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Viable Calves Produced by Somatic Cell Nuclear Transfer Using Meiotic-Blocked Oocytes

BEM, Tiago H. C. De; CHIARATTI, Marcos R.; ROCHETTI, Raquel; BRESSAN, Fabiana F.; SANGALLI, Juliano R.; MIRANDA, Moyses S.; PIRES, Pedro R. L.; SCHWARTZ, Katia R. L.; SAMPAIO, Rafael V.; FANTINATO-NETO, Paulo; PIMENTEL, Jose R. V.; PERECIN, Felipe; SMITH,
Fonte: MARY ANN LIEBERT INC Publicador: MARY ANN LIEBERT INC
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
95.85%
Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585 +/- 34,775 vs. 595,579 +/- 31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179 +/- 45,617 vs. 498,771 +/- 33,231) and blastocysts (816,627 +/- 40...

Contribuição ao estudo da hematologia de bezerros da raça nelore, originados por meio da técnica de transferência nuclear de célula somática (TNCS) - Clonagem; Contribution to the study of hematology of Nelore calves produced by somatic cell nuclear transfer

Komninou, Eliza Rossi
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 22/08/2008 PT
Relevância na Pesquisa
125.99%
A presente pesquisa teve a finalidade de estudar a hematologia de bezerros clonados da raça Nelore, originados por meio da técnica de transferência nuclear de células somáticas (TNCS) por meio da avaliação do quadro eritrocitário, da dinâmica dos tipos de hemoglobina e do metabolismo do ferro destes animais durante o primeiro mês de vida. O delineamento experimental envolveu a colheita de 260 amostras de sangue e soro sanguíneo de 20 bezerros distribuídas nos seguintes momentos: imediatamente após o nascimento, 12 horas após o nascimento, 1 dia de vida, 2 , 3 , 4, 5, 7, 10, 15, 20 e 30 dias de vida. Os animais foram divididos em quatro grupos experimentais: 12 bezerros obtidos por meio da técnica de TNCS pelos laboratórios A e 8, 4 por meio de fertilização in vitro (FIV) e 4 por monta natural (MN). A ocorrência de anemia de grau moderado a grave, do tipo normocítico e normocrômico, foi observada em 100 % (5/5) dos 5 bezerros clonados pelo Laboratório A, enquanto a incidência nos bezerros clonados pelo Laboratório B foi igual a 14,2 % (1/7), nos bezerros obtidos por fertilização in vitro foi igual 50,0 % (2/4) e em bezerros obtidos por monta natural foi igual a 50,0 % (2/4). A avaliação do eritrograma dos bezerros cio nados pelo Laboratório A evidenciou que a anemia instalou-se gradualmente a partir das 12 horas de vida atingindo ao final da primeira semana...

Chemically Assisted Enucleation Results in Higher G6PD Expression in Early Bovine Female Embryos Obtained by Somatic Cell Nuclear Transfer

Saraiva, Naiara Zoccal; Oliveira, Clara Slade; Drummond Tetzner, Tatiane Almeida; de Lima, Marina Ragagnin; de Melo, Danilas Salinet; Meo Niciura, Simone Cristina; Garcia, Joaquim Mansano
Fonte: Mary Ann Liebert, Inc. Publicador: Mary Ann Liebert, Inc.
Tipo: Artigo de Revista Científica Formato: 425-435
ENG
Relevância na Pesquisa
85.99%
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Despite extensive efforts, low efficiency is still an issue in bovine somatic cell nuclear transfer (SCNT). The hypothesis of our study was that the use of cytoplasts produced by chemically assisted enucleation (EN) would improve nuclear reprogramming in nuclear transfer (NT)-derived embryos because it results in lower damage and higher cytoplasm content than conventional EN. For that purpose, we investigated the expression of two X-linked genes: X inactive-specific transcript (XIST) and glucose 6-phosphate dehydrogenase (G6PD). In the first experiment, gene expression was assessed in day-7 female blastocysts from embryonic cell NT (ECNT) groups [conventional, ECNT conv; chemically assisted, ECNT deme (demecolcine)]. Whereas in the ECNT conv group, only one embryo (25%; n = 4) expressed XIST transcripts, most embryos showed XIST expression (75%; n = 4) in the ECNT deme group. However, no significant differences in transcript abundance of XIST and G6PD were found when comparing the embryos from all groups. In a second experiment using somatic cells as nuclear donors, we evaluated gene expression profiles in female SCNT-derived embryos. No significant differences in relative abundance (RA) of XIST transcripts were observed among the groups. Nonetheless...

