Valproic Acid EnhancesIn VitroDevelopment and Oct-3/4 Expression of Miniature Pig Somatic Cell Nuclear Transfer Embryos

Miyoshi, Katsuhiko; Mori, Hironori; Mizobe, Yamato; Akasaka, Eri; Ozawa, Akio; Yoshida, Mitsutoshi

doi:10.1089/cell.2009.0032

Cited by 71 publications

(66 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The production of cloned mice from both ME and AE cytoplasts also support our previous findings on the beneficial effects of VPA treatments for mouse cloning in the B6CBAF1 strain (Costa-Borges et al 2010). These beneficial effects, corroborated in miniature pig SCNT embryos (Miyoshi et al 2010), have recently been questioned by Ono et al (2010), who compared the effects of different HDACis on cloning BD129F1 mice. The discrepancy of results in the mouse species can be probably attributed to strain-specific differences, as not all HDACis are equally effective in all genetic backgrounds (Kishigami et al 2007, Van Thuan et al 2009).…”

Section: Discussionsupporting

confidence: 84%

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid

Costa-Borges

González²,

Santaló³

et al. 2011

Reproduction

View full text Add to dashboard Cite

Mouse recipient cytoplasts for somatic cell nuclear transfer (SCNT) are routinely prepared by mechanical enucleation (ME), an invasive procedure that requires expensive equipment and considerable micromanipulation skills. Alternatively, oocytes can be enucleated using chemically assisted (AE) or chemically induced (IE) enucleation methods that are technically simple. In this study, we compared the reprogramming potential and developmental capacity of cloned embryos generated by ME, AE, and IE procedures and treated with the histone deacetylase inhibitor valproic acid. A rapid and almost complete deacetylation of histone H3 lysine 14 in the somatic nucleus followed by an equally rapid and complete re-acetylation after activation was observed after the injection of a cumulus cell nucleus into ME and AE cytoplasts. In contrast, histone deacetylation occurred at a much lower level in IE cytoplasts. Despite these differences, the cloned embryos generated from the three types of cytoplasts developed into blastocysts of equivalent total and inner cell mass mean cell numbers, and the rates of blastocyst formation and embryonic stem cell derivation were similar among the three groups. The cloned embryos produced from ME and AE cytoplasts showed an equivalent rate of full-term development, but no offspring could be obtained from the IE group, suggesting a lower reprogramming capacity of IE cytoplasts. Our results demonstrate the usefulness of AE in mouse SCNT procedures, as an alternative to ME. AE can facilitate oocyte enucleation and avoid the need for expensive microscope optics, or for potentially damaging Hoechst staining and u.v. irradiation, normally required in ME procedures.

show abstract

Section: Discussionsupporting

confidence: 84%

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid

Costa-Borges

González²,

Santaló³

et al. 2011

Reproduction

View full text Add to dashboard Cite

show abstract

“…In other words, the present system using pOEIN transfectants would be useful for screening drugs supporting in vivo development of SCNT embryos. With this system, we have recently demonstrated that valproic acid, a histone deacetylase inhibitor, is beneficial for prolonged expression of Oct-3/4 as well as for increasing the blastocyst formation rates of SCNT embryos [59]. In addition, our system is helpful for establishment of ES cell lines by long-term serial passage of ICM-derived cells, since pluripotent-state ES cells can be simply and noninvasively monitored under a fluorescent microscope.…”

