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1.
Phytopathology ; 110(1): 80-84, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31631804

RESUMO

The pepper cultivar Yellow Lantern, one of the spiciest pepper varieties, is a local germplasm of Capsicum chinense, cultivated exclusively on Hainan Island, China. However, this variety is susceptible to viral diseases that severely affect its production. In this study, we report that pepper veinal mottle virus (PVMV) is associated with foliar chlorosis and rugosity symptoms in Yellow Lantern. To verify this correlation, we constructed a full-length cDNA clone of a PVMV isolate named HNu. The virus progeny derived from the cDNA clone replicated and moved systemically in the pepper, inducing the same symptoms as those induced by PVMV-HNu in Yellow Lantern peppers in the field. The results support that PVMV-HNu is the causal agent of foliar chlorosis and rugosity disease in Yellow Lantern. This knowledge will help in the diagnosis and prevention of disease caused by PVMV. Furthermore, the cDNA clone serves as a reverse genetic tool to study the molecular pathogenesis of PVMV.


Assuntos
Capsicum , Doenças das Plantas , Potyvirus/genética , Capsicum/virologia , China , Clonagem de Organismos , DNA Complementar/genética , Doenças das Plantas/virologia , Potyvirus/fisiologia
2.
Yi Chuan ; 41(12): 1099-1109, 2019 Dec 20.
Artigo em Chinês | MEDLINE | ID: mdl-31857281

RESUMO

Somatic cell nuclear transfer (SCNT) is the only reproductive engineering technique that can confer genomic totipotency on somatic cell. SCNT is of great significance for animal germplasm conservation, animal husbandry development, and biomedical research. Although many research advances have been made in this technology, the developmental rate of SCNT mammalian embryos is very low, which seriously limits the application of SCNT in animal husbandry and biomedicine. The primary reason for the low efficiency of cloned embryos is somatic cell reprogramming errors or incomplete reprogramming. These errors or incompleteness present as the abnormal expression of imprinted gene Xist, abnormal DNA methylation, and abnormal histone modification. In this review, we summarize the main factors that influence the low development efficiency of mammalian cloned embryos to provide theoretical reference for the research and practice of improving somatic cell cloning efficiency.


Assuntos
Reprogramação Celular , Epigênese Genética , Técnicas de Transferência Nuclear , Animais , Clonagem de Organismos , Metilação de DNA , Embrião de Mamíferos , Desenvolvimento Embrionário , Mamíferos
3.
Rev. bioét. derecho ; (47): 141-157, nov. 2019.
Artigo em Espanhol | IBECS | ID: ibc-184871

RESUMO

La clonación y transgénesis animal son prácticas biotecnológicas en auge, para nada exentas de problemáticas éticas en lo que respecta al uso que hacen de los animales no humanos. En este artículo se examinan los diversos ámbitos de aplicación de la clonación animal (médico-farmacéutico, industria alimentaria, recreación de especies extintas, clonación de animales de compañía e industria artística y deportiva) y se revisan los principales argumentos éticos que cuestionan la clonación y la transgénesis animal desde una perspectiva antiespecista. Esta perspectiva sostiene que los animales no humanos son merecedores de consideración moral como sujetos de vidas significativas, y no únicamente como medios para la realización de fines humanos


La clonació i transgènesi animal són pràctiques biotecnològiques creixents i no exemptes de problemàtiques ètiques pel que fa a l'ús que fan dels animals no humans. En aquest article s'examinen els diversos àmbits d'aplicació de la clonació animal (metge-farmacèutic, indústria alimentària, recreació d'espècies extintes, clonació d'animals de companyia i indústria artística i esportiva) i es revisen els principals arguments ètics que qüestionen la clonació i la transgènesi animal des d'una perspectiva antiespecista. Aquesta perspectiva sosté que els animals no humans són mereixedors de consideració moral com a subjectes de vides significatives, i no únicament com a mitjans per a la realització de finalitats humanes


Animal cloning and animal transgenesis are growing biotechnological practices, not at all exempt from ethical problems regarding the use they make of non-human animals. This article examines the different areas of application of animal cloning (medical-pharmaceutical, food industry, recreating of extinct species, cloning of companion animals and the art and sport industries) and reviews the main ethical arguments that question cloning and animal transgenesis from an antispeciesist perspective. This perspective argues that non-human animals deserve moral consideration as subjects of meaningful lives, and not only as means for the achievement of human ends


