Cloned Calves Produced by Transfer of Reconstituted Embryos Derived from Fibroblast Cells of a Female Fetus.

Sakaguchi, Minoru; Yotsushima, Kenji; Kakei, Tomoko; Nakahara, Hiroyuki; Takahashi, Seiya; Imai, Hiroshi; Izaike, Yoshiaki

doi:10.1262/jrd.46.265

Cited by 5 publications

(4 citation statements)

References 11 publications

(17 reference statements)

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“…Fibroblast cell lines were established as previously described [30,31]. L1 and L2 were obtained from ear skin tissue of a Japanese black bull (L1) or cow (L2), while L3 was obtained from lung tissue of a female fetus (approximately at 5.5 months of pregnancy).…”

Section: Donor Cellsmentioning

confidence: 99%

Treatment with a Histone Deacetylase Inhibitor after Nuclear Transfer Improves the Preimplantation Development of Cloned Bovine Embryos

Akagi

Matsukawa

Mizutani

et al. 2011

Journal of Reproduction and Development

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Abstract. We examined the effects of treatment with histone deacetylase inhibitors (HDACi), trichostatin A (TSA) and scriptaid (SCR), on the blastocyst formation rate in bovine somatic cell nuclear transferred (SCNT) embryos derived from fibroblast cells. Three fibroblast cell lines (L1, L2 and L3) were used as somatic cell donors to produce SCNT embryos (L1, L2 and L3 embryos, respectively). In Experiment 1, we compared the in vitro developmental competence of L1 embryos treated with various concentrations of TSA for different time periods following chemical activation. Embryos treated with 5 nM TSA for 20 h showed a significantly increased blastocyst formation rate compared with untreated controls. In Experiment 2, we examined the effect of TSA (5 nM) treatment of L1, L2 and L3 embryos as well as the effect of treatment of L1, L2 and L3 embryos with various concentrations of SCR on in vitro developmental competence. It was found that 5 nM TSA treatment significantly increased the blastocyst formation rate in L1 and L3 embryos but did not have an influence on the development of L2 embryos. On the other hand, 5 nM SCR treatment significantly increased the blastocyst formation rates of L1 and L2 embryos compared with controls. However, there was no significant increase in the blastocyst formation rate of L3 embryos when they were treated with SCR. In Experiment 3, acetylation of H4K12 was examined in donor cells and pronuclear-stage L1, L2 and L3 embryos treated with 5 nM TSA or 5 nM SCR by immunostaining. The level of H4K12 acetylation was different among donor cells. The staining intensities in the TSA-treated L1 and L3 embryos and SCR-treated L2 embryos were significantly higher than those of untreated embryos. These results suggest that HDACi treatment of bovine SCNT embryos improves the blastocyst formation rate; however, the optimal treatment conditions may differ among donor cell lines. Key words: Bovine, Nuclear transfer, Histone deacetylase inhibitor (J. Reprod. Dev. 57: [120][121][122][123][124][125][126] 2011) ive cloned offspring have been produced in many species by somatic cell nuclear transfer (SCNT), although the cloning efficiency is still very low. It has been reported that, in SCNT, embryo losses occur at various stages, and the anomalies in fetal and perinatal stages have been observed [1][2][3][4][5][6]. Because such abnormalities of clones are not transmitted to their progenies [7][8][9][10][11][12], most of the developmental problems of clones are believed to be the results of epigenetic defects [13]. In fact, several studies have revealed abnormal epigenetic modifications such as DNA methylation and histone modifications in SCNT embryos [14][15][16][17].A wide variety of histone deacetylase inhibitors (HDACi) of both natural and synthetic origin has been reported [18]. Trichostatin A (TSA), which is a natural product isolated from Streptomyces hygroscopicus [19], is one of the most frequently used drugs for various studies as a selective HDACi. Recently, it has been reported from two la...

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Section: Donor Cellsmentioning

confidence: 99%

Treatment with a Histone Deacetylase Inhibitor after Nuclear Transfer Improves the Preimplantation Development of Cloned Bovine Embryos

Akagi

Matsukawa

Mizutani

et al. 2011

Journal of Reproduction and Development

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“…However, there were no significant differences in the cleavage rate or the rate of development to the blastocyst stage between the two groups, which might have resulted from the different electrofusion conditions between them. In our previous studies (Sakaguchi et al . 2000; Takahashi et al .…”

Section: Discussionmentioning

confidence: 83%

Nuclear transfer using a bovine mammary epithelial cell line (BMEC)

