Somatic Cell Nuclear Transfer Protocols Research Articles

Reduction of H3K9 methylation by G9a inhibitors improves the development of mouse SCNT embryos

Removal of somatic histone H3 lysine 9 trimethylation (H3K9me3) from the embryonic genome can improve the efficiency of mammalian cloning using somatic cell nuclear transfer (SCNT). However, this strategy involves the injection of histone demethylase mRNA into embryos, which is limiting because of its invasive and labor-consuming nature. Here, we report that treatment with an inhibitor of G9a (G9ai), the major histone methyltransferase that introduces H3K9me1/2 in mammals, greatly improved the development of mouse SCNT embryos. Intriguingly, G9ai caused an immediate reduction of H3K9me1/2, a secondary loss of H3K9me3 in SCNT embryos, and increased the birth rate of cloned pups about 5-fold (up to 3.9%). G9ai combined with the histone deacetylase inhibitor trichostatin A further improved this rate to 14.5%. Mechanistically, G9ai and TSA synergistically enhanced H3K9me3 demethylation and boosted zygotic genome activation. Thus, we established an easy, highly effective SCNT protocol that would enhance future cloning research and applications.

Prostasomes as interlocutor for zygotic epigenome health: role of prostasomesecretome in maintenance of zygotic epigenome

Prostasomes as transgenerational epigenetic tool box has not been established, its role in transferring small RNA, lncRNA as paternal messages is yet to be explored. The present study was undertaken to establish role of prostasomes in modulating testicular and vaginal environmental cues such as protein and RNA messages through to zygote and thereby actin gas interlocutor of paternal genome. The results from the study found out prostasomes fusion with spermatozoa takes place at the time of maturation and capacitation wherein it causes downregulation of ROS (Protein and mRNA) in terms of decreased NADPH and NOS activity significantly (p<0.01), it was also found to modulate mitochondrial membrane potential significantly(p<0.01). Prostasome supplementation lead to increased survival of spermatozoa in sp-TALP medium and was found to be superior to cryopreserved and fresh semen when used in routine in vitro fertilization (IVF) experiment. The study further envisages to establish prostasomes as repository of novel biomarkers for infertility in male and female and as diagnostic tool in in vitro fertilization and somatic cell nuclear transfer protocol and thereby establishing prostasomes as one of the fundamental vesicles influencing transgenerational transfer of RNA and protein to zygotic genome thereby influencing zygotic genome health.

Somatic Cell Nuclear Transfer in Rabbits.

Somatic cell nuclear transfer (SCNT) is a technology that enables differentiated somatic cells to acquire a totipotent state, thus making it of great value in developmental biology, biomedical research, and agricultural applications. Rabbit cloning associated with transgenesis has the potential to improve the applicability of this species for disease modeling, drug testing, and production of human recombinant proteins. In this chapter, we introduce our SCNT protocol for the production of live cloned rabbits.

Cattle Cloning by Somatic Cell Nuclear Transfer.

Cloning by somatic cell Nuclear Transfer (SCNT) is a powerful technology capable of reprograming terminally differentiated cells to totipotency for generating whole animals or pluripotent stem cells for use in cell therapy, drug screening, and other biotechnological applications. However, the broad usage of SCNT remains limited due to its high cost and low efficiency in obtaining live and healthy offspring. In this chapter, we first briefly discuss the epigenetic constraints responsible for the low efficiency of SCNT and current attempts to overcome them. We then describe our bovine SCNT protocol for delivering live cloned calves and addressing basic questions about nuclear reprogramming. Other research groups can benefit from our basic protocol and build up on it to improve SCNT in the future. Strategies to correct or mitigate epigenetic errors (e.g., correcting imprinting loci, overexpression of demethylases, chromatin-modifying drugs) can integrate the protocol described here.