Assuntos
Animais , Clonagem de Organismos/ética , Clonagem de Organismos/veterinária , Biotecnologia/ética , Direitos dos Animais/legislação & jurisprudência , Sistemas CRISPR-Cas , Proteína 9 Associada à CRISPR , Pesquisa Biomédica/ética , Animais de Laboratório
5.
Anim Reprod Sci ; 208: 106125, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31405460

RESUMO

Somatic cell nuclear transfer (SCNT) is an important technique for producing cloned animals. It, however, is inefficient when there is use of SCNT for cloned animal production. Cytoplasm injection cloning technology (CICT) was developed to overcome the inefficiencies of SCNT use of this purpose. The use of CICT involves additional cytoplasm fusing with enucleated oocytes to restore the cytoplasmic volume, thus improving the in vitro developmental competence and quality of cloned embryos. In this study, there was application of CICT in cats to improve the in vitro developmental competence of cloned embryos, as well as the production of the offspring. The results of this study were that fusion rate of the cloned embryos with use of the CICT method was greater than that with SCNT (80.0 ± 4.8% compared with 67.8 ± 11.3%, respectively), and more blastocysts developed with use of CICT than SCNT (20.0 ± 2.0% compared with 13.5 ± 5.0%, respectively). The 62 cloned embryos that were produced with use of CICT were transferred into five estrous synchronized recipients, and 151 cloned embryos produced using SCNT were transferred to 13 estrous-synchronized recipients. After the embryo transfer, there was birth from surrogate mothers of one live-born kitten that resulted using SCNT compared with three live-born kittens using CICT. The number of CICT-cloned embryos born was greater than that of SCNT-cloned embryos (4.8 ± 2.3% compared with 0.7 ± 1.3%, P < 0.05). These results indicate that the CICT technique can be used to produce cloned kittens, including endangered feline species.


Assuntos
Gatos , Clonagem de Organismos/veterinária , Citoplasma , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/fisiologia , Animais , Técnicas de Cultura Embrionária/veterinária , Transferência Embrionária/veterinária , Espécies em Perigo de Extinção , Feminino , Técnicas de Transferência Nuclear/veterinária , Gravidez
6.
Anim Reprod Sci ; 208: 106136, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31405474

RESUMO

Hand-made cloning (HMC) is a method of choice for somatic cell nuclear transfer (SCNT). There is 20% to 50% of cytoplasm lost during manual enucleation of oocytes with HMC. To compensate, two enucleated demicytoplasts, instead of one, are fused with each donor cell, which leads to cytoplasm pooling from two different demicytoplasts. In this study, effects of using one, instead of two demicytoplasts (controls) was examined, for production of embryos using HMC. Use of one demicytoplast decreased blastocyst development (12.7 ±â€¯1.98% compared with 47.6 ±â€¯3.49%, P < 0.001), total cell number (TCN, 167.6 ± 14.66 compared with 335.9 ± 58.96, P < 0.01), apoptotic index (2.11 ± 0.38 compared with 3.43±0.38, P < 0.05) but did not significantly alter inner cell mass:trophectoderm cell number ratio (0.17 ± 0.01 compared with 0.19 ± 0.02) and the global content of H3K9ac and H3K27me3 of blastocysts, compared to controls. There were gene expression alterations in pluripotency- (SOX2 and NANOG but not OCT4), epigenetic- (DNMT1 but not DNMT3a and HDAC1), apoptosis- (CASPASE3 but not BCL-2 and BAX), trophectoderm- (CDX2), development- (G6PD but not GLUT1) and cell cycle check point control-related related genes (P53) compared with controls. Transfer of cloned blastocysts from one demicytoplast (n = 8) to recipients resulted in a live calf birth that after 12 days died whereas, with transfer of control blastocysts (n = 14) there was birth of a healthy calf. In conclusion, use of one, instead of two demicytoplasts for HMC, compromises in vitro developmental competence, and alters expression of several important genes affecting embryo development.