Section: Discussionmentioning

confidence: 99%

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

Miyoshi

Mori

Mizobe

et al. 2009

Journal of Reproduction and Development

Self Cite

View full text Add to dashboard Cite

Abstract. The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4-6 days of culture) and 50.0% of blastocysts (at 5-6 days of culture), whereas none of the 1-cell to 16-cell embryos at 1-5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4-6 days after SCNT and that this noninvasive monitoring system using Oct-3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei. Key words: Enhanced green fluorescent protein, In vitro development, Nuclear transfer, Oct-3/4 promoter, Reprogramming (J. Reprod. Dev. 55: [661][662][663][664][665][666][667][668][669] 2009) uccessful production of cloned animals using somatic cell nuclear transfer (SCNT) has been reported in several mammalian species [1][2][3][4][5][6][7][8][9][10][11][12]. However, the low cloning efficiency associated with development of SCNT embryos to offspring remains the major obstacle to widespread use of this technology in a number of animal science and biomedical applications. A method for evaluating the developmental ability of SCNT embryos before being transferred into recipient females should be explored to optimize the procedures of SCNT and overcome the low cloning efficiency. Evaluation of the in vitro developmental ability of an SCNT embryo to the blastocyst stage would be considered one of the methods for predicting its in vivo development, but it has been found that this does not often assure successful development to term of SCNT embryos [13]. After SCNT, the gene expression pattern in somatic cells is reprogrammed to mimic that in preimplantation embryos [14]. However, there is a difference in gene expression pattern between SCNT embryos and in vitro-fertilized embryos, suggesting that the low efficiency of SCNT is associated with incomplete reprogramming in the donor nuclei transferred into recipient oocytes [15]. Therefore, evaluation of the reprogramming level in the donor nuclei appears to be most essential for predicting the in vivo developmental ability of SCNT embryos.Oct-3/4 is a transcription factor of the Pit-Oct-Unc family [16,17], and...

show abstract

“…4C, lane 8) may be ascribed to failure to express their α-GalT mRNA at a level detectable by RT-PCR, as previously pointed out. This phenomenon may be related to the failure of proper reprogramming of donor nuclei after SCNT, since gene activation of embryonic and pluripotency-related genes is sometimes suppressed [43,44]. In our preliminary data, expression of α-GalT mRNA increased gradually with progression towards the blastocyst stage (unpublished results).…”

Section: Discussionmentioning

confidence: 67%

Successful Suppression of Endogenous α-1,3-Galactosyltransferase Expression by RNA Interference in Pig Embryos Generated In Vitro

Chi

Shinohara

Yokomine

et al. 2012

Journal of Reproduction and Development

Self Cite

View full text Add to dashboard Cite

Abstract. RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B 4 , a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of doublestranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation. Key words: Blastocyst, α-1,3-Galactosyltransferase, Pig, RNAi, Somatic cell nuclear transfer (J. Reprod. Dev. 58: [69][70][71][72][73][74][75][76] 2012) S uppression of endogenous target gene expression by gene targeting has been proven as a powerful tool for exploration of the biological function of a gene of interest. Recently, attempts to use antisense technology, now termed RNA interference (RNAi), as a convenient and valuable tool for understanding the in vivo function of a gene of interest have been made [1][2][3][4][5][6][7][8]. RNAi is a multiple-step process that involves the generation of 21-25 nt small interfering (si) RNA and results in degradation of the homologous RNA [2]. Since siRNA can be easily synthesized chemically, it is easy to study the role of endogenous genes by injecting double-stranded (ds) RNA or siRNA into early embryos. However, in the former cases, the effectiveness of dsRNA introduced into cultured cells is known to be retained for a relatively short time (less than one week) [5][6][7][8][9]. In contrast, use of siRNA should be more beneficial for conferring long-term expression of siRNA in transfected cells. In fact, this technology (which is termed "transgenic RNAi") was found to be effective in knocking down ta...

show abstract

Valproic Acid EnhancesIn VitroDevelopment and Oct-3/4 Expression of Miniature Pig Somatic Cell Nuclear Transfer Embryos

Cited by 71 publications

References 45 publications

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

Successful Suppression of Endogenous <i>α-1,3-Galactosyltransferase</i> Expression by RNA Interference in Pig Embryos Generated <i>In Vitro</i>

Contact Info

Product

Resources

About

Valproic Acid EnhancesIn VitroDevelopment and Oct-3/4 Expression of Miniature Pig Somatic Cell Nuclear Transfer Embryos

Cited by 71 publications

References 45 publications

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

Successful Suppression of Endogenous <i>&alpha;-1,3-Galactosyltransferase</i> Expression by RNA Interference in Pig Embryos Generated <i>In Vitro</i>

Contact Info

Product

Resources

About

Successful Suppression of Endogenous <i>α-1,3-Galactosyltransferase</i> Expression by RNA Interference in Pig Embryos Generated <i>In Vitro</i>