IMPROVING THE EFFICIENCY OF BOVINE SOMATIC CELL NUCLEAR TRANSFER PROTOCOL BY ESTABLISHMENT OF CULTURE MEDIUM CHANGING SYSTEM

Bovine cloning is expected as a promising technology for endangered genome conservation and the production of transgenic cloned cows to manufacture human recombinant proteins. However, cloned bovine embryos often resulted in poor development, thus reducing the success of producing cloned offspring. One of the reasons is based on culture medium, which directly relates to the development of cloned bovine embryos. This study aimed to establish culture medium changing system to improve the efficiency of bovine somatic cell nuclear transfer protocol. Our results showed that using the culture medium of Takahashi and First 1992 produced the cloned bovine blastocyst at the highest rate (9.4%, P < 0.05) among different modified synthetic oviduct fluid (mSOF) media. In addition, the rate of embryos developed to the hatching blastocyst was highest in the culture group within transfer embryos to the fresh medium with 5% FBS on day 3 and day 5 (18.5%) compared to the other treatments. Finally, we found that cloned embryos in the group of partial replacement presented significantly higher blastocyst formation rate and quality compared to the group of full replacement (27.6% and 15.6%, respectively, P < 0.05), and it also showed the highest number of cells in blastocyst (about 116 cells). In conclusion, using mSOF and changing the fresh medium supplemented with 5% FBS on day 3 following partial replacement on day 5 improved the preimplantation development of cloned bovine embryos and promoted the blastocyst quality.

Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues.

Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.

Global DNA methylation: role, status and genome-wide approaches to study epigenetic mark in cloned embryos

Somatic cell nuclear transfer (SCNT) technique has been proving its worth for more than two decades now as over 20 different species have been successfully cloned. SCNT protocol for cloning is well established but efficiency in terms of live birth rate is still low. Epigenetic abnormality following nuclear reprogramming is considered as the main culprit behind its low efficiency. DNA methylation is one of the most important epigenetic modifications that directly or indirectly regulate gene expression pattern, development and genome stability. Embryos produced through SCNT are found to express abnormal DNA methylation profile in comparison with in vivo or in vitro produced embryos. In order to improve DNA methylation profile in cloned embryos, a complete database of whole genome is required to find out specific faulty targets. Many techniques including low throughput and high throughput approach has been used to profile DNA methylation pattern in bovine embryos throughout the developmental stages. In the present review, we have compiled the overall status of global DNA methylation, the effect of aberrant DNA methylation on development and evolution in methodologies used for profiling global DNA methylome in cloned embryos.

Cloning of Monkeys by Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) is a promising method to establish genetically modified monkeys with identical genetic background as models in biomedical research. We have recently cloned monkeys by optimization of the SCNT protocols and inclusion of the epigenetic modulator. Here, we describe the protocol for generation of cloned monkeys by somatic cell nuclear transfer.

Generation of goats by nuclear transfer: a retrospective analysis of a commercial operation (1998-2010).

At LFB USA, Inc., the ultimate use for transgenic cloned goats is for the production of recombinant human protein therapeutics in their milk. This retrospective analysis of the Somatic Cell Nuclear Transfer (SCNT) program, spanning from 1998 to 2010, examined parameters potentially affecting the outcomes and efficiencies in this commercial operation. Over 37,000 + ova were utilized in the SCNT protocol producing a total of 203 cloned goats. Fifty one (51) clones were produced from non-transfected (transgenic and non-transgenic animal donor) cell lines and 152 clones were produced from transfected cell lines. Comparisons and summaries of (a) transfected versus non-transfected cell lines, (b) relationship of SCNT parameters to offspring produced, (c) skin versus fetal cells, (d) fresh versus cryopreserved cells, (e) parameters from all cell lines used versus those producing SCNT offspring, (f) variation among cell sources, (g) methods of SCNT parturition management and effects on live offspring, and lastly (h) SCNT variation by program are reported. Findings indicate that (a) non-transfected cell lines were more efficient versus transfected cell lines in generating viable cloned offspring on a per reconstructed embryo transferred basis, (b) transfected fetal fibroblasts had improved efficiency versus transfected skin fibroblasts, (c) the percentage of non-transfected cell lines that produced offspring was statistically higher than transfected cell lines, (d) and induction of parturition improved the percentage of viable offspring. In summary, this retrospective analysis on the SCNT process has identified certain parameters for improved efficiency in producing viable cloned goats in a commercial setting.