Assuntos
Búfalos/embriologia , Búfalos/genética , Clonagem de Organismos/veterinária , Citoplasma/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , RNA Mensageiro/metabolismo , Animais , Clonagem de Organismos/métodos , Transferência Embrionária/veterinária , Epigênese Genética , RNA Mensageiro/genética
7.
Bioethics ; 33(9): 1085-1090, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31437866

RESUMO

In a recent publication Tom Douglas and Katrien Devolder have proposed a new account of genetic parenthood, building on the work of Heidi Mertes. Douglas and Devolder's account aims to solve, among other things, the question of who are the genetic parents of an individual created through somatic cell nuclear transfer (i.e. cloning): (a) the nuclear DNA provider or (b) the progenitors of the nuclear DNA provider. Such a question cannot be answered by simply appealing to the folk account of genetic parenthood, according to which the genetic parents of an individual are those individuals who produced the egg and sperm, respectively, which fused to create the embryo. It cannot be so as in cloning there is no fertilization as such. In this article I critically examine Douglas and Devolder's new account of genetic parenthood and demonstrate that it is vulnerable to counterexamples that exploit the lack of a condition specifying that genetic parents should cause a child's coming into existence.


Assuntos
Pais , Técnicas de Reprodução Assistida , Criança , Clonagem de Organismos , Fertilização , Humanos , Masculino , Técnicas de Transferência Nuclear
8.
Pesqui. vet. bras ; 39(7): 485-491, July 2019. tab
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1040717

RESUMO

In order for successful extra-uterine adaptation to occur, it is necessary for the neonate to be able to establish its respiratory functions effectively, guaranteeing efficient oxygenation and good vitality. Respiratory disorders are the major cause of death during the neonatal period in cattle, and this mortality is even more significant when it comes to calves originated by in vitro fertilization (FIV) or animal cloning (CA). Blood gas analysis assesses acid-base balance changes effectively, and when associated with the neonate's clinical examination, provides subsidies for accurate diagnosis and early treatment of neonatal maladaptation. The objective of this study was to study neonates born from artificial insemination (IA) and to compare them to calves conceived by FIV and CA, regarding blood gas and clinical examination. For that, 20 AI calves, 15 FIV calves, and 15 cloned calves were evaluated immediately after calving and at 6, 12, 24 and 48 hours of life. At all experimental times, venous blood samples were collected for blood gas and clinical examination was performed. In the postpartum evaluation, Apgar score and column length and respiratory amplitude measurements were used. IVF animals showed no alterations, resembling Group IA calves. The calves from CA showed more pronounced acidosis postpartum than expected physiological acidosis mixed for neonates, with decreasing values of bicarbonate (HCO3-), and base excess (BE) and the increase in carbon dioxide pressure (PCO2) when compared to the other groups. This disorder may have reflected lower mean values of Apgar scores and increased heart and respiratory rates. Intensive follow-up of these neonates is suggested, with monitoring by clinical and hemogasometric examination for early diagnosis of this condition and treatment based on oxygen therapy and bicarbonate replacement.(AU)


Para que ocorra adaptação extra-uterina bem sucedida é necessário que o neonato consiga estabelecer suas funções respiratórias de maneira eficaz, garantindo oxigenação eficiente e boa vitalidade. Distúrbios respiratórios são os maiores causadores de óbito durante o período neonatal em bovinos, e essa mortalidade é ainda mais expressiva quando se trata de bezerros originados por fertilização in vitro (FIV) ou clonagem animal (CA). A hemogasometria avalia alterações do equilíbrio ácido-básico de forma eficaz, e quando associada ao exame clínico do neonato, fornece subsídios para diagnóstico acurado e tratamento precoce da má adaptação neonatal. O objetivo deste trabalho foi estudar recém-nascidos bovinos originados por inseminação artificial (IA) e compará-los a bezerros concebidos por FIV e CA, no que se refere a hemogasometria e exame clínico. Para isso, foram utilizados 20 bezerros IA, 15 bezerros FIV e 15 bezerros clonados que foram avaliados imediatamente após o parto e com 6, 12, 24 e 48 horas de vida. Em todos os momentos experimentais foram colhidas amostras de sangue venoso para hemogasometria e foi realizado o exame clínico. Na avaliação pós-parto foram utilizados escore Apgar e mensurações de comprimento de coluna e amplitude respiratória. Os animais FIV não demonstraram alterações, assemelhando-se aos bezerros do Grupo IA. Os bezerros provenientes de CA apresentaram acidose pós-parto mais acentuada do que a acidose mista fisiológica esperada para neonatos, evidenciada pela diminuição dos valores de bicarbonato (HCO3-) e excesso de bases (EB) e pelo aumento de pressão parcial de dióxido de carbono (PCO2) quando comparados aos demais grupos. Esse distúrbio pode ter refletido em valores médios menores de escore Apgar e no aumento das frequências cardíaca e respiratória. Sugere-se acompanhamento intensivo desses neonatos, com monitoramento por meio do exame clínico e hemogasométrico para diagnóstico precoce dessa condição e tratamento baseado em oxigenioterapia e reposição de bicarbonato.(AU)


