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1.
Theriogenology ; 177: 151-156, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34700072

RESUMO

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report the telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, the donor cells, and their age-matched naturally produced counterparts by Terminal Restriction Fragment (TRF) length analysis and real-time Q PCR T/S ratio methods. Genomic DNA was extracted from venous blood collected from 6 cloned animals and their age-matched counterparts. Using the southern blot technique, digested DNA was blotted onto a positively charged nylon membrane, and its hybridization was carried out using telomere (TTAGGG)n specific, DIG-labeled hybridization probe (Roche Diagnostics, Germany) at 42 °C for 4 h. Stringent washes were carried out at the same temperature, followed by a chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated three times, and the data, presented as mean ± SEM, were analyzed using a two-sample t-test (MINITAB statistical software, Minitab ltd, CV3 2 TE, UK). No difference was found in the mean telomere length of cloned camels when compared to their naturally produced age-matched counterparts. However, the telomere length was more (P < 0.05) than that of the somatic cells used for producing the SCNT embryos. A moderate positive Pearson correlation coefficient (r = 0.6446) was observed between the telomere lengths estimated by TRF and Q PCR T/S ratio method. In conclusion, this is the first study wherein we are reporting telomere length in naturally produced and cloned dromedary camels produced by somatic cell nuclear transfer. We found that telomere lengths in cloned camels were similar to their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.


Assuntos
Camelus , Clonagem de Organismos , Animais , Clonagem de Organismos/veterinária , Transferência Embrionária/veterinária , Técnicas de Transferência Nuclear/veterinária , Telômero/genética
2.
BMC Plant Biol ; 21(1): 405, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34488640

RESUMO

BACKGROUND: Clones provide a sensitive method for evaluating genotypic stability and detecting genotype-environment (G × E) interactions because of non-additive genetic effects among clones and there being no genetic effect among ramets of an ortet. With this study, we aimed to confirm and expand earlier findings, estimate stability parameters, and provide accurate estimates of clonal repeatabilities and genetic gains for a triploid breeding program of P. tomentosa Carr. RESULTS: Six 5-year-old clonal trials established in Northern China were used to determine the clonal variation, clone × site interactions, and the stability parameters of fiber properties of wood and growth traits. 360 trees from ten hybrid clones were collected from six sites. The clonal and site effects had a highly significant effect (P < 0.001) for all studied traits. While the clone × site interactions had a highly significant effect (P < 0.001) on fiber length (FL), coarseness (C), and tree growth (tree height [H], diameter at breast height [DBH] and stem volume [SV]), and a moderate effect (P < 0.05) on fiber width (FW) and fiber length/width (FL/W). For FL and SV, most of the triploid hybrid clones had higher reaction norms to the improvement in growth conditions and higher phenotypic plasticity. The estimated clonal repeatability of FW (0.93) was slightly higher than for FL (0.89), FL/W (0.83), C (0.91), DBH (0.76), H (0.85), and SV (0.80). Three clonal testing sites were sufficient to estimate quantitative parameters of fiber properties, however, more clonal testing sites would help improve the accuracy of quantitative parameters of the growth traits. CONCLUSIONS: Our results highlight that accurate estimation of quantitative parameters for growth traits in triploid hybrid clones of P. tomentosa requires more clonal testing sites than the fiber properties.


Assuntos
Clonagem de Organismos , Genótipo , Populus/crescimento & desenvolvimento , Populus/genética , Triploidia , Madeira , Interação Gene-Ambiente , Variação Genética , Melhoramento Vegetal/métodos
3.
Int J Mol Sci ; 22(14)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34299109

RESUMO

The domestic goat (Capra aegagrus hircus), a mammalian species with high genetic merit for production of milk and meat, can be a tremendously valuable tool for transgenic research. This research is focused on the production and multiplication of genetically engineered or genome-edited cloned specimens by applying somatic cell nuclear transfer (SCNT), which is a dynamically developing assisted reproductive technology (ART). The efficiency of generating the SCNT-derived embryos, conceptuses, and progeny in goats was found to be determined by a variety of factors controlling the biological, molecular, and epigenetic events. On the one hand, the pivotal objective of our paper was to demonstrate the progress and the state-of-the-art achievements related to the innovative and highly efficient solutions used for the creation of transgenic cloned does and bucks. On the other hand, this review seeks to highlight not only current goals and obstacles but also future challenges to be faced by the approaches applied to propagate genetically modified SCNT-derived goats for the purposes of pharmacology, biomedicine, nutritional biotechnology, the agri-food industry, and modern livestock breeding.


