Meta-analysis of melatonin treatment and porcine somatic cell nuclear transfer embryo development

Abstract Porcine somatic cell nuclear transfer (SCNT) plays an important role in many areas of research. However, the low efficiency of SCNT in porcine embryos limits its applications. Porcine embryos contain high concentrations of lipid, which makes them vulnerable to oxidative stress. Some studies have used melatonin to reduce reactive oxygen species damage. At present there are many reports concerning the effect of exogenous melatonin on porcine SCNT. Some studies suggest that the addition of melatonin can increase the number of blastocyst cells, while others indicate that melatonin can reduce the number of blastocyst cells. Therefore, a meta-analysis was carried out to resolve the contradiction. In this study, a total of 63 articles from the past 30 years were analyzed, and six papers were finally selected. Through the analysis, it was found that the blastocyst rate was increased by adding exogenous melatonin. Melatonin had no effect on cleavage rate or the number of blastocyst cells, but did decrease the number of apoptotic cells. This result is crucial for future research on embryo implantation.

Meta-analysis of melatonin treatment and porcine somatic cell nuclear transfer embryo development

Porcine somatic cell nuclear transfer (SCNT) plays an important role in many areas of research. However, the low efficiency of SCNT in porcine embryos limits its applications. Porcine embryos contain high concentrations of lipid, which makes them vulnerable to oxidative stress. Some studies have used melatonin to reduce reactive oxygen species damage. At present there are many reports concerning the effect of exogenous melatonin on porcine SCNT. Some studies suggest that the addition of melatonin can increase the number of blastocyst cells, while others indicate that melatonin can reduce the number of blastocyst cells. Therefore, a meta-analysis was carried out to resolve the contradiction. In this study, a total of 63 articles from the past 30 years were analyzed, and six papers were finally selected. Through the analysis, it was found that the blastocyst rate was increased by adding exogenous melatonin. Melatonin had no effect on cleavage rate or the number of blastocyst cells, but did decrease the number of apoptotic cells. This result is crucial for future research on embryo implantation.(AU)

Nellore cloned calf viability during the postnatal period / Viabilidade de bezerros nelores clonados durante o período pós natal imediato

This study evaluated the viability of Nellore cloned calves derived from somatic cell nuclear transfer (SCNT) and compare their viability with animals of the same breed derived from in vitro fertilization (IVF). Thus, two groups were formed. Group I (GI) consisted of 10 calves derived from SCNT and group II (GII) consisted of 10 calves derived from IVF. The differences detected between the groups were in the physical examination of the respiratory tract in GI, which represented the most common clinical-pathological disturbances. The Apgar index score indicated that 80% of GI animals were depressed and all had pale mucous membranes. Thus, anemia was reported in GI. In GII, this started at 12 h of life and was probably caused by an iron deficiency. Moreover, total calcium and ionized calcium levels were higher in GI immediately after birth. These alterations probably resulted in a high incidence of mortality in GI, reaching 90% of the calves, whereas mortality was only 20% for the calves in GII. In conclusion, cloned calves, which were derived from SCNT, had physiological and metabolic alterations after delivery, leading to a higher mortality rate during the perinatal period.(AU)

O objetivo deste trabalho foi avaliar a viabilidade de bezerros da raça Nelore oriundos da técnica da transferência nuclear de células somáticas (TNCS), no período pós natal imediato, comparando-a com animais desta mesma raça, oriundos de fertilização in vitro (FIV). Para tanto, os animais foram alocados em dois grupos, a saber: Grupo I (GI) - 10 animais frutos de TNCS; e, Grupo II (GII) - 10 animais oriundos de FIV. Nos respectivos bezerros, todos obtidos por cesariana, foram realizadas as avaliações físicas, escore de APGAR, bem como coleta de amostras de sangue nos momentos 0 (ao nascimento), às 2, 4, 6 e 12 horas de vida, a fim de avaliar os resultados de eritrograma, análises bioquímicas e hormonais, comparando-os entre os grupos e momentos. Nos animais que vieram a óbito foi realizada a necropsia para investigar a causa mortis. As diferenças observadas foram em relação aos achados clínico-patológicos, envolvendo, principalmente, o sistema respiratório caracterizado por bradpneia associada à dispneia, e a presença de edema e atelectasia pulmonar observadas no GI. Ademais, após a colostragem notou-se que 80% dos animais avaliados não foram capazes de manter a glicemia sendo mais evidentes nos animais do GII, possivelmente devido à hiperinsulinemia que se manifestou neste grupo ao longo de todo o período experimental. ...(AU)