Assuntos
Animais , Bovinos , Índice de Apgar , Acidose Respiratória/veterinária , Gasometria/veterinária , Ventilação Voluntária Máxima , Animais Recém-Nascidos/sangue , Inseminação Artificial/veterinária , Fertilização In Vitro/veterinária , Clonagem de Organismos/veterinária
9.
Theriogenology ; 135: 25-32, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31195358

RESUMO

Somatic cell nuclear transfer (SCNT), using transgenic donor cells, is a highly efficient method for producing transgenic embryos. We compared the developmental competence, quality and gene expression of transgenic embryos produced by Hand-made cloning from buffalo fetal fibroblasts (BFFs) containing human insulin gene, with non-transgenic embryos produced from BFFs (Controls). The expression vector (pAcISUBC), constructed by inserting human insulin gene between DNA fragments containing mammary gland-specific buffalo ß-lactoglobulin (buBLG) promoter and terminator buBLG 3'UTR regions into pAcGFP-N1 vector, was used for obtaining the 11 kb insert for transfection of BFFs by nucleofection. Presence of the transgene in embryos was confirmed by examining GFP expression by RT-PCR and immunofluorescence. The blastocyst rate was lower (P < 0.05) for transgenic embryos than for controls (35.7 ±â€¯1.8% vs 48.7 ±â€¯2.4%). The apoptotic index was higher (P < 0.05) for transgenic than for control blastocysts which, in turn, was higher (P < 0.05) than for IVF counterparts (6.9 ±â€¯0.9, 3.8 ±â€¯0.5 and 1.8 ±â€¯0.3, respectively). The total cell number was similar for transgenic and non-transgenic blastocysts (143.2 ±â€¯17.0 and 137.2 ±â€¯7.6, respectively). The expression level of pro-apoptotic genes BAX and BID but not that of CASP3 and CASP9, and cell cycle check point control-related gene P53 was higher (P < 0.05), and that of development- (IGF-1R and G6PD) and pluripotency-related gene NANOG was lower (P < 0.05) in transgenic than in control embryos. The expression level of epigenetic-related genes DNMT1, DNMT3a and HDAC1 and pluripotency-related gene OCT4 was similar in the two groups. The expression level of BAX, BID, CASP9, P53, DNMT1 and DNMT3a was higher (P < 0.05) and that of OCT4, NANOG IGF-1R and G6PD was lower (P < 0.05) in cloned transgenic than in IVF blastocysts whereas, that of CASP3 and HDAC1 was similar between the two groups. In conclusion, these results suggest that transgenic embryos produced by SCNT have lower developmental competence and quality, and altered gene expression compared to non-transgenic embryos.


Assuntos
Búfalos/embriologia , Búfalos/genética , Insulina/genética , Técnicas de Transferência Nuclear/veterinária , Animais , Clonagem de Organismos , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos/metabolismo , Fertilização In Vitro/veterinária , Regulação da Expressão Gênica , Humanos , Organismos Geneticamente Modificados
10.
Theriogenology ; 135: 85-93, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31203092

RESUMO

This document discusses recent developments in cloning of husbandry animals through somatic cell nuclear transfer, particularly with a view on improvements in their efficacy. Commercial developments in North and South America, Australia-New Zealand, and China are noted. The regulations and safety aspects surrounding the use of clones and their offspring for the purpose of food production are discussed. It is generally considered that foods from offspring of clones are no different than similar foods from conventional animals, yet besides safety, also ethical and animal welfare considerations come into play at the policy level. The related topic of detection and traceability of clones is discussed, which covers both molecular and documentary methods.