Akagi

Takahashi

Ohkoshi

et al. 2002

Animal Science Journal

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The developmental potential of nuclei from a bovine mammary epithelial cell line (BMEC) in nuclear transfer was investigated. For nuclear transfer donors, BMEC cells (passage 15) were cultured for 4-5 days after seeding at cell densities of 1.0 ¥ 10 5 cells/cm 2 (high-density group) or 0.8 ¥ 10 4 cells/cm 2 (low-density group). First, the effective electric stimulation for fusion of enucleated oocytes with BMEC cells was examined. Fusion rates reached maximum with two DC pulses of 30 V/150 m m for 20 m s in the high-density group and with two DC pulses of 25 V/150 m m for 10 m s in the low-density group. The fusion rate (37.5%) in the high-density group was significantly ( P < 0.005) lower than in the low-density group (71.4%). Second, the in vitro developmental potential of nuclear transfer embryos derived from BMEC cells was examined. In the high-density and low-density groups, 18.8% and 24.1% of fused oocytes, respectively, developed to blastocyst stage. The results of this study indicate that nuclei from BMEC cells support the development of nuclear transfer embryos to the blastocyst stage and that the efficiency of oocyte-cell fusion is affected by the culture conditions of the donor BEMC cells before nuclear transfer.

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“…These characteristics make them suitable as nuclear donors. In fact, the production of nuclear transferred offspring using fibroblasts from a female fetus as donor has been described for cattle [11]. In chickens, however, our preliminary study failed to yield snt-GGCs from intact fibroblasts cultured in vitro because of an inability to form a 'pearl chain' in an alternating electrical field during electrofusion (data not shown).…”

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confidence: 76%

Conditioning of Karyoplasts for Producing Somatic Nuclear Transferred Gonadal Germ Cells in Domestic Chickens

Kohara

Minematsu

Aikawa

et al. 2008

Journal of Reproduction and Development

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Abstract. The objective of this study was to establish a protocol for generating karyoplasts that can be used to produce somatic nuclear transferred gonadal germ cells (snt-GGCs) in domestic chickens. Karyoplasts were produced by centrifuging cultured fibroblasts from 10-day-old chick embryos at 10,000 × g in the presence of 1.0 μg/ml cytochalasin B. The number of karyoplasts was significantly (P<0.05) higher and the diameters of the karyoplasts were significantly (P<0.05) smaller when fibroblasts were centrifuged for 60 min than for 10 or 30 min. It was possible to generate sntGGCs by electrofusion of GGCs with karyoplasts produced from cryopreserved or serum-starved fibroblasts. These results indicate that karyoplasts generated from 10-day-old chick embryos can be used to produce snt-GGCs even after cryopreservation and serum starvation of the fibroblasts. Key words: Chicken, Cryopreservation, Gonadal germ cell, Karyoplast, Nuclear transfer, Serum starvation (J. Reprod. Dev. 54: [221][222][223][224] 2008) ince the birth of Dolly the sheep [1], the first mammal derived from the transfer of a somatic nucleus, somatic nuclear transfer technology has become key in reproductive and developmental biology. However, somatic nuclear transfer technology developed for mammals cannot be applied directly to avian species owing to major differences in the basic reproductive physiology between these two classes. An alternative strategy of producing somatic nuclear transferred offspring in avian species by using avian primordial germ cells (PGCs) or gonadal germ cells (GGCs) has been described by Tajima [2].PGCs are undifferentiated germ cells that subsequently differentiate into spermatogonia in the testis or into oogonia in the ovary. In avian, PGCs originate from the epiblast [3] and appear in the area pellucida [4]. After development of the vascular system, PGCs circulate along with embryonic blood cells and finally migrate into the embryonic gonads [5]. After migration, PGCs are called GGCs. Avian germline chimeras have been produced by transfer of cryopreserved PGCs [6] or GGCs [7] into recipient embryos. When a somatic nucleus is introduced into an enucleated PGC or GGC, which is then transferred into the bloodstream of a recipient embryo, the recipient theoretically produces sperm or eggs originating from the somatic nucleus.Somatic nuclear transferred PGCs (snt-PGCs) have been produced from PGCs and embryonic blood cells (EBCs) by electrofusion [8,9] and virus-stimulated fusion [9]. Ishiguro et al.[10] described a method for producing inter-genus somatic nuclear transferred GGCs (snt-GGCs) from chickens and quails. In all the reports mentioned here, EBCs were used as nuclear donors, but limited information is available as to whether cells other than EBCs can be used for producing snt-PGCs or snt-GGCs.Fibroblasts can be recovered readily from adult chickens and embryos, cultured in vitro and cryopreserved. These characteristics make them suitable as nuclear donors. In fact, the production of nuclear transferred of...

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Cloned Calves Produced by Transfer of Reconstituted Embryos Derived from Fibroblast Cells of a Female Fetus.

Cited by 5 publications

References 11 publications

Treatment with a Histone Deacetylase Inhibitor after Nuclear Transfer Improves the Preimplantation Development of Cloned Bovine Embryos

Treatment with a Histone Deacetylase Inhibitor after Nuclear Transfer Improves the Preimplantation Development of Cloned Bovine Embryos

Nuclear transfer using a bovine mammary epithelial cell line (BMEC)

Conditioning of Karyoplasts for Producing Somatic Nuclear Transferred Gonadal Germ Cells in Domestic Chickens

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