Lessons Learned from Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) has been an area of interest in the field of stem cell research and regenerative medicine for the past 20 years. The main biological goal of SCNT is to reverse the differentiated state of a somatic cell, for the purpose of creating blastocysts from which embryonic stem cells (ESCs) can be derived for therapeutic cloning, or for the purpose of reproductive cloning. However, the consensus is that the low efficiency in creating normal viable offspring in animals by SCNT (1–5%) and the high number of abnormalities seen in these cloned animals is due to epigenetic reprogramming failure. In this review we provide an overview of the current literature on SCNT, focusing on protocol development, which includes early SCNT protocol deficiencies and optimizations along with donor cell type and cell cycle synchrony; epigenetic reprogramming in SCNT; current protocol optimizations such as nuclear reprogramming strategies that can be applied to improve epigenetic reprogramming by SCNT; applications of SCNT; the ethical and legal implications of SCNT in humans; and specific lessons learned for establishing an optimized SCNT protocol using a mouse model.

Combined refinements to somatic cell nuclear transfer methods improve porcine embryo development.

The discovery of how to utilize CRISPR (clustered, regularly interspaced, short, palindromic repeats)-Cas (CRISPR-associated) systems for genome modification has accelerated development of the field of genome editing, especially in large animals such as pigs. The low efficiency of somatic cell nuclear transfer (SCNT) is now becoming a major obstacle in the production of genome-edited animals via cell-mediated approaches and improving efficacy of this technique is crucial. In this study, we propose a few simple modifications to a zona-free SCNT protocol that are effective to produce numerous high-quality blastocysts. To refine the SCNT protocol we modified the following steps/factors: 1) culture medium for SCNT embryos, 2) chemical treatment to prevent precocious activation of the manipulated/reconstructed oocytes and 3) donor cell serum starvation treatment. Although changes in each of these steps only resulted in small improvements, the combination of all modifications altogether significantly enhanced developmental competence of SCNT embryos. Our modified method yielded approximately three times greater blastocyst formation rates. Moreover, resulting blastocysts had roughly twice as many cells as compared to blastocysts produced by the conventional SCNT method. With these significant in vitro improvements, our refined SCNT method is potentially suited for use in the production of genome edited pigs.