Epigenetic characterization of the H19/IGF2 locus in calf clones placenta / Caracterização epigenética do locus H19/IGF2 na placenta de bezerros clones

Somatic Cell Nuclear Transfer (SCNT-Cloning) is a promising technique in many areas and is based on genetically identical individuals. However, its efficiency is low. Studies suggest that the leading cause is inadequate epigenetic reprogramming. This study aimed to characterize the methylation pattern of the exon 10 regions of the IGF2 gene and the Imprinting Control Region (ICR) of the H19 gene in the placenta of cloned calves. For this study, female and male cloned calves presenting different phenotypes were used. Genomic DNA from these animals' placenta was isolated, then treated with sodium bisulfite and amplified to the ICR/H19 and IGF2 loci. PCR products were cloned into competent bacteria and finally sequenced. A significant difference was found between controls and clones with healthy phenotypes for the ICR/H19 region. In this region, controls showed a hemimethylated pattern, as predicted in the literature due to this region has an imprinted control, while clones were showed less methylated. For the IGF2, no significant differences were found between controls and clones. These results suggest that different genomic regions in the genome may be independently reprogrammed and that failures in reprogramming the DNA methylation patterns of imprinted genes may be one of the causes of the low efficiency of SCNT.(AU)

A Transferência Nuclear de Células Somáticas (TNCS-Clonagem) é uma técnica promissora em várias áreas, e se baseia na produção de indivíduos geneticamente idênticos. No entanto, sua eficiência é baixa. Estudos sugerem que a principal causa seja uma reprogramação epigenética inadequada. O objetivo desse trabalho é caracterizar o padrão de metilação da região éxon 10 do gene IGF2 e da Região Controladora de Imprinting (ICR) do gene H19 na placenta de bezerros clonados. Para a execução do trabalho foram selecionados clones bovinos fêmeas e machos, apresentando diferentes fenótipos. O DNA da placenta desses animais foi extraído, e em seguida foi tratado com bissulfito de sódio e amplificado para os loci ICR/H19 e IGF2. Os produtos da PCR foram clonados em bactérias competentes e, por fim, sequenciados. Foi encontrada uma diferença significativa entre os controles e os clones com fenótipos saudáveis para a região da ICR/H19. Nesta região, os controles tiveram um padrão hemimetilado, como previsto pela literatura, devido essa região ser imprinted. Enquanto os clones encontravam-se menos metilados. Para a região do éxon 10 do IGF2, não foi encontrada diferença significativa entre controles e clones. Estes resultados sugerem que as diferentes regiões do genoma podem se reprogramar independente umas das outras e que falhas na reprogramação do padrão de metilação do DNA de genes imprinted podem ser uma das causas da baixa eficiência da TNCS.(AU)

Epigenetic characterization of the H19/IGF2 locus in calf clones placenta / Caracterização epigenética do locus H19/IGF2 na placenta de bezerros clones

Somatic Cell Nuclear Transfer (SCNT-Cloning) is a promising technique in many areas and is based on genetically identical individuals. However, its efficiency is low. Studies suggest that the leading cause is inadequate epigenetic reprogramming. This study aimed to characterize the methylation pattern of the exon 10 regions of the IGF2 gene and the Imprinting Control Region (ICR) of the H19 gene in the placenta of cloned calves. For this study, female and male cloned calves presenting different phenotypes were used. Genomic DNA from these animals' placenta was isolated, then treated with sodium bisulfite and amplified to the ICR/H19 and IGF2 loci. PCR products were cloned into competent bacteria and finally sequenced. A significant difference was found between controls and clones with healthy phenotypes for the ICR/H19 region. In this region, controls showed a hemimethylated pattern, as predicted in the literature due to this region has an imprinted control, while clones were showed less methylated. For the IGF2, no significant differences were found between controls and clones. These results suggest that different genomic regions in the genome may be independently reprogrammed and that failures in reprogramming the DNA methylation patterns of imprinted genes may be one of the causes of the low efficiency of SCNT.(AU)