Assuntos
Clonagem de Organismos/veterinária , Técnicas de Transferência Nuclear/veterinária , Bem-Estar do Animal , Animais , Animais Domésticos , Animais Geneticamente Modificados , Clonagem de Organismos/métodos , Alimentos
11.
J Vet Sci ; 20(3): e31, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31161749

RESUMO

This study examined the effects of a caffeine treatment to improve nuclear reprogramming in porcine cloned embryos. Embryonic development and the expression of genes related to pluripotency (POU5F1, SOX2, NANOG, and CDX2) were compared after caffeine supplementation during manipulation at different concentrations (0, 1.25, 2.5, and 5.0 mM) and after varying the delayed activation time (control, 1, 2, and 4 h) after fusion. Caffeine added to media during manipulation produced a higher rate of development to blastocysts in the 1.25 mM group than in the other concentration groups (22.8% vs. 16.1%, 16.2%, and 19.2%; p < 0.05). When caffeine was added during the 4 h delayed activation, the 1.25 mM caffeine concentration produced a significantly higher rate of development than those in the other 4 h-activation-delayed caffeine concentration groups (22.4% vs. 9.4%, 14.0%, and 11.1%; p < 0.05). On the other hand, no significant improvement over that in the control group was observed when caffeine was supplemented during both the manipulation period and delayed activation period (16.0% vs. 15.2%), respectively. The levels of POU5F1, SOX2, and NANOG expression in blastocysts were significantly higher in the delayed activation caffeine group (4 h, 1.25 mM) than in the control group (1 h, 0 mM; p < 0.05). In conclusion, a caffeine treatment at 1.25 mM during delayed activation for 4 h can improve the preimplantation development of porcine somatic cell nuclear transfer embryos by activating nuclear reprogramming.


Assuntos
Cafeína/farmacologia , Reprogramação Celular/efeitos dos fármacos , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Animais , Blastocisto/metabolismo , Clonagem de Organismos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Transferência Nuclear , Suínos
12.
Zygote ; 27(3): 166-172, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31171048

RESUMO

SummaryRabbits play an important role in people's lives due to their high nutritional value and high-quality hair that can be used as raw material for textiles. Furthermore, rabbits are an important animal model for human disease, as genome-edited animals are particularly valuable for studying gene functions and pathogenesis. Somatic cell nuclear transfer (SCNT) is an important technique for producing genome-edited animals and it has great value in saving endangered species and in clone stem cell therapy. However, the low efficiency of SCNT limits its application, with the selection of suitable rabbit oocytes being crucial to its success. In the present study, we collected oocytes from ovarian follicles and stained them with 26 µM brilliant cresyl blue (BCB). We then matured the oocytes in vitro and used them for SCNT. Comparison of the BCB-positive oocytes with BCB-negative oocytes and the control group showed that the BCB-positive group had a significantly higher maturation rate (81.4% vs. 48.9% and 65.3% for the negative and control groups, respectively), cleavage rate (86.6% vs. 67.9% and 77.9%), blastocyst rate (30.5% vs. 12.8% and 19.6%), total number of blastocysts (90±7.5 vs. 65.3±6.3 and 67.5±5.7), and inner cell mass (ICM)/ trophectoderm (TE) index (42.3±4.2 vs. 30.2±2.1 and 33.9±5.1) (P<0.05). The BCB-positive group had a significantly lower apoptosis index (2.1±0.6 vs. 8.2±0.9 and 6.7±1.1 for the negative and control groups, respectively) (P<0.05). These findings demonstrate that BCB-positive oocytes have a higher maturation ability and developmental competence in vitro, indicating that BCB staining is a reliable method for selecting oocytes to enhance the efficiency of SCNT.


Assuntos
Blastocisto/citologia , Fertilização In Vitro/métodos , Oócitos/citologia , Oxazinas/química , Coloração e Rotulagem/métodos , Animais , Células Cultivadas , Clonagem de Organismos , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Feminino , Técnicas de Maturação in Vitro de Oócitos , Técnicas de Transferência Nuclear , Oócitos/química , Folículo Ovariano/citologia , Coelhos
13.
Zygote ; 27(3): 143-152, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31182178

RESUMO

SummaryMuch effort has been devoted to improving the efficiency of animal cloning. The aim of this study was to investigate the effect of BRG1 contained in Xenopus egg extracts on the development of cloned mouse embryos. The results showed that mouse NIH/3T3 cells were able to express pluripotent genes after treatment with egg extracts, indicating that the egg extracts contained reprogramming factors. After co-injection of Xenopus egg extracts and single mouse cumulus cells into enucleated mouse oocytes, statistically higher pronucleus formation and development rates were observed in the egg Extract- co-injected group compared with those in the no egg extract-injected (NT) group (38-66% vs 18-34%, P<0.001). Removal of BRG1 protein from Xenopus egg extracts was conducted, and the BRG1-depleted extracts were co-injected with single donor cells into recipient oocytes. The results showed that the percentages of pronucleus formation were significantly higher in both BRG1-depleted and BRG1-intact groups than that in the nuclear transfer (NT) group (94, 64% vs 50%, P<0.05). Furthermore, percentages in the BRG1-depleted group were even higher than in the BRG1-intact group (94% vs 64%). More confined expression of Oct4 in the inner cell mass (ICM) was observed in the blastocyst derived from the egg extract-injected groups. However, Nanog expression was more contracted in the ICM of cloned blastocysts in the BRG1-depleted group than in the BGR1-intact group. Based on the present study, BRG1 might not play an essential role in reprogramming, but the factors enhancing pronucleus formation and development of cloned mouse embryos are contained in Xenopus egg extracts.


Assuntos
Blastocisto/citologia , Extratos Celulares/química , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Xenopus laevis/metabolismo , Animais , Blastocisto/metabolismo , Clonagem de Organismos/métodos , Células do Cúmulo/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células NIH 3T3 , Técnicas de Transferência Nuclear , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Oócitos/citologia , Proteínas de Ligação a RNA/genética
14.
Zygote ; 27(3): 111-117, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31182179

RESUMO

SummaryIntraspecific and interspecific cloning via somatic cell nuclear transfer (iSCNT) is a biotechnique with great possibilities for wild mammals because it allows the maintenance of biodiversity by recovering species, nuclear reprogramming for the production of pluripotency-induced cells, and studies related to embryonic development. Nevertheless, many areas in cloning, especially those associated with wild mammals, are still in question because of the difficulty in obtaining cytoplasmic donor cells (or cytoplasts). Conversely, donor cell nuclei (or karyoplasts) are widely obtained from the skin of living or post-mortem individuals and often maintained in somatic cell banks. Moreover, the creation of karyoplast-cytoplast complexes by fusion followed by activation and embryo development is one of the most difficult steps that requires further clarification to avoid genetic failures. Although difficult, cloning different species, such as wild carnivores and ungulates, can be successful via iSCNT with embryo development and the birth of offspring. Thus, novel research in the area that contributes to the conservation of biodiversity and knowledge of the physiology of species continues. The present review presents the failures and successes that occurred with the application of the technique in wild mammals, with the goal of helping future work on cloning via iSCNT.


Assuntos
Clonagem de Organismos/métodos , Transferência Embrionária/métodos , Embrião de Mamíferos/embriologia , Técnicas de Transferência Nuclear , Animais , Núcleo Celular/metabolismo , Embrião de Mamíferos/citologia , Feminino , Mamíferos/classificação , Mamíferos/embriologia , Oócitos/citologia , Oócitos/metabolismo , Gravidez
15.
Zygote ; 27(3): 137-142, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31036094

RESUMO

SummaryChemical oocyte enucleation holds the potential to ease somatic cell nuclear transfer (SCNT), although high enucleation rates remain limited to micromanipulation-based approaches. Therefore, this study aimed to test mitomycin C (MMC) for use in bovine functional chemical oocyte enucleation. Incubation of denuded eggs in 10 µg ml-1 MMC for different periods did not affect most maturation rates (0.5 h: 85.78%A, 1.0 h: 72.77%B, 1.5 h: 83.87%A, and 2.0 h: 82.05%A) in comparison with non-treated controls (CTL; 85.77%A). Parthenogenetic development arrest by MMC was efficient at cleavage (CTL: 72.93%A, 0.5 h: 64.92%A,B, 1.0 h: 60.39%B,C, 1.5 h: 66.35%A,B, and 2.0 h: 53.84%C) and blastocyst stages (CTL: 33.94%A, 0.5 h: 7.58%B, 1.0 h: 2.47%C, 1.5 h: 0.46%C, and 2.0 h: 0.51%C). Blastocysts were obtained after nuclear transfer (NT) using MMC enucleation [NT(MMC): 4.54%B] but at lower rates than for the SCNT control [NT(CTL): 26.31%A]. The removal of the meiotic spindle after MMC incubation fully restored SCNT blastocyst development [NT(MMC+SR): 24.74%A]. Early pregnancies were obtained by the transfer of NT(MMC) and NT(MMC+SR) blastocysts to synchronized recipients. In conclusion, MMC leads to functional chemical oocyte enucleation during SCNT and further suggests its potential for application towards technical improvements.


Assuntos
Blastocisto/efeitos dos fármacos , Núcleo Celular/metabolismo , Clonagem de Organismos/métodos , Mitomicina/farmacologia , Técnicas de Transferência Nuclear/normas , Oócitos/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/farmacologia , Blastocisto/citologia , Blastocisto/metabolismo , Bovinos , Clonagem de Organismos/veterinária , Transferência Embrionária , Desenvolvimento Embrionário , Feminino , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Oócitos/metabolismo , Partenogênese , Gravidez
16.
Int J Dev Biol ; 63(3-4-5): 123-130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058291

RESUMO

Somatic cell nuclear transfer (SCNT) has a broad spectrum of potential applications, including rescue of endangered species, production of transgenic animals, drug production, and regenerative medicine. Unfortunately, the efficiency of SCNT is still disappointingly low. Many factors affecting cloning procedures have been described in several previous reviews; here we review the most effective improvements in SCNT, with a special emphasis on the effect of mitochondrial defects on SCNT embryo/ foetus development, an issue never touched upon before.


Assuntos
Técnicas de Transferência Nuclear/tendências , Animais , Animais Geneticamente Modificados , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologia , Reprogramação Celular/efeitos dos fármacos , Clonagem de Organismos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desmetilases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Técnicas de Transferência Nuclear/efeitos adversos , Protaminas/metabolismo , Medicina Regenerativa , Inativação do Cromossomo X/genética
17.
Int J Mol Sci ; 20(11)2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31142052

RESUMO

Somatic cell nuclear transfer (SCNT) is a very powerful technique used to produce genetically identical or modified animals. However, the cloning efficiency in mammals remains low. In this study, we aimed to explore the effects of vitamin C (Vc)-treated donor cells on cloned embryos. As a result, Vc treatment relaxed the chromatin of donor cells and improved cloned embryo development. RNA sequencing was adopted to investigate the changes in the transcriptional profiles in early embryos. We found that Vc treatment increased the expression of genes involved in the cell-substrate adherens junction. Gene ontology (GO) analysis revealed that Vc treatment facilitated the activation of autophagy, which was deficient in cloned two-cell embryos. Rapamycin, an effective autophagy activator, increased the formation of cloned blastocysts (36.0% vs. 25.6%, p < 0.05). Abnormal expression of some coding genes and long non-coding RNAs in cloned embryos was restored by Vc treatment, including the zinc-finger protein 641 (ZNF641). ZNF641 compensation by means of mRNA microinjection improved the developmental potential of cloned embryos. Moreover, Vc treatment rescued some deficient RNA-editing sites in cloned two-cell embryos. Collectively, Vc-treated donor cells improved the development of the cloned embryo by affecting embryonic transcription. This study provided useful resources for future work to promote the reprogramming process in SCNT embryos.


Assuntos
Ácido Ascórbico/farmacologia , Blastocisto/efeitos dos fármacos , Clonagem de Organismos/métodos , Oócitos/efeitos dos fármacos , Transcriptoma , Vitaminas/farmacologia , Animais , Autofagia , Blastocisto/metabolismo , Bovinos/genética , Células Cultivadas , Feminino , Masculino , Técnicas de Transferência Nuclear , Oócitos/metabolismo
18.
PLoS One ; 14(4): e0213737, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30995216

RESUMO

In this study, a modified method of handmade cloning (m-HMC), which had been originally developed in sheep, was used for somatic cell nuclear transfer (SCNT) in the dromedary camel. The unique feature of m-HMC over current SCNT methods lies in the use of a simple device (a finely drawn micropipette made of Pasteur pipette) for chemically-assisted enucleation of oocytes under a stereomicroscope with improved efficiency and ease of operation. Using this system, the throughput of cloned embryo reconstitution was increased over 2-fold compared to the control SCNT method (c-NT). Stepwise measurement of reactive oxygen species (ROS) revealed that method, steps, and duration of SCNT all influenced oxidative activity of oocytes, but their impact were not similar. Specifically, UV-assisted oocyte enucleation was identified as the major source of ROS production, which explained significantly higher total ROS of reconstituted embryos in c-NT compared to m-HMC. Fusion efficiency (95.3±3.3 vs. 75.4±7.6%) and total efficiency of blastocyst development (22.5±3.0 vs. 14.1±4.3%) were significantly higher in m-HMC compared to c-NT, respectively, and blastocysts of transferable quality were obtained in similar rates (41.9±8.2 vs. 48.0±15.2%, respectively). Significance differences were observed in total cell number (155.3±13.6 vs. 123.6±19.5) and trophectoderm (145±9.5 vs. 114.3±15.2), but not inner cell mass (10.3±4.1 vs. 9.3±5.3) counts between blastocysts developed in c-NT compared to m-HMC, respectively. However, expression of key developmental genes (POU5F1, KLF4, SOX2, MYC, and CDX2) was comparable between blastocysts of both groups. The introduced m-HMC method might be a viable approach for efficient production of dromedary camel clones for research and commercial utilization.