Differential In Vivo Binding Dynamics of Somatic and Oocyte-specific Linker Histones in Oocytes and During ES Cell Nuclear Transfer

Becker, Matthias; Becker, Antje; Miyara, Faiçal; Han, Zhiming; Kihara, Maki; Brown, David T.; Hager, Gordon L.; Latham, Keith; Adashi, Eli Y.; Misteli, Tom
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em /08/2005 EN
Relevância na Pesquisa
86.09%
The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional...

Epigenetic Reprogramming by Somatic Cell Nuclear Transfer in Primates

Sparman, Michelle; Dighe, Vikas; Sritanaudomchai, Hathaitip; Ma, Hong; Ramsey, Cathy; Pedersen, Darlene; Clepper, Lisa; Nighot, Prashant; Wolf, Don; Hennebold, Jon; Mitalipov, Shoukhrat
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/2009 EN
Relevância na Pesquisa
86.02%
We recently demonstrated that somatic cells from adult primates could be reprogrammed into a pluripotent state by somatic cell nuclear transfer. However, the low efficiency with donor cells from one monkey necessitated the need for large oocyte numbers. Here, we demonstrate nearly threefold higher blastocyst development and embryonic stem (ES) cell derivation rates with different nuclear donor cells. Two ES cell lines were isolated using adult female rhesus macaque skin fibroblasts as nuclear donors and oocytes retrieved from one female, following a single controlled ovarian stimulation. In addition to routine pluripotency tests involving in vitro and in vivo differentiation into various somatic cell types, primate ES cells derived from reprogrammed somatic cells were also capable of contributing to cells expressing markers of germ cells. Moreover, imprinted gene expression, methylation, telomere length, and X-inactivation analyses were consistent with accurate and extensive epigenetic reprogramming of somatic cells by oocyte-specific factors.

Chemically Assisted Enucleation Results in Higher G6PD Expression in Early Bovine Female Embryos Obtained by Somatic Cell Nuclear Transfer

Saraiva, Naiara Zoccal; Oliveira, Clara Slade; Tetzner, Tatiane Almeida Drummond; de Lima, Marina Ragagnin; de Melo, Danilas Salinet; Niciura, Simone Cristina Méo; Garcia, Joaquim Mansano
Fonte: Mary Ann Liebert, Inc. Publicador: Mary Ann Liebert, Inc.
Tipo: Artigo de Revista Científica
Publicado em /10/2012 EN
Relevância na Pesquisa
85.99%
Despite extensive efforts, low efficiency is still an issue in bovine somatic cell nuclear transfer (SCNT). The hypothesis of our study was that the use of cytoplasts produced by chemically assisted enucleation (EN) would improve nuclear reprogramming in nuclear transfer (NT)–derived embryos because it results in lower damage and higher cytoplasm content than conventional EN. For that purpose, we investigated the expression of two X-linked genes: X inactive-specific transcript (XIST) and glucose 6-phosphate dehydrogenase (G6PD). In the first experiment, gene expression was assessed in day-7 female blastocysts from embryonic cell NT (ECNT) groups [conventional, ECNT conv; chemically assisted, ECNT deme (demecolcine)]. Whereas in the ECNT conv group, only one embryo (25%; n=4) expressed XIST transcripts, most embryos showed XIST expression (75%; n=4) in the ECNT deme group. However, no significant differences in transcript abundance of XIST and G6PD were found when comparing the embryos from all groups. In a second experiment using somatic cells as nuclear donors, we evaluated gene expression profiles in female SCNT-derived embryos. No significant differences in relative abundance (RA) of XIST transcripts were observed among the groups. Nonetheless...

Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Ka
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
86%
Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

Reshaping the Transcriptional Frontier: Epigenetics and Somatic Cell Nuclear Transfer

LONG, CHARLES R.; WESTHUSIN, MARK E.; GOLDING, MICHAEL C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
86.02%
Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos...