A Transferência Nuclear de Células Somáticas (TNCS-Clonagem) é uma técnica promissora em várias áreas, e se baseia na produção de indivíduos geneticamente idênticos. No entanto, sua eficiência é baixa. Estudos sugerem que a principal causa seja uma reprogramação epigenética inadequada. O objetivo desse trabalho é caracterizar o padrão de metilação da região éxon 10 do gene IGF2 e da Região Controladora de Imprinting (ICR) do gene H19 na placenta de bezerros clonados. Para a execução do trabalho foram selecionados clones bovinos fêmeas e machos, apresentando diferentes fenótipos. O DNA da placenta desses animais foi extraído, e em seguida foi tratado com bissulfito de sódio e amplificado para os loci ICR/H19 e IGF2. Os produtos da PCR foram clonados em bactérias competentes e, por fim, sequenciados. Foi encontrada uma diferença significativa entre os controles e os clones com fenótipos saudáveis para a região da ICR/H19. Nesta região, os controles tiveram um padrão hemimetilado, como previsto pela literatura, devido essa região ser imprinted. Enquanto os clones encontravam-se menos metilados. Para a região do éxon 10 do IGF2, não foi encontrada diferença significativa entre controles e clones. Estes resultados sugerem que as diferentes regiões do genoma podem se reprogramar independente umas das outras e que falhas na reprogramação do padrão de metilação do DNA de genes imprinted podem ser uma das causas da baixa eficiência da TNCS.(AU)

Avaliação dos efeitos da melatonina sobre a produção de embriões bovinos obtidos por fecundação in vitro e transferência nuclear de células somáticas / Evaluation of melatonin effects on the production of bovine embryos obtained by in vitro fertilization and somatic cell nuclear transfer

Na reprodução assistida, o estresse oxidativo pode provocar efeitos deletérios sobre as taxas de produção de embriões. O objetivo do presente estudo foi avaliar o efeito da suplementação com o antioxidante melatonina (MEL) sobre a produção de embriões bovinos obtidos por fecundação in vitro(FIV) e transferência nuclear de células somáticas (TNCS). Os resultados mostraram que a MEL (10-7 M) no meio de maturação ou de cultivo embrionário in vitro não afetou a taxa de clivagem ou de produção de blastocistos na FIV ou na TNCS, e também não afetou a prenhez, a taxa de nascimento ou o peso dos bezerros na TNCS. O tratamento das células doadoras de núcleo com MEL (10-9M) também não levou a um aumento na produção de embriões, prenhez ou taxa de nascimento na TNCS. Assim, conclui-se que a MEL não foi capaz de aumentar a eficiência da reprodução assistida bovina, nas condições experimentais utilizadas.(AU)

n assisted reproduction, oxidative stress can cause deleterious effects on embryo production rates. The aim of the present study was to evaluate the effect of melatonin supplementation (MEL) on the production of bovine embryos obtained by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). The findings of this work demonstrated that melatonin enrichment (10-7 M) of the in vitro maturation (IVM) medium or in the embryo culture medium does not affect both cleavage and blastocyst rates in IVF or SCNT, and does not affect pregnancy, birth rate or weight calf in SCNT either. The treatment of nucleus donor cells with melatonin (10-9M) could not also improve embryo production, pregnancy or birth rate in SCNT. According to these work it is concluded that melatonin is not able toameliorate bovine assisted reproduction efficiency, under experimental conditions used.(AU)

Avaliação dos efeitos da melatonina sobre a produção de embriões bovinos obtidos por fecundação in vitro e transferência nuclear de células somáticas / Evaluation of melatonin effects on the production of bovine embryos obtained by in vitro fertilization and somatic cell nuclear transfer