Assuntos
Blastocisto/metabolismo , Camelus/embriologia , Clonagem de Organismos , Desenvolvimento Embrionário , Técnicas de Transferência Nuclear , Animais , Blastocisto/citologia , Feminino
19.
Theriogenology ; 132: 1-11, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30981084

RESUMO

The genome editors CRISPR/Cas9 (clustered regularly interspaced short palindromicrepeats/Cas9 nuclease-null) and TALENs (transcription activator-like effector nuclease) are popularly used for targeted modification of the mammalian genome. To date, few comparative studies have been carried out to investigate the differences between the use of CRISPR/Cas9 and TALENs in genome editing for goat breeding. Here, we compared CRISPR/Cas9 and TALEN technologies at multiple levels for generating a knock out (KO) of the Alpas cashmere goat myostatin (MSTN) gene, which negatively regulates the proliferation and differentiation of skeletal muscle cells. The electrotransfection efficiency observed using CRISPR/Cas9 was 8.1% more than that observed using TALEN for generating MSTN KO cells. In addition, the cutting efficiency of CRISPR/Cas9 for editing exon 1 of the MSTN gene was higher than that of TALENs. However, the off-target effects of the CRISPR/Cas9 system were also higher than those of TALENs. Further, we found that the frequency of obtaining MSTN-/- mutations by CRISPR/Cas9 was 8.5 times higher than that by TALEN. The CRISPR/Cas9-edited colonies involved longer deletions (up to 117 bp) than the TALEN-edited colonies (up to 13 bp). Remarkably, when embryos used to generate cloned goat via somatic cell nuclear transfer were compared, we found that the TALEN MSTN KO embryos easily developed to 8 cells and their cleavage rate was significantly higher than that of CRISPR/Cas9-edited embryos. Finally, we produced a MSTN KO lamb using CRISPR/Cas9, which suggested that a high level of targeted gene modification could be achieved in goat using CRISPR/Cas9. Taken together, our study indicates that although TALEN enables a variety of genome modifications and may have some advantages over CRISPR/Cas9, the latter provides a significant advantage by permitting precise and efficient gene editing. Thus, CRISPR/Cas9 has more potential to become a robust gene-engineering tool for application in the breeding of farm animals.


Assuntos
Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Deleção de Genes , Cabras/genética , Miostatina/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Animais , Sequência de Bases , Clonagem de Organismos , DNA/genética , Embrião de Mamíferos , Edição de Genes , Engenharia Genética , Cabras/embriologia
20.
Med Sci (Paris) ; 35(3): 266-270, 2019 Mar.
Artigo em Francês | MEDLINE | ID: mdl-30931913

RESUMO

Analysing the data recently presented by Jiankui He and assuming that it is authentic shows that the goal of abolishing the expression of CCR5 may have been reached for one of the resulting twins, although this remains to be proven. However, the canonical delta32 mutation has not been achieved. The various preliminary experiments and controls give some confidence that major off-target modifications have not occurred; again, this is difficult to exclude. Clearly, the requirements of perfect technical mastery of the process have not been met, to say nothing of the requirements for complete transparency and full societal approval.


Assuntos
Bioética , Sistemas CRISPR-Cas/genética , Pesquisas com Embriões , Edição de Genes/ética , Má Conduta Científica , Bioética/tendências , China , Clonagem de Organismos/ética , Clonagem de Organismos/métodos , Congressos como Assunto , Resistência à Doença/genética , Pesquisas com Embriões/ética , Deleção de Genes , HIV , Infecções por HIV/genética , Humanos , Recém-Nascido , Invenções/ética , Mutação , Receptores CCR5/genética , Má Conduta Científica/ética
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