24 Evaluation of Latrunculin A for the Activation of Hand-Made Cloning (HMC) Porcine Embryos

Somatic cell nuclear transfer (SCNT) is an important biotechnological tool. However, production rates of viable offspring remain low. One possible cause of this low efficiency is chromosomal losses during early activation process (Liu et al. 2015 Cell. Reprogram. 17, 463–471). The use of actin inhibitors that block second polar body extrusion during activation protocols might be a strategy to avoid such losses. The objective of this work was to compare the efficiency of the use of 2 actin inhibitors during the activation of hand-made cloning (HMC) porcine embryos. One of the compounds used was latrunculin A (LatA), which joins directly to actin monomers, preventing their assembly to the filaments. The other was cytochalasin B (CB), which is commonly used for activation protocols. It binds to the growing actin filaments and prevents their elongation. For this purpose, in vitro-matured cumulus–oocyte compexes were deprived of their cumulus and zonae pellucidae cells by mechanical and enzymatic treatments. Oocytes were randomly distributed in 2 experimental groups (HMC) and 2 parthenogenetic control groups (PA). For HMC groups, oocytes were bisected using a microblade and the resulting hemioocytes were stained with Hoechst 33342 and observed under UV light to identify those that had lost the metaphase II plate. Adult skin fibroblasts from primary cultures were used as nuclear donors. For nuclear transfer, 2 hemicytoplasts were fused to a donor cell by an electric pulse of 1.42 kV/cm for 30 μs. After 2 h of nuclear reprogramming, the reconstituted embryos were activated by an electric pulse of 1.2 kV/cm for 80 μs and incubated with cycloheximide (CHX, 10 μg mL−1 , 3 h) in combination with one of the actin inhibitors: LatA 2 μM (CHX-LatA goup) or CB 2.5 μg mL−1 (CHX-CB group). The PA groups were subjected to the same activation treatments (PA-CHX+LatA and PA-CHX+CB groups). All embryos were cultured in SOFaa medium, using an adaptation of the well-of-the-well (WOW) system (microwells), in a humidified atmosphere with 5% CO2 in air at 39°C. Cleavage, morulae, and blastocysts rates were evaluated at Days 2, 4, and 7-8, respectively. At least 3 replicates were performed per group. Results are presented in Table 1. Our results demonstrate that the production of embryos by HMC activated with CHX-LatA is as efficient as that with CHX-CB, the protocol currently used in SCNT protocols. Further research is needed to study its effect on chromosomal complements and long-term development. Table 1.Effect of activation with cycloheximide (CHX) and latrunculin A (LatA) on in vitro development of hand-made cloning (HMC) porcine embryos (% ± SD in parentheses)

Comparison of Developmental Efficiency of Murine Somatic Cell Nuclear Transfer Protocol

The Somatic cell nuclear transfer (SCNT) method can be applied to various fields such as species conservation, regenerative medicine, farming industries and drug production. However, the efficiency using SCNT is very low for many reasons. One of the troubles of SCNT is that it is highly dependent on the researcher’s competence. For that reason, four somatic cell nuclear injection methods were compared to evaluate the effect of hole-sealing process and existence of cytochalasin B (CB) on efficiency of murine SCNT protocol. As a results, the microinjection with the hole-sealing process, the oocyte plasma membrane is inhaled with injection pipette, in HCZB with CB was presented to be the most efficient for the reconstructed in SCNT process. In addition, we demonstrated that the oocytes manipulated in Hepes-CZB medium (HCZB) with CB does not affect the developmental rate and the morphology of the blastocyst during the pre-implantation stage. For this reason, we suggest the microinjection involving hole-sealing in HCZB with CB could improve SCNT process efficiency.

Effects of Trichostatin A and PXD101 on the In Vitro Development of Mouse Somatic Cell Nuclear Transfer Embryos.

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with aberrant epigenetic nuclear reprogramming. It has been demonstrated that treatment with histone deacetylase inhibitors (HDACis) enhances developmental potential of SCNT embryos. Previous studies in many species revealed that treatment of SCNT embryos with trichostatin A (TSA)-an HDACi-significantly enhances the in vitro development of SCNT embryos. In this study, we compared two different SCNT protocols with TSA and investigate, for the first time, the effect of another new HDACi, PXD101 (belinostat), on in vitro development of mouse SCNT embryo. Rates of blastocyst development in mouse SCNT embryos treated with either 5 nM TSA during (6 hours) and after (4 hours) activation (39.1%) or with 50 nM PXD101 during (6 hours) and after (4 hours) activation (40.2%) were significantly higher than those of nontreated SCNT embryos (11.5%) and both treatments also significantly improved the subsequent establishment of NT-ESCs in comparison with the nontreated group (38.1% and 40.9% vs. 11.8%). In conclusion, we optimized the TSA concentration and treatment timing and, for the first time, investigated the effect of PXD101 on mouse development of SCNT embryos and establishment of NT-ESCs.

A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.