Na reprodução assistida, o estresse oxidativo pode provocar efeitos deletérios sobre as taxas de produção de embriões. O objetivo do presente estudo foi avaliar o efeito da suplementação com o antioxidante melatonina (MEL) sobre a produção de embriões bovinos obtidos por fecundação in vitro(FIV) e transferência nuclear de células somáticas (TNCS). Os resultados mostraram que a MEL (10-7 M) no meio de maturação ou de cultivo embrionário in vitro não afetou a taxa de clivagem ou de produção de blastocistos na FIV ou na TNCS, e também não afetou a prenhez, a taxa de nascimento ou o peso dos bezerros na TNCS. O tratamento das células doadoras de núcleo com MEL (10-9M) também não levou a um aumento na produção de embriões, prenhez ou taxa de nascimento na TNCS. Assim, conclui-se que a MEL não foi capaz de aumentar a eficiência da reprodução assistida bovina, nas condições experimentais utilizadas.

n assisted reproduction, oxidative stress can cause deleterious effects on embryo production rates. The aim of the present study was to evaluate the effect of melatonin supplementation (MEL) on the production of bovine embryos obtained by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). The findings of this work demonstrated that melatonin enrichment (10-7 M) of the in vitro maturation (IVM) medium or in the embryo culture medium does not affect both cleavage and blastocyst rates in IVF or SCNT, and does not affect pregnancy, birth rate or weight calf in SCNT either. The treatment of nucleus donor cells with melatonin (10-9M) could not also improve embryo production, pregnancy or birth rate in SCNT. According to these work it is concluded that melatonin is not able toameliorate bovine assisted reproduction efficiency, under experimental conditions used.

Uso da clonagem por transferência nuclear na conservação e multiplicação de mamíferos silvestres / Use of cloning by nuclear transfer in the conservation and multiplication of wild mammals

A clonagem por transferência nuclear de células somáticas (TNCS) desempenha um importante papel na conservação e na multiplicação de mamíferos silvestres, especialmente quando associada à TNCS interespecífica. Contudo, a clonagem ainda possui uma baixa eficiência, sendo necessária uma extensa busca por elucidações mais precisas das metodologias empregadas em suas etapas de preparo do carioplastos (células doadoras de núcleo), obtenção dos citoplastos (oócitos receptores), e reconstrução embrionária. Assim, o objetivo desta revisão é abordar os aspectos relevantes das etapas da clonagem aplicadas a mamíferos silvestres, descrevendo seus resultados promissores e avanços alcançados.(AU)

Cloning by somatic cell nuclear transfer (SCNT) plays an important role in the conservation and multiplication of wild mammals, especially when associated with interspecific SCNT. Nevertheless, cloning still has a low efficiency, requiring an extensive search for more precise elucidations of the methodologies used in its stages of preparation of the karyoplasts (nucleus donor cells), production of cytoplasts (receptor oocytes), and embryonic reconstruction. Thus, the purpose of this review is to address the relevant aspects of the cloning steps applied to wild mammals, describing their promising results and advances.(AU)

Uso da clonagem por transferência nuclear na conservação e multiplicação de mamíferos silvestres / Use of cloning by nuclear transfer in the conservation and multiplication of wild mammals

A clonagem por transferência nuclear de células somáticas (TNCS) desempenha um importante papel na conservação e na multiplicação de mamíferos silvestres, especialmente quando associada à TNCS interespecífica. Contudo, a clonagem ainda possui uma baixa eficiência, sendo necessária uma extensa busca por elucidações mais precisas das metodologias empregadas em suas etapas de preparo do carioplastos (células doadoras de núcleo), obtenção dos citoplastos (oócitos receptores), e reconstrução embrionária. Assim, o objetivo desta revisão é abordar os aspectos relevantes das etapas da clonagem aplicadas a mamíferos silvestres, descrevendo seus resultados promissores e avanços alcançados.

Cloning by somatic cell nuclear transfer (SCNT) plays an important role in the conservation and multiplication of wild mammals, especially when associated with interspecific SCNT. Nevertheless, cloning still has a low efficiency, requiring an extensive search for more precise elucidations of the methodologies used in its stages of preparation of the karyoplasts (nucleus donor cells), production of cytoplasts (receptor oocytes), and embryonic reconstruction. Thus, the purpose of this review is to address the relevant aspects of the cloning steps applied to wild mammals, describing their promising results and advances.

Forty years of assisted reproduction research in non-domestic, wild and endangered mammals / 40 anos de pesquisa em reprodução de mamíferos não-domésticos, selvagens e em extinção

For nearly forty years, at three institutions, our team conducted studies to advance the use of ARTs for propagating threatened and endangered mammalian species. The initial studies began in 1978 in the Department of Obstetrics and Gynecology at the University of Alabama in Birmingham, moving in the mid-1980s to The Center for Reproduction of Endangered Species (CREW) at the Cincinnati Zoo and lastly at the Audubon Center for Research in Endangered Species (ACRES) in New Orleans from 1996 to 2015. Our collaborative endeavors with more than two dozen zoological and academic institutions resulted in the births of ET offspring in two non-human primate species, four non-domestic bovid species and seven non-domestic felid species, six of which were interspecies transfers (Table 1). Origin of embryos that were successfully transferred ranged from those flushed from the uterus of mated females (baboon and bovids) to those generated by IVF, ICSI, and SCNT (gorilla and non-domestic felids). Additionally, embryos of five species underwent cryopreservation (baboon, common eland, African wildcat, caracal, black-footed cat) before successful transfer.(AU)

Forty years of assisted reproduction research in non-domestic, wild and endangered mammals / 40 anos de pesquisa em reprodução de mamíferos não-domésticos, selvagens e em extinção

For nearly forty years, at three institutions, our team conducted studies to advance the use of ARTs for propagating threatened and endangered mammalian species. The initial studies began in 1978 in the Department of Obstetrics and Gynecology at the University of Alabama in Birmingham, moving in the mid-1980’s to The Center for Reproduction of Endangered Species (CREW) at the Cincinnati Zoo and lastly at the Audubon Center for Research in Endangered Species (ACRES) in New Orleans from 1996 to 2015. Our collaborative endeavors with more than two dozen zoological and academic institutions resulted in the births of ET offspring in two non-human primate species, four non-domestic bovid species and seven non-domestic felid species, six of which were interspecies transfers (Table 1). Origin of embryos that were successfully transferred ranged from those flushed from the uterus of mated females (baboon and bovids) to those generated by IVF, ICSI, and SCNT (gorilla and non-domestic felids). Additionally, embryos of five species underwent cryopreservation (baboon, common eland, African wildcat, caracal, black-footed cat) before successful transfer.

Thirty years of assisted reproductive technology in the domestic cat: A selected summary / Trinta anos de tecnologia de reprodução assistida no gato doméstico: Um resumo selecionado

The first kittens to be born after embryo transfer (ET) in the cat (Schriver and Kraemer, 1978) were reported four decades ago. The births of kittens after in vitro fertilization (IVF)/ET (Goodrowe et al., 1988) and after embryo cryopreservation/ET (Dresser et al., 1988) were described 10 years later. In an early report it was established that embryo donors had the ability ot exhibit repeated ovarian stimulatory response to multiple gonadotropin treatments (porcine FSH; Dresser et al., 1987). After studies on gonadotropin-induced hyperstimulation of follicular development for recovery and ET of fresh and cryopreserved in vivo derived uterine stage embryos from mated donors (Dresser et al., 1988; Pope et al., 1989), we transitioned into developing methods for in vitro production of domestic cat embryos for the purpose of applying the technology to support conservation of threatened and endangered felid species (Pope et al., 1993). Since then, techniques for the in vitro production of cat embryos have been developed sufficiently to allow births of kittens after transfer of embryos derived by an assortment of in vitro techniques, including cryopreservation (Pope et al., 1994; Gómez et al., 2003a; Pope et al., 2012a, b; Galiguis et al., 2014), intracytoplasmic sperm injection (ICSI; Pope et al., 1998; Gómez et al., 2000; Pope et al., 2012a), gender selection (Pope et al., 2009) and somatic cell nuclear transfer (SCNT) (Gómez et al., 2004; 2008; 2009).(AU)

New tools for cell reprogramming and conversion: Possible applications to livestock

Somatic cell nuclear transfer and iPS are both forms of radical cell reprogramming able to transform a fully differentiated cell type into a totipotent or pluripotent cell. Both processes, however, are hampered by low efficiency and, in the case of iPS, the application to livestock species is uncertain. Epigenetic manipulation has recently emerged as an efficient and robust alternative method for cell reprogramming. It is based upon the use of small molecules that are able to modify the levels of DNA methylation with 5-azacitidyne as one of the most widely used. Among a number of advantages, it includes the fact that it can be applied to domestic species including pig, dog and cat. Treated cells undergo a widespread demethylation which is followed by a renewed methylation pattern induced by specific chemical stimuli that lead to the desired phenotype. A detailed study of the mechanisms of epigenetic manipulation revealed that cell plasticity is achieved through the combined action of a reduced DNA methyl transferase activity with an active demethylation driven by the TET protein family. Surprisingly the same combination of molecular processes leads to the transformation of fibroblasts into iPS and regulate the epigenetic changes that take place during early development and, hence, during reprogramming following SCNT. Finally, it has recently emerged that mechanic stimuli in the form of a 3D cell rearrangement can significantly enhance the efficiency of epigenetic reprogramming as well as of maintenance of pluripotency. Interestingly these mechanic stimuli act on the same mechanisms both in epigenetic cell conversion with 5-Aza-CR and in iPS. We suggest that the balanced combination of epigenetic erasing, 3D cell rearrangement and chemical induction can go a long way to obtain ad hoc cell types that can fully exploit the current exiting development brought by gene editing and animal cloning in livestock production.

New tools for cell reprogramming and conversion: Possible applications to livestock

Somatic cell nuclear transfer and iPS are both forms of radical cell reprogramming able to transform a fully differentiated cell type into a totipotent or pluripotent cell. Both processes, however, are hampered by low efficiency and, in the case of iPS, the application to livestock species is uncertain. Epigenetic manipulation has recently emerged as an efficient and robust alternative method for cell reprogramming. It is based upon the use of small molecules that are able to modify the levels of DNA methylation with 5-azacitidyne as one of the most widely used. Among a number of advantages, it includes the fact that it can be applied to domestic species including pig, dog and cat. Treated cells undergo a widespread demethylation which is followed by a renewed methylation pattern induced by specific chemical stimuli that lead to the desired phenotype. A detailed study of the mechanisms of epigenetic manipulation revealed that cell plasticity is achieved through the combined action of a reduced DNA methyl transferase activity with an active demethylation driven by the TET protein family. Surprisingly the same combination of molecular processes leads to the transformation of fibroblasts into iPS and regulate the epigenetic changes that take place during early development and, hence, during reprogramming following SCNT. Finally, it has recently emerged that mechanic stimuli in the form of a 3D cell rearrangement can significantly enhance the efficiency of epigenetic reprogramming as well as of maintenance of pluripotency. Interestingly these mechanic stimuli act on the same mechanisms both in epigenetic cell conversion with 5-Aza-CR and in iPS. We suggest that the balanced combination of epigenetic erasing, 3D cell rearrangement and chemical induction can go a long way to obtain ad hoc cell types that can fully exploit the current exiting development brought by gene editing and animal cloning in livestock production.(AU)

Thirty years of assisted reproductive technology in the domestic cat: A selected summary / Trinta anos de tecnologia de reprodução assistida no gato doméstico: Um resumo selecionado

The first kittens to be born after embryo transfer (ET) in the cat (Schriver and Kraemer, 1978) were reported four decades ago. The births of kittens after in vitro fertilization (IVF)/ET (Goodrowe et al., 1988) and after embryo cryopreservation/ET (Dresser et al., 1988) were described 10 years later. In an early report it was established that embryo donors had the ability ot exhibit repeated ovarian stimulatory response to multiple gonadotropin treatments (porcine FSH; Dresser et al., 1987). After studies on gonadotropin-induced hyperstimulation of follicular development for recovery and ET of fresh and cryopreserved in vivo derived uterine stage embryos from mated donors (Dresser et al., 1988; Pope et al., 1989), we transitioned into developing methods for in vitro production of domestic cat embryos for the purpose of applying the technology to support conservation of threatened and endangered felid species (Pope et al., 1993). Since then, techniques for the in vitro production of cat embryos have been developed sufficiently to allow births of kittens after transfer of embryos derived by an assortment of in vitro techniques, including cryopreservation (Pope et al., 1994; Gómez et al., 2003a; Pope et al., 2012a, b; Galiguis et al., 2014), intracytoplasmic sperm injection (ICSI; Pope et al., 1998; Gómez et al., 2000; Pope et al., 2012a), gender selection (Pope et al., 2009) and somatic cell nuclear transfer (SCNT) (Gómez et al., 2004; 2008; 2009).