Potential of Adipose-Derived Mesenchymal Stem Cells and Skeletal Muscle-Derived Satellite Cells for Somatic Cell Nuclear Transfer Mediated Transgenesis in Arbas Cashmere Goats

Ren, Yu; Wu, Haiqing; Ma, Yuzhen; Yuan, Jianlong; Liang, Hao; Liu, Dongjun
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 03/04/2014 EN
Relevância na Pesquisa
86.13%
Somatic cell nuclear transfer is used to generate genetic models for research and new, genetically modified livestock varieties. Goat fetal fibroblast cells (gFFCs) are the predominant nuclear donors in Cashmere goat transgenic cloning, but have disadvantages. We evaluated the potential of goat adipose-derived mesenchymal stem cells (gADSCs) and goat skeletal muscle-derived satellite cells (gMDSCs) for somatic cell nuclear transfer, evaluating their proliferation, pluripotency, transfection efficiency and capacity to support full term development of embryos after additive gene transfer or homologous recombination. gADSCs and gMDSCs were isolated by enzyme digestion and differentiated into neurocytes, myotube cells and insulin-producing cells. Neuron-specific enolase, fast muscle myosin and insulin expression were determined by immunohistochemistry. Following somatic cell nuclear transfer with donor cells derived from gADSCs, gMDSCs and gFFCs, transfection and cloning efficiencies were compared. Red fluorescent protein levels were determined by quantitative PCR and western blotting. 5-Methylcytosine, H4K5, H4K12 and H3K18 were determined immunohistochemically. gADSCs and gMDSCs were maintained in culture for up to 65 passages, whereas gFFCs could be passaged barely more than 15 times. gADSCs and gMDSCs had higher fluorescent colony forming efficiency and greater convergence (20%) and cleavage (10%) rates than gFFCs...

Factors Affecting the Development of Somatic Cell Nuclear Transfer Embryos in Cattle

AKAGI, Satoshi; MATSUKAWA, Kazutsugu; TAKAHASHI, Seiya
Fonte: The Society for Reproduction and Development Publicador: The Society for Reproduction and Development
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
86.07%
Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.

Embryonic Development following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A; Inoue, Azusa; Zhang, Yi
Fonte: Elsevier BV Publicador: Elsevier BV
Tipo: Artigo de Revista Científica
EN_US
Relevância na Pesquisa
126.03%
Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by IVF but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells, and its removal by ectopic expression of the H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency.

Use of adult mesenchymal stem cells isolated from bone marrow and blood for somatic cell nuclear transfer in pigs

Faast, R.; Harrison, S.; Beebe, L.; McIlfatrick, S.; Ashman, R.; Nottle, M.
Fonte: Mary Ann Liebert, Inc. Publishers Publicador: Mary Ann Liebert, Inc. Publishers
Tipo: Artigo de Revista Científica
Publicado em //2006 EN
Relevância na Pesquisa
105.94%
Mesenchymal stem cells (MSCs) isolated from bone marrow were used to examine the hypothesis that a less differentiated cell type could increase adult somatic cell nuclear transfer (SCNT) efficiencies in the pig. SCNT embryos were produced using a fusion before activation protocol described previously and the rate at which these developed to the blastocyst stage compared with that using fibroblasts obtained from ear tissue from the same animal. The use of bone marrow MSCs did not increase cleavage rates compared with adult fibroblasts. However, the percentage of embryos that developed to the blastocyst stage was almost doubled, providing support for the hypothesis that a less differentiated cell can increase cloning efficiencies. As MSCs are relatively difficult to isolate from the bone marrow of live animals, a second experiment was undertaken to determine whether MSCs could be isolated from the peripheral circulation and used for SCNT. Blood MSCs were successfully isolated from four of the five pigs sampled. These cells had a similar differentiation capacity and marker profile to those isolated from bone marrow but did not result in increased rates of development. This is the first study to our knowledge, to report that MSCs can be derived from peripheral blood and used for SCNT for any species. These cells can be readily obtained under relatively sterile conditions compared with adult fibroblasts and as such...

Heteroplasmy in bovine fetuses produced by intra and inter subspecific somatic cell nuclear transfer: Neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood.

Hiendleder, S.; Zakhartchenko, V.; Wenigerkind, H.; Reichenbach, H.D.; Bruggerhoff, K.; Prelle, K.; Brem, G.; Stojkovic, M.; Wolf, E.
Fonte: Soc Study Reproduction Publicador: Soc Study Reproduction
Tipo: Artigo de Revista Científica
Publicado em //2003 EN
Relevância na Pesquisa
86.03%
Varying degrees of mitochondrial DNA (mtDNA) heteroplasmy have been observed in nuclear transfer embryos, fetuses, and offspring, but the mechanisms leading to this condition are unknown. We have generated a clone of 12 bovine somatic cell nuclear transfer fetuses, using nuclear donor cells, recipient oocytes, and recipient heifers with defined mtDNA genotypes, to study nuclear-mitochondrial interactions and the origins of mtDNA heteroplasmy. Embryos were reconstructed from granulosa cells with Bos taurus mtDNA type A and recipient oocytes collected from three different maternal lineages with B. taurus mtDNA type B, B. taurus mtDNA type C, or B. indicus mtDNA. Sequence differences in the control region (CR) of B. taurus mtDNAs ranged from 6 to 11 nucleotides and differences between B. taurus and B. indicus CRs from 45 to 50 nucleotides. Fetuses were recovered from recipient heifers with B. taurus mtDNA type B on Day 80 after nuclear transfer (eight B. taurus A/B, two B. taurus A/C, and two B. taurus A/B. indicus). Agarose gel analysis of the CR by polymerase chain reaction-based restriction fragment length polymorphism failed to detect nuclear donor mtDNA in 11 investigated tissues of 10 viable fetuses and in DNA samples of two fetuses in resorption (one B. taurus A/B and one B. taurus A/C). A more sensitive analysis of 1801 plasmid clones with CR inserts derived from tissues of a B. taurus A/B. indicus fetus detected no or very low levels of heteroplasmy (0.5–0.7%). However...

Somatic cell nuclear transfer in the sheep induces placental defects that likely precede fetal demise

Fletcher, C.; Roberts, C.; Hartwich, K.; Walker, S.; McMillen, I.
Fonte: Bio Scientifica Ltd Publicador: Bio Scientifica Ltd
Tipo: Artigo de Revista Científica
Publicado em //2007 EN
Relevância na Pesquisa
105.92%
The efficiency of cloning by somatic cell nuclear transfer (SCNT) is poor in livestock with ~5% of transferred cloned embryos developing to term. SCNT is associated with gross placental structural abnormalities. We aimed to identify defects in placental histology and gene expression in failing ovine cloned pregnancies to better understand why so many clones generated by SCNT die in utero. Placentomes from SCNT pregnancies (n = 9) and age matched, naturally mated controls (n = 20) were collected at two gestational age ranges (105–134 days and 135–154 days; term = 147 days). There was no effect of cloning on total placental weight. However, cloning reduced the number of placentomes at both gestational ages (105–134 days: control 55.0 ± 4.2, clone 44.7 ± 8.0 and 135–154 days: control 72.2 ± 5.1, clone 36.6 ± 5.1; P < 0.001) and increased the mean individual placentome weight (105–134 days: control 10.6 ± 1.3 g, clone 18.6 ± 2.8 g and 135–154 days: control 6.6 ± 0.6 g, clone 7.0 ± 2.0 g; P < 0.02). Placentomes from cloned pregnancies had a significant volume of shed trophoblast and fetal villous hemorrhage, absent in controls, at both gestational age ranges (P < 0.001) that was shown to be apoptotic by activated caspase-3 immunoreactivity. Consequently...

Cytochalasin B and trichostatin A treatment postactivation improves In Vitro development of porcine somatic cell nuclear transfer embryos

Beebe, L.; McIlfatrick, S.; Nottle, M.
Fonte: Mary Ann Liebert, Inc. Publishers Publicador: Mary Ann Liebert, Inc. Publishers
Tipo: Artigo de Revista Científica
Publicado em //2009 EN
Relevância na Pesquisa
105.93%
Somatic cell nuclear transfer (SCNT) is a useful technique for the production of transgenic pigs that can be used for biomedical research. However, the efficiency of SCNT in pigs is low. In this study, we examined the effect of two postactivation treatments, cytochalasin B (CB) and trichostatin A (TSA), on the in vitro development of porcine SCNT embryos. Treating porcine parthenotes with 7.5μg/mL CB for 3h after electrical activation was effective in preventing the extrusion of the second polar body in 65% of the oocytes compared to 17% in the control group. Treating SCNT embryos with CB for 3h after electrical activation significantly increased the average blastocyst cell number compared to the control group (CB treatment 51, Control 34, p<0.05). Treatment of porcine SCNT embryos with CB for 3h and 50nM TSA for 24h after electrical activation resulted in a threefold increase in blastocyst rate (CB+TSA 64%, CB 20%, p<0.05) and an increase in the average blastocyst cell number (CB+TSA 63, CB 46, p<0.05), compared to CB treatment alone. These results show that treatment with TSA and CB significantly improves the in vitro morphological development and quality of porcine SCNT embryos.; Luke F.S. Beebe, Stephen J. McIlfatrick...

The development and ultrastructure of intergeneric nuclear transfer embryos using ovine ooplasm.

Hamilton, Hamish MacDonald
Fonte: Universidade de Adelaide Publicador: Universidade de Adelaide
Tipo: Tese de Doutorado
Publicado em //2005
Relevância na Pesquisa
106.14%
This thesis encompasses work that aimed to further understand genomic reprogramming, an event crucial in obtaining development in cloned embryos produced by somatic cell nuclear transfer (SCNT). An increasing number of different mammalian species have been cloned using nuclear transfer technology since Dolly the cloned sheep was first successfully produced. However, the biological mechanisms involved in the process of nuclear reprogramming are yet to be fully described. At the centre of this study was an intergeneric SCNT model, which was implemented to determine whether reprogramming factors are conserved across genera. The interaction between donor nucleus and recipient ooplasm was characterised with regard to developmental potential, timing of genome activation, nucleolus formation, and expression of significant proteins. In initial studies, fusion parameters of the intergeneric SCNT procedure were optimised for the ovine cytoplast and porcine donor granulosa cell. Cell fusion and lysis percentages were determined over a range of electrical pulse voltage, duration and repetition. The optimal electrofusion settings were a single DC pulse of 1.5 kV/cm for 20 usec following a 2 sec 400 kHz alignment pulse. In addition, it was demonstrated that ovine oocytes were sensitive to electric stimulation to the extreme that oocyte activation would occur no matter how low the voltage. The practical significance was that it would not be possible to implement a fusion before activation protocol. The ability of the ooplasm of one species to replicate chromosomes and support early embryo cleavage was determined in a preliminary experiment where intergeneric embryos were produced by SCNT using bovine and ovine foetal fibroblasts...

Production of homozygous α-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer; Production of homozygous alpha-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer

Nottle, M.; Beebe, L.; Harrison, S.; McIlfatrick, S.; Ashman, R.; O'Connell, P.; Salvaris, E.; Fisicaro, N.; Pommey, S.; Cowan, P.; d'Apice, A.
Fonte: Blackwell Munksgaard Publicador: Blackwell Munksgaard
Tipo: Artigo de Revista Científica
Publicado em //2007 EN
Relevância na Pesquisa
105.99%
We report here our experience regarding the production of double or homozygous Gal knockout (Gal KO) pigs by breeding and somatic cell nuclear transfer (SCNT). Large White × Landrace female heterozygous Gal KO founders produced using SCNT were mated with Hampshire or Duroc males to produce a F1 generation. F1 heterozygous pigs were then bred to half-sibs to produce a F2 generation which contained Gal KO pigs. To determine the viability of mating Gal KO pigs with each other, one female F2 Gal KO pig was bred to a half-sib and subsequently a full-sib Gal KO. F1 and F2 heterozygous females were also mated to F2 Gal KO males. All three types of matings produced Gal KO pigs. To produce Gal KO pigs by SCNT, heterozygous F1s were bred together and F2 fetuses were harvested to establish primary cultures of Gal KO fetal fibroblasts. Gal KO embryos were transferred to five recipients, one of which became pregnant and had a litter of four piglets. Together our results demonstrate that Gal KO pigs can be produced by breeding with each other and by SCNT using Gal KO fetal fibroblasts.; Mark B. Nottle, Luke F. S. Beebe, Sharon J. Harrison, Stephen M. McIlfatrick, Rodney J. Ashman, Phillip J. O’Connell, Evelyn J. Salvaris, Nella Fisicaro, Sandra Pommey...

Uso de agentes modificadores de cromatina na transferência nuclear de células somáticas em bovinos; Use of chromatin modifying agents in bovine somatic cell nuclear transfer

Sangalli, Juliano Rodrigues
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 13/12/2011 PT
Relevância na Pesquisa
105.96%
Embora a transferência nuclear de células somáticas (TNCS) seja uma ferramenta promissora, seu amplo uso é impedido devido às altas taxas de mortalidade durante o desenvolvimento dos animais clonados. Acredita-se que a reprogramação epigenética anormal seja a principal causa desta baixa eficiência. Nós hipotetizamos que agentes modificadores de cromatina (AMCs) atingindo a acetilação das histonas e a metilação do DNA poderiam alterar a configuração da cromatina e torná-la mais facilmente reprogramável. Deste modo, fibroblastos bovinos foram tratados com 5-aza-2\'-deoxicitidina (AZA) mais tricostatina A (TSA) ou hidralazina (HH) mais ácido valpróico (VPA) enquanto, em outro experimento, zigotos bovinos clonados foram tratados com TSA. O tratamento dos fibroblastos com AZA+TSA ou HH+VPA aumentou a acetilação das histonas, mas não afetou o nível de metilação do DNA. Entretanto, o tratamento com HH+VPA diminuiu a viabilidade/proliferação celular. O uso destas células como doadoras de núcleo não mostrou efeitos positivos sobre o desenvolvimento pré- e pós-implantação. Em relação ao tratamento dos zigotos clonados com TSA, o tratamento destes mostrou um aumento nos padrões de acetilação das histonas...

Interspecies Somatic Cell Nuclear Transfer Is Dependent on Compatible Mitochondrial DNA and Reprogramming Factors

Jiang, Yan; Kelly, Richard; Peters, Amy; Fulka, Helena; Dickinson, Adam; Mitchell, Daniel A.; St. John, Justin C.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 27/04/2011 EN
Relevância na Pesquisa
86.06%
Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore...

Multipotent cell types in primary fibroblast cell lines used to clone pigs using somatic cell nuclear transfer

Harrison, S.J.; Beebe, L.F.S.; Vassiliev, I.; McIlfatrick, S.M.; Nottle, M.B.
Fonte: OMICS Publishing Group Publicador: OMICS Publishing Group
Tipo: Artigo de Revista Científica
Publicado em //2015 EN
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125.99%
We have previously demonstrated that the use of porcine mesenchymal stem cells (MSCs) isolated from the bone marrow can increase the proportion of somatic cell nuclear transfer (SCNT) embryos that develop to the blastocyst stage compared with adult fibroblasts obtained from the same animal. The aim of the present study was to determine if MSCs are also present in primary cultures of adult fibroblasts which are commonly used for cloning live animals. To do this we chose a primary culture of adult fibroblasts that we had previously used to clone pigs. Single cell clones were isolated using low-density plating. After seven days of culture 63% of colonies displayed typical fibroblast morphology, while the remainder appeared cobblestone-like in appearance. Two of the 57 clones that displayed fibroblast morphology differentiated into adipocytes but not chondrocytes or osteocytes (uni-potent clones). Three of the 33 cobblestone-like clones differentiated into chondrocytes only, while 3 differentiated into adipocytes and chondrocytes but not osteocytes (bi-potent clones). One of the bi-potent cobblestone-like clones was then used for SCNT and in vitro development compared with a fibroblast-like clone which did not differentiate. Both cell types produced blastocysts at similar rates. In conclusion we have identified uni-potent and bi-potent cell types in primary cultures of adult fibroblasts used previously to clone live piglets.; Sharon J. Harrison...