A Simplified Approach for Oocyte Enucleation in Mammalian Cloning

Despite its success in almost all farm and laboratory animals, somatic cell nuclear transfer (SCNT) is still a low-efficiency technique. In this investigation, we determined the impact of each enucleation step on oocyte viability (assessed by parthenogenetic activation): Hoechst (HO) staining, cytochalasin B, ultraviolet (UV) exposure, and demecolcine. Our data showed that of all the factors analyzed, UV exposure impaired oocyte development (cleavage, 59% for untreated oocytes vs. 8% UV exposed; blastocyst stage, 32% untreated vs. 0% UV exposed). A minor toxicity was detected following demecolcine treatment (cleavage, 62%; blastocyst stage, 13%). Next, we compared HO/UV (canonical) and demecolcine-assisted enucleation (DAE), with a straight removal of metaphase chromosomes without any chemical or physical aid (straight enucleation). DAE improved the preimplantation development of sheep cloned embryos compared to HO/UV enucleation (cleavage, 38% vs. 19%; blastocysts, 17% vs. 4%), yet straight enucleation resulted in the highest cleavage and blastocysts rates (61% and 30%, respectively). We concluded that: (1) UV exposure harms sheep oocyte and embryo development; (2) DAE may represent an alternative approach, especially for unskilled operators; and (3) straight enucleation remains, in our estimation, the most reliable and least harmful protocol for SCNT.

Nuclear transfer technique affects mRNA abundance, developmental competence and cell fate of the reconstituted sheep oocytes

The effect of technical steps of somatic cell nuclear transfer (SCNT) on different aspects of cloned embryo development was investigated in sheep. In vitro-matured oocytes were enucleated in the presence or absence of zona and reconstituted by three different SCNT techniques: conventional zona-intact (ZI-NT), standard zona-free (ZF-NT) and intracytoplasmic nuclear injection (ICI-NT). Stepwise alterations in nuclear remodeling events and in mRNA abundances, throughput and efficiency of cloned embryo development and cell allocation of the resulted blastocysts were assessed. Early signs of nuclear remodeling were observed as soon as 2 h post-reconstitution (hpr) for fusion-based methods of nuclear transfer (ZI-NT and ZF-NT) but were not observable until 4 hpr with the ICI-NT method. The relative mRNA abundances of HSP90AA1 (HSP90), NPM2 and ATPase genes were not affected by i) presence or absence of zona, ii) oocyte enucleation method and iii) nuclear transfer method. After reconstitution, however, the relative mRNA contents of POU5F1 (OCT4) with the ZI-NT and ZF-NT methods and of PAPOLA (PAP) with ZF-NT were significantly lower than those for the ICI-NT method. Zona removal doubled the throughput of cloned blastocyst development for the ZF-NT technique compared with ZI-NT and ICI-NT. Cleavage rate was not affected by the SCNT protocol, whereas blastocyst yield rate in ICI-NT technique (17.0±1.0%) was significantly (P<0.05; ANOVA) higher than in ZF-NT (7.1±1.5%) but not in the ZI-NT group (11.2±3.3%). Despite the similarities in total cell number, SCNT protocol changed the distribution of cells in the blastocysts, as ZF-NT-cloned blastocysts had significantly smaller inner cell mass than ZI-NT. These results indicate that technical aspects of cloning may result in the variety of cloning phenotypes.