The transformational impact of site-specific DNA modifiers on biomedicine and agriculture

The development of genetically modified livestock has been dependent on incremental technological advances such as embryo transfer, homologous recombination, and somatic cell nuclear transfer (SCNT). This development rate has increased exponentially with the advent of targeted gene modifiers such as zinc finger nucleases, TAL-effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR-Cas). CRISPR-Cas based systems in particular have broad applicability, and have low technical and economic barriers for their implementation. As a result, they are having, and will continue to have, a transformational impact in the field of gene editing in domestic animals. With these advances also comes the responsibility to properly apply this technology so it has a beneficial effect throughout all levels of society.

The transformational impact of site-specific DNA modifiers on biomedicine and agriculture

The development of genetically modified livestock has been dependent on incremental technological advances such as embryo transfer, homologous recombination, and somatic cell nuclear transfer (SCNT). This development rate has increased exponentially with the advent of targeted gene modifiers such as zinc finger nucleases, TAL-effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR-Cas). CRISPR-Cas based systems in particular have broad applicability, and have low technical and economic barriers for their implementation. As a result, they are having, and will continue to have, a transformational impact in the field of gene editing in domestic animals. With these advances also comes the responsibility to properly apply this technology so it has a beneficial effect throughout all levels of society.(AU)

Alternative strategies for nuclear reprogramming in Somatic Cell Nuclear transfer (SCNT)

Twenty Years passed by since the production of Dolly the sheep, but despite significant technical progress has been achieved in the manipulation procedures, the proportion of offspring following transfer of SCNT embryos has remained almost unchanged in farm animals. Remarkable progress has been obtained instead in laboratory animals, particularly by Japanese Groups, in the mouse. However, the nuclear reprogramming strategies tested in mouse do not always work in farm animals, and others are difficult to be implemented, for require complicated molecular biology tools unavailable yet in large animals. In this review we put in contest the previous work done in farm and laboratory animals with recent achievements obtained in our laboratory, and we also indicate a road map to increase the reliability of SCNT procedures.

Alternative strategies for nuclear reprogramming in Somatic Cell Nuclear transfer (SCNT)

Twenty Years passed by since the production of Dolly the sheep, but despite significant technical progress has been achieved in the manipulation procedures, the proportion of offspring following transfer of SCNT embryos has remained almost unchanged in farm animals. Remarkable progress has been obtained instead in laboratory animals, particularly by Japanese Groups, in the mouse. However, the nuclear reprogramming strategies tested in mouse do not always work in farm animals, and others are difficult to be implemented, for require complicated molecular biology tools unavailable yet in large animals. In this review we put in contest the previous work done in farm and laboratory animals with recent achievements obtained in our laboratory, and we also indicate a road map to increase the reliability of SCNT procedures.(AU)

Probability, odds and random chance: the difficult task of modulating the epigenetic profile of cloned embryos

Since the beginning of modern embryology, scientists have wondered about how a small number of totipotent embryonic cells can become an individual with a wide variety of organs and tissues with distinct functions. Also, the idea of generating a cloned animal using a nucleus from a donor cell is not recent. However, it has taken years of research to achieve this goal, especially regarding mechanisms of cell reprogramming required to return a differentiated cell to totipotency. Cloning by somatic cell nuclear transfer (SCNT) has been a valuable tool to understand epigenetic mechanisms related to cellular reprogramming. However, cloning efficiency is still low, with a low percentage of embryos resulting in healthy animals. The high attrition rate is associated with incomplete or abnormal epigenetic reprogramming, such that many cloned embryos have DNA methylation patterns different than controls, resulting in faulty gene expression and subsequent developmental failures. Attempts to improve genome reprogramming by modulation of oocyte quality and/or somatic cell plasticity, thereby increasing cloning efficiency and preventing detrimental effects on development, have proven ineffective. The recent development of DNA editing techniques may facilitate an improved understanding of cellular reprogramming and the role of DNA methylation in development. These novel tools may lead to new means to modulate epigenetic programming and inheritance, and hold great promise to assist in epigenetic remodeling of the donor nucleus. Such strategies are likely to improve the odds for successful cloning.