating stimulus is a large increase in the concentration of intracellular-free calcium and numerous physical and chemical treatments have been found to induce calcium changes that initiate the events of oocyte activation. Although live cloned offspring have been produced in a number of species, the overall efficiencies of the nuclear transfer procedures described thus far are unacceptably low and phenotypic anomalies are common. With the aim of improving these efficiencies, researchers are developing artificial activation treatments which induce oocyte responses that mimic those induced by fertilizing sperm. One strategy is to replicate the pattern of calcium change more closely. Another strategy is to couple an activating stimulus with treatments that inhibit maturation (or M-phase) promoting factor (MPF) activity, which regulates meiotic progression in oocytes. This paper reviews what is understood of calcium release at fertilization and describes the treatments that have been used to induce oocyte activation artificially in parthenogenetic and nuclear transfer studies. The relative effectiveness of the strategies employed to mimic the oocyte's response to sperm are discussed. Key words: Oocyte activation, Fertilization, Parthenogenesis, Nuclear transfer (J. Reprod. Dev. 48: 2002) o produce a viable embryo at fertilization, the s p e r m n o t o n l y p r o v i d e s t h e m a l e chromosomal complement, but also the stimulus that initiates development. The mechanism by which the activating signal is transmitted from the sperm to the oocyte is not fully clarified, but it is clear that this signal triggers a large increase in the concentration of intracellular-free calcium. In fertilized sea urchin and Xenopus eggs, the calcium increase takes the form of a propagating wave that originates at the point of sperm attachment and traverses the egg at a velocity of 5 to 10 µm/sec

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Production of Cloned Goats after Nuclear Transfer Using Adult Somatic Cells1

Cited by 141 publications

References 30 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

In vitro maturation, apoptotic gene expression and incidence of numerical chromosomal abnormalities following cryotop vitrification of sheep cumulus-oocyte complexes

Calcium Release at Fertilization: Artificially Mimicking the Oocyte's Response to Sperm.

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