27 PSAMMAPLIN A INCREASES DEVELOPMENT AND QUALITY OF MOUSE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Among the many biological and technical factors affecting the success rate of mouse somatic cell nuclear transfer (SCNT), faulty reprogramming of the differentiated donor nucleus to a totipotent embryonic state by the recipient oocyte seems key. Accordingly, treatment of SCNT embryos with epigenetic modifiers such as valproic acid (VPA), a histone deacetylase inhibitor (HDACi), enhances cloning efficiency. Psammaplin A (PsA) is a natural and potent DNA methyltransferase inhibitor and HDACi that has never been used in nuclear reprogramming studies. The purpose of our study was to determine the effect of PsA on the development and quality of mouse SCNT embryos, and to compare it to that of VPA. To this aim, mechanically enucleated oocytes from B6CBAF1 female mice were reconstructed with cumulus cell nuclei, activated, and cultured in the presence of the epigenetic modifier. Embryos that reached the blastocyst stage were differentially stained for counting inner cell mass (ICM) and trophectoderm cells. Alternatively, 2-cell embryos were transferred to CD1 recipient females to assess full-term development. In a first set of experiments, embryos were exposed to different concentrations of PsA (5, 10, and 20 µM) or VPA (2 and 4 mM) for 1 to 2 h after reconstruction and 6 h of activation (total 8–9 h). We found that 10 µM PsA and 2 mM VPA significantly increased blastocyst rates (37.3 and 31 v. 23.3% for the control group), although no differences were found in blastocyst quality (10.4–13.6 ICM cells). In a second set of experiments, we studied the effect of treatment duration by incubating the embryos in 10 µM PsA or 2 mM VPA for 8 to 9, 16, or 24 h after reconstruction. With PsA, all treatments showed equivalent blastocyst rates (35.2–43.3%), which were significantly higher than in the control group (20%), but only treatments for 16 and 24 h yielded blastocysts with significantly higher numbers of ICM cells (16.3 and 18.5 v. 10 for the control group). With VPA, treatments for 8 to 9 h and 16 h were equivalent in terms of blastocyst rates (34.0 and 32.5%) and significantly higher than the control group, but only VPA 16 h yielded blastocysts with a significantly higher number of ICM cells (15.6). In a third set of experiments, we studied the full-term development of embryos treated with 10 µM PsA or 2 mM VPA for 16 h and we found that both treatments, but especially the PsA treatment, resulted in higher birth rates than those obtained in the control group, although the differences were not statistically significant (1.79 and 0.86 v. 0.46%). Finally, when the actin polymerization inhibitor latrunculin A was used instead of cytochalasin B in the SCNT protocol during oocyte micromanipulation and activation, we obtained a 3-fold increase in the birth rate of embryos treated with PsA (5.29%). In conclusion, PsA enhances development and quality of mouse SCNT embryos, to a greater extent than VPA, and when combined with the use of latrunculin A instead of cytochalasin B, it results in an 11.5-fold increase in full-term development. Support from MEC AGL-2011-23784, 2009-SGR-282, and PIF-UAB Fellowships is acknowledged.

Somatic Cell Nuclear Transfer Cloning: Practical Applications and Current Legislation

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

Comparison between the Effects of Valproic Acid and Trichostatin A on the In Vitro Development, Blastocyst Quality, and Full-Term Development of Mouse Somatic Cell Nuclear Transfer Embryos

Reprogramming of differentiated nuclei into a totipotent embryonic state following somatic cell nuclear transfer (SCNT) is not efficient. Previous studies in the hybrid B6D2F1 mouse strain revealed that a transient treatment of the SCNT embryos with the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) significantly enhance the potential of the cloned embryos to develop in vitro and to term. Here, we compare two different SCNT protocols with TSA and explore, for the first time, the effect of another HDACi, valproic acid (VPA), on the in vitro development, blastocyst quality, and full-term development of mouse B6CBAF1 cloned embryos. Rates of blastocyst development in SCNT embryos treated with either 5 nM TSA during and after activation (31.8%) or with 100 nM TSA or 2 mM VPA before and during activation (34.5 and 38.3%, respectively) were clearly superior to those of nontreated SCNT embryos (22.9-25.1%). These increased in vitro development rates of the HDACi-treated embryos were correlated with an increased level of histone H3 lysine 14 acetylation and an improved blastocyst quality, as judged by the increased number of total and ICM cells in comparison to the nontreated embryos (30-35% increase). Treatment of SCNT embryos with TSA or VPA also allowed the obtention of viable cloned mice, whereas none could be produced from untreated SCNT embryos. In conclusion, we have demonstrated for the first time that VPA can improve the in vitro and full-term development of B6CBAF1 SCNT embryos, at a similar level as TSA. Our findings may open new opportunities to improve cloning efficiencies in other mouse strains or species.