Assuntos
Animais Geneticamente Modificados/genética , Clonagem de Organismos/veterinária , Embrião de Mamíferos/citologia , Engenharia Genética/veterinária , Técnicas de Transferência Nuclear/veterinária , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Cabras
4.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299380

RESUMO

Artificial activation of oocytes is an important step for successful parthenogenesis and somatic cell nuclear transfer (SCNT). Here, we investigated the initiation of DNA synthesis and in vivo development of canine PA embryos and cloned embryos produced by treatment with 1.9 mM 6-dimethylaminopurine (6-DMAP) for different lengths of time. For experiments, oocytes for parthenogenesis and SCNT oocytes were cultured for 4 min in 10 µM calcium ionophore, and then divided into 2 groups: (1) culture for 2 h in 6-DMAP (DMAP-2h group); (2) culture for 4 h in DMAP (DMAP-4h group). DNA synthesis was clearly detected in all parthenogenetic (PA) embryos and cloned embryos incorporated BrdU 4 h after activation in DMAP-2h and DMAP-4h groups. In vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rates of PA embryos in DMAP-2h were 34%, which was significantly higher than those in DMAP-4h (6.5%, p < 0.05). However, in SCNT, there was no significant difference in pregnancy rate (DMAP-2h: 41.6% vs. DMAP-4h: 33.3%) and implantation rates (DMAP-2h: 4.94% vs. DMAP-4h: 3.19%) between DMAP-2h and DMAP-4h. In conclusion, the use of DMAP-2h for canine oocyte activation may be ideal for the in vivo development of PA zygotes, but it was not more effective in in vivo development of canine reconstructed SCNT oocytes. The present study demonstrated that DMAP-2h treatment on activation of canine parthenogenesis and SCNT could effectively induce the onset of DNA synthesis during the first cell cycle.


Assuntos
Adenina/análogos & derivados , Replicação do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Adenina/farmacologia , Animais , Clonagem de Organismos/métodos , Cães , Transferência Embrionária/métodos , Feminino , Técnicas de Transferência Nuclear , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Gravidez
5.
Biomolecules ; 11(6)2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199637

RESUMO

Endogenous retroviruses (ERVs), previously viewed as deleterious relics of ancestral retrovirus infections, are silenced in the vast majority of cells to minimize the risk of retrotransposition. Counterintuitively, bursts of ERV transcription usually occur during maternal-to-zygotic transition (MZT) in preimplantation embryos; this is regarded as a major landmark event in the zygotic genome activation (ZGA) process, indicating that ERVs play an active part in ZGA. Evolutionarily, the interaction between ERVs and hosts is mutually beneficial. The endogenization of retrovirus sequences rewires the gene regulatory network during ZGA, and ERV repression may lower germline fitness. Unfortunately, owing to various limitations of somatic cell nuclear transfer (SCNT) technology, both developmental arrest and ZGA abnormalities occur in a high percentage of cloned embryos, accompanied by ERV silencing, which may be caused by the activation failure of upstream ERV inducers. In this review, we discuss the functions and regulation of ERVs during the ZGA process and the feasibility of temporal control over ERVs in cloned embryos via exogenous double homeobox (DUX). We hypothesize that further accurate characterization of the ERV-rewired gene regulatory network during ZGA may provide a novel perspective on the development of preimplantation embryos.


Assuntos
Clonagem de Organismos , Desenvolvimento Embrionário , Retrovirus Endógenos/metabolismo , Genoma , Técnicas de Transferência Nuclear , Zigoto/metabolismo , Animais
6.
Nat Plants ; 7(7): 899-905, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34211132

RESUMO

Plant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system1, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector subarray plasmids, to assemble DddA-derived cytosine base editor (DdCBE)2 plasmids and used the resulting DdCBEs to efficiently promote point mutagenesis in mitochondria and chloroplasts. Our DdCBEs induced base editing in lettuce or rapeseed calli at frequencies of up to 25% (mitochondria) and 38% (chloroplasts). We also showed DNA-free base editing in chloroplasts by delivering DdCBE mRNA to lettuce protoplasts to avoid off-target mutations caused by DdCBE-encoding plasmids. Furthermore, we generated lettuce calli and plantlets with edit frequencies of up to 99%, which were resistant to streptomycin or spectinomycin, by introducing a point mutation in the chloroplast 16S rRNA gene.


Assuntos
Brassica napus/genética , Clonagem de Organismos/métodos , DNA de Cloroplastos , DNA Mitocondrial , Edição de Genes/métodos , Alface/genética , Melhoramento Vegetal/métodos , Produtos Agrícolas/genética
7.
Theranostics ; 11(15): 7391-7424, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34158857

RESUMO

The normal development and maturation of oocytes and sperm, the formation of fertilized ova, the implantation of early embryos, and the growth and development of foetuses are the biological basis of mammalian reproduction. Therefore, research on oocytes has always occupied a very important position in the life sciences and reproductive medicine fields. Various embryo engineering technologies for oocytes, early embryo formation and subsequent developmental stages and different target sites, such as gene editing, intracytoplasmic sperm injection (ICSI), preimplantation genetic diagnosis (PGD), and somatic cell nuclear transfer (SCNT) technologies, have all been established and widely used in industrialization. However, as research continues to deepen and target species become more advanced, embryo engineering technology has also been developing in a more complex and sophisticated direction. At the same time, the success rate also shows a declining trend, resulting in an extension of the research and development cycle and rising costs. By studying the existing embryo engineering technology process, we discovered three critical nodes that have the greatest impact on the development of oocytes and early embryos, namely, oocyte micromanipulation, oocyte electrical activation/reconstructed embryo electrofusion, and the in vitro culture of early embryos. This article mainly demonstrates the efforts made by researchers in the relevant technologies of these three critical nodes from an engineering perspective, analyses the shortcomings of the current technology, and proposes a plan and prospects for the development of embryo engineering technology in the future.


Assuntos
Clonagem de Organismos , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Técnicas de Transferência Nuclear , Oócitos/metabolismo , Animais
8.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072531

RESUMO

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.


Assuntos
Técnicas de Reprogramação Celular , Clonagem de Organismos , Embrião de Mamíferos , Desenvolvimento Embrionário , Técnicas de Transferência Nuclear , Animais , Apoptose/genética , Biomarcadores , Bovinos , Clonagem de Organismos/métodos , Metilação de DNA , Implantação do Embrião , Epigênese Genética , Feminino , Fibroblastos , Expressão Gênica , Histonas/metabolismo , Gravidez , Sensibilidade e Especificidade , Doadores de Tecidos
9.
Anim Genet ; 52(5): 608-620, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34182591

RESUMO

Cloned animals are prone to abnormal phenotypes such as enlarged tongue, fetal oversize, and progeria. In the present study, whole-genome bisulfite sequencing and mRNA sequencing were performed on tongue and biceps femoris muscles of cloned piglets with and without macroglossia, in an attempt to elucidate the epigenetic causes of the macroglossia phenotype. We identified 14 958 and 18 752 differentially methylated regions in the tongue and biceps femoris muscles, respectively, of macroglossia piglets and these correspond to 4574 and 4772 differentially methylated genes compared with the control group (piglets without macroglossia). Larger methylation difference was found in tongue muscle than in biceps femoris muscle. In total, 114 genes in tongue and 72 genes in biceps femoris muscles were found to be differentially expressed between the two groups. Of these differentially expressed genes in tongue muscle, 31 were also differentially methylated genes, among which DIO3 and ZIC1 were imprinting or predicted imprinting genes. These two and another six overlapping genes (ALDH1A2, MKX, MAB21L2, CA3, RANBP3L, and MYL10) are crucial factors involved in embryonic development or tissue and organ development. GO enrichment analysis suggested possible alteration of these processes. Our study provides novel molecular insights into the formation of macroglossia in cloned pigs.


Assuntos
Clonagem de Organismos , Metilação de DNA , Músculos Isquiossurais , Macroglossia/genética , Sus scrofa/genética , Língua , Animais , Epigênese Genética , Epigenoma , Fenótipo , Suínos
10.
Bioethics ; 35(7): 680-687, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34128235

RESUMO

In a recent article Thomas Douglas and Katrien Devolder propose a theory of genetic parenthood according to which a human child can have only two genetic human parents. I argue this theory is arbitrarily narrow and fails to account for cases such as hybrids, cloning, chimerism, twinning, parthenogenesis, mitochondrial replacement techniques, and more. I propose an alternate theory of genetic parenthood, one that is prima facie consistent with our commonsense intuitions about genetic parenthood and relevant to a right to procreative autonomy.


Assuntos
Pais , Técnicas de Reprodução Assistida , Criança , Clonagem de Organismos , Humanos , Reprodução
11.
Stem Cell Res Ther ; 12(1): 283, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980321

RESUMO

BACKGROUND: The global health emergency of COVID-19 has necessitated the development of multiple therapeutic modalities including vaccinations, antivirals, anti-inflammatory, and cytoimmunotherapies, etc. COVID-19 patients suffer from damage to various organs and vascular structures, so they present multiple health crises. Mesenchymal stem cells (MSCs) are of interest to treat acute respiratory distress syndrome (ARDS) caused by SARS-CoV-2 infection. MAIN BODY: Stem cell-based therapies have been verified for prospective benefits in copious preclinical and clinical studies. MSCs confer potential benefits to develop various cell types and organoids for studying virus-human interaction, drug testing, regenerative medicine, and immunomodulatory effects in COVID-19 patients. Apart from paving the ways to augment stem cell research and therapies, somatic cell nuclear transfer (SCNT) holds unique ability for a wide range of health applications such as patient-specific or isogenic cells for regenerative medicine and breeding transgenic animals for biomedical applications. Being a potent cell genome-reprogramming tool, the SCNT has increased prominence of recombinant therapeutics and cellular medicine in the current era of COVID-19. As SCNT is used to generate patient-specific stem cells, it avoids dependence on embryos to obtain stem cells. CONCLUSIONS: The nuclear transfer cloning, being an ideal tool to generate cloned embryos, and the embryonic stem cells will boost drug testing and cellular medicine in COVID-19.


Assuntos
COVID-19 , Animais , Clonagem Molecular , Clonagem de Organismos , Células-Tronco Embrionárias , Humanos , Estudos Prospectivos , SARS-CoV-2
12.
Nat Commun ; 12(1): 2989, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34017000

RESUMO

The allogeneic transplantation of primordial germ cells (PGCs) derived from somatic cells overcomes the limitation of avian cloning. Here, we transdifferentiate chicken embryo fibroblasts (CEFs) from black feathered Langshan chickens to PGCs and transplant them into White Plymouth Rock chicken embryos to produce viable offspring with characteristics inherited from the donor. We express Oct4/Sox2/Nanog/Lin28A (OSNL) to reprogram CEFs to induced pluripotent stem cells (iPSCs), which are further induced to differentiate into PGCs by BMP4/BMP8b/EGF. DNA demethylation, histone acetylation and glycolytic activation elevate the iPSC induction efficiency, while histone acetylation and glycolytic inhibition facilitate PGCs formation. The induced PGCs (iPGCs) are transplanted into the recipients, which are self-crossed to produce 189/509 somatic cells derived chicken with the donor's characteristics. Microsatellite analysis and genome sequencing confirm the inheritance of genetic information from the donor. Thus, we demonstrate the feasibility of avian cloning from somatic cells.


Assuntos
Transdiferenciação Celular/genética , Clonagem de Organismos/métodos , Células Germinativas/transplante , Células-Tronco Pluripotentes Induzidas/fisiologia , Criação de Animais Domésticos/métodos , Animais , Proteína Morfogenética Óssea 4/genética , Células Cultivadas , Embrião de Galinha/citologia , Galinhas , Fator de Crescimento Epidérmico/genética , Estudos de Viabilidade , Fibroblastos/fisiologia , Células Germinativas/fisiologia , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição SOXB1/genética , Transplante Homólogo/métodos
13.
Science ; 372(6539): 223, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33859013
14.
Dokl Biochem Biophys ; 496(1): 48-51, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33689075

RESUMO

Somatic Cell Nuclear Transfer (SCNT) technique was used to produce the first viable cloned cattle offspring in Russia. Whole-genome SNP genotyping confirmed that the cloned calf was identical to the fibroblast cell line that was used for SCNT. CRISPR/Cas9 approach was subsequently used to knock out genes for beta-lactoglobulin gene (PAEP) and the beta-lactoglobulin-like protein gene (LOC100848610) in the fibroblast cells. Gene editing (GE) efficiency was 4.4% for each of these genes. We successfully obtained single-cell-derived fibroblast colonies containing PAEP and LOC100848610 knockouts, which will be used to produce beta-lactoglobulin-deficient cattle.


Assuntos
Animais Geneticamente Modificados/genética , Sistemas CRISPR-Cas , Bovinos/genética , Clonagem de Organismos/métodos , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Edição de Genes/métodos , Animais , Animais Geneticamente Modificados/embriologia , Bovinos/embriologia , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Técnicas de Inativação de Genes/métodos , Técnicas de Transferência Nuclear
15.
Theriogenology ; 166: 21-28, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33667861

RESUMO

Our aim was to establish an efficient culture system to produce embryos by SCNT of the endangered Vietnamese I pig. Reducing the serum concentration from 10.0% to 0.2% during culture efficiently synchronized I pig fibroblasts used as donor cells at the G0/G1 stage. Oocyte maturation in a defined porcine oocyte medium (POM) supplemented with EGF and gonadotrophins resulted in higher cleavage and blastocyst rates compared with a non-defined POM containing pig follicular fluid (but without EGF) and both the defined and non-defined variants of NCSU-37. For embryo culture PZM3 and PZM5 media were superior to NCSU-37, in terms of the percentage of cleaved embryos. Addition of serum to PZM3 medium on Day 5 of culture (Day 0 = SCNT) improved blastocyst development. When SCNT embryos were transferred at the blastocyst stage, 7 of 11 recipients became pregnant. However, live offspring were not obtained. In conclusion, we established a system for the production of I pig embryos by SCNT and achieved blastocyst production rate at 26.4% by improving culture systems for donor cells, oocytes and embryos culture. Transfer of embryos resulted in pregnancies; however, live offspring were not obtained.


Assuntos
Blastocisto , Técnicas de Transferência Nuclear , Animais , Grupo com Ancestrais do Continente Asiático , Ciclo Celular , Clonagem de Organismos/veterinária , Meios de Cultura , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Feminino , Humanos , Técnicas de Transferência Nuclear/veterinária , Oócitos , Gravidez , Suínos
16.
J Biol Chem ; 296: 100497, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33675752

RESUMO

The CRISPR/Cas9 system has been used in a wide range of applications in the production of gene-edited animals and plants. Most efforts to insert genes have relied on homology-directed repair (HDR)-mediated integration, but this strategy remains inefficient for the production of gene-edited livestock, especially monotocous species such as cattle. Although efforts have been made to improve HDR efficiency, other strategies have also been proposed to circumvent these challenges. Here we demonstrate that a homology-mediated end-joining (HMEJ)-based method can be used to create gene-edited cattle that displays precise integration of a functional gene at the ROSA26 locus. We found that the HMEJ-based method increased the knock-in efficiency of reporter genes by eightfold relative to the traditional HDR-based method in bovine fetal fibroblasts. Moreover, we identified the bovine homology of the mouse Rosa26 locus that is an accepted genomic safe harbor and produced three live-born gene-edited cattle with higher rates of pregnancy and birth, compared with previous work. These gene-edited cattle exhibited predictable expression of the functional gene natural resistance-associated macrophage protein-1 (NRAMP1), a metal ion transporter that should and, in our experiments does, increase resistance to bovine tuberculosis, one of the most detrimental zoonotic diseases. This research contributes to the establishment of a safe and efficient genome editing system and provides insights for gene-edited animal breeding.


Assuntos
Bovinos/genética , Clonagem de Organismos , Resistência à Doença , Edição de Genes , Loci Gênicos , Tuberculose Bovina/genética , Animais , Bovinos/microbiologia , Reparo do DNA por Junção de Extremidades , Tuberculose Bovina/metabolismo
17.
Zygote ; 29(5): 331-336, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33685548

RESUMO

The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.


Assuntos
MicroