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1.
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
2.
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
3.
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
4.
Reprod Domest Anim ; 54(9): 1258-1264, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31283039

RESUMO

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC-derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC-derived SCNT embryos showed a higher (p < .05) developmental competence to the blastocyst stage (47.8%) than FF-derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p < .05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p < .05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell-oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC-derived SCNT embryos showed higher blastocyst formation (48.4%) than FF-derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.


Assuntos
Células-Tronco Germinativas Adultas , Clonagem de Organismos/veterinária , Técnicas de Transferência Nuclear/veterinária , Suínos/embriologia , Animais , Clonagem de Organismos/métodos , Demecolcina/farmacologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Feto/citologia , Fibroblastos/citologia , Moduladores de Tubulina/farmacologia
5.
J Anim Sci ; 97(9): 3786-3794, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31353395

RESUMO

Microchimerism is defined as the presence of a small population of cells or DNA in 1 organism originated from a genetically different organism. It is well established that this phenomenon occurs in humans and mice as cells are exchanged between mother and fetus during gestation. Currently, no information is available about the presence of maternal microchimerism in goats, and the only published study is limited to an evaluation of fetal and fetal-fetal microchimerism in blood samples following natural breeding. In order to determine whether bidirectional fetal-maternal cell or DNA trafficking occurs in goats, we assessed: 1) fetal microchimerism in surrogates that gave birth to somatic cell nuclear transfer (SCNT)-derived transgenic offspring (n = 4), 2) maternal microchimerism following natural breeding of SCNT-derived transgenic does with a nontransgenic buck (n = 4), and 3) fetal-fetal microchimerism in nontransgenic twins of transgenic offspring (n = 3). Neomycin-resistance gene (NEO) gene was selected as the marker to detect the presence of the αMHC-TGF-ß1-Neo transgene in kidney, liver, lung, lymph node, and spleen. We found no detectable maternal or fetal-fetal microchimerism in the investigated tissues of nontransgenic offspring. However, fetal microchimerism was detected in lymph node tissue of one of the surrogate dams carrying a SCNT pregnancy. These results indicate occurrence of cell trafficking from fetus to mother during SCNT pregnancies. The findings of this study have direct implications on the use and disposal of nontransgenic surrogates and nontransgenic offspring.


Assuntos
Quimerismo , Cabras/genética , Animais , Animais Geneticamente Modificados , DNA/genética , Feminino , Feto , Cabras/fisiologia , Técnicas de Transferência Nuclear/veterinária , Parto , Gravidez
6.
EMBO J ; 38(14): e101260, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31304630

RESUMO

Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens.


Assuntos
Perfilação da Expressão Gênica/métodos , Switching de Imunoglobulina , Imunoglobulina G/genética , Células T Matadoras Naturais/metabolismo , Nódulos Linfáticos Agregados/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Galactosilceramidas/administração & dosagem , Galactosilceramidas/imunologia , Interleucina-4/genética , Camundongos , Células T Matadoras Naturais/citologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Técnicas de Transferência Nuclear , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteínas com Domínio T/genética , Vacinação
7.
Reprod Domest Anim ; 54(9): 1195-1205, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31228864

RESUMO

As a natural plant-derived antitoxin, resveratrol possesses several pharmacological activities. This study aimed to evaluate the effects of resveratrol addition on nuclear maturation, oocyte quality during in vitro maturation (IVM) of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer (SCNT). Our experiments showed that the treatment of porcine oocytes with 5 µM resveratrol during IVM resulted in the highest rate of the first polar body extrusion. Treatment of oocytes with resveratrol had no influence on cytoskeletal dynamics, whereas it significantly increased glucose uptake ability compared to the control oocytes. Oocytes matured with 5 µM resveratrol displayed significantly lower intracellular reactive oxygen species (ROS) levels and higher relative mRNA expression levels of the genes encoding such antioxidant enzymes as catalase (CAT) and superoxide dismutase 1 (SOD1). In addition, resveratrol also prevented onset and progression of programmed cell death in porcine oocytes, which was confirmed by significant upregulation of the anti-apoptotic B-cell lymphoma 2 (BCL-2) gene and significant downregulation of the pro-apoptotic BCL2-associated X (BAX) gene. Furthermore, the blastocyst rates and the blastocyst cell numbers in cloned embryos derived from the oocytes that had matured in the presence of 5 µM resveratrol were significantly increased. In conclusion, supplementation of IVM medium with 5 µM resveratrol improves the quality of porcine oocytes by protecting them from oxidative damage and apoptosis, which leads to the production of meiotically matured oocytes exhibiting enhanced developmental potential following SCNT.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Transferência Nuclear/veterinária , Resveratrol/farmacologia , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Blastocisto , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes bcl-2 , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/efeitos dos fármacos , RNA Mensageiro , Espécies Reativas de Oxigênio/metabolismo , Suínos , Proteína X Associada a bcl-2/genética
8.
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
9.
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
10.
BMB Rep ; 52(8): 482-489, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31234956

RESUMO

Reproductive biotechnology has developed rapidly and is now able to overcome many birth difficulties due to infertility or the transmission of genetic diseases. Here we introduce the next generation of assisted reproductive technologies (ART), such as mitochondrial replacement technique (MRT) or genetic correction in eggs with micromanipulation. Further, we suggest that the transmission of genetic information from somatic cells to subsequent generations without gametes should be useful for people who suffer from infertility or genetic diseases. Pluripotent stem cells (PSCs) can be converted into germ cells such as sperm or oocytes in the laboratory. Notably, germ cells derived from nuclear transfer embryonic stem cells (NT-ESCs) or induced pluripotent stem cells (iPSCs) inherit the full parental genome. The most important issue in this technique is the generation of a haploid chromosome from diploid somatic cells. We hereby examine current science and limitations underpinning these important developments and provide recommendations for moving forward. [BMB Reports 2019; 52(8): 482-489].


Assuntos
Células-Tronco Embrionárias/citologia , Células Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Humanos , Técnicas de Transferência Nuclear
11.
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
12.
Nat Commun ; 10(1): 2852, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253764

RESUMO

Cytosine base editors (CBEs) enable programmable C-to-T conversion without DNA double-stranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications. However, all reported cases only involved C-to-T substitution at a single targeted genomic site. Whether C-to-T substitution is effective in multiple sites/loci has not been verified in large animals. Here, by using pigs, an important animal for agriculture and biomedicine, as the subjective animal, we showed that CBEs could efficiently induce C-to-T conversions at multiple sites/loci with the combination of three genes, including DMD, TYR, and LMNA, or RAG1, RAG2, and IL2RG, simultaneously, at the embryonic and cellular levels. CBEs also could disrupt genes (pol gene of porcine endogenous retrovirus) with dozens of copies by introducing multiple premature stop codons. With the CBEs, pigs carrying single gene or multiple gene point mutations were generated through embryo injection or nuclear transfer approach.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Mutação Puntual , Suínos/genética , Desaminase APOBEC-1 , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR , DNA/genética , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Genoma , Técnicas de Transferência Nuclear/veterinária , RNA Guia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
13.
Int J Dev Biol ; 63(6-7): 287-293, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250912

RESUMO

During somatic cell nuclear transfer (SCNT), egg activation is required to initiate embryonic development. In zebrafish cloning, the reconstructed egg is activated by exposing it to hypotonic water. Egg activation using water-only is not capable of activating the same intracellular calcium release as fertilization which is required for proper embryonic development. Here we test whether the use of soluble sperm extract (SSE) can properly modulate the activation of reconstructed eggs during SCNT. We microinjected SSE from genomic-inactivated zebrafish sperm into unfertilized eggs and reconstructed eggs right after somatic cell nuclear transfer. We also evaluated the most effective approach for SSE microinjection. Microinjection of SSE (with 0.68 mg/ml of protein concentration) into non-activated eggs through the micropyle induced parthenogenetic development beyond the blastula stage, whereas all water-only activated eggs failed to enter the cleavage period. Microinjection of SSE at 1 mg/ml of protein concentration into non-activated reconstructed egg improved the developmental rate of cloned embryos in comparison to non-injected control clones. The cumulative survival time of cloned embryos injected with SSE was significantly longer than reconstructed eggs activated following sham injection (P<0.01). No significant difference was found among controls (P=0.32). SSE benefits both parthenogenesis and the survival cloned embryos which have never been reported in zebrafish. Further work is necessary to define the functional component(s) of SSE as well as the physiological pathway, to understand its principle of action and advance the utilization of SSE in cloning.


Assuntos
Embrião não Mamífero/citologia , Desenvolvimento Embrionário/genética , Técnicas de Transferência Nuclear , Óvulo/citologia , Partenogênese , Injeções de Esperma Intracitoplásmicas/métodos , Espermatozoides/citologia , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Embrião não Mamífero/fisiologia , Masculino , Óvulo/fisiologia , Espermatozoides/fisiologia , Peixe-Zebra
14.
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
15.
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
16.
Theriogenology ; 135: 164-168, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31216507

RESUMO

Though blastocyst production in vitro has been successful in several animal species, a culture system to produce viable and normal canine blastocysts in vitro remains to be established. In this study, we report the development of an in vitro culture system for canine preimplantation embryos produced via parthenogenetic activation (PA) and somatic cell nucleus transfer (SCNT). Our results show that the medium developed by us, named "Qingdao Agricultural University's (QAU)-4 medium", successfully breaks the developmental arrest observed at the eight-cell stage in canine embryos grown in other culture systems. The blastocysts produced in QAU-4 displayed normal blastocyst structures, including a clear inner cell mass and blastocyst cavity. We also found that blastocyst formation in PA embryos cultured in QAU-4 medium was quite high, though this was not so in the case of SCNT embryos. However, supplementation of QAU-4 medium with 100 nM of scriptaid caused a sharp increase in blastocyst formation in SCNT embryos. After culture, hatched blastocysts were also observed to successfully adhere to collagen-coated dishes, where further growth and differentiation occurred. To our knowledge, this is the first in vitro canine preimplantation embryo culture system that can successfully produce canine blastocysts.


Assuntos
Blastocisto/fisiologia , Cães/embriologia , Técnicas de Cultura Embrionária/veterinária , Técnicas de Transferência Nuclear/veterinária , Animais , Biomarcadores , Sobrevivência Celular , Células-Tronco Embrionárias/fisiologia , Feminino , Masculino , Partenogênese
17.
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
18.
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
19.
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
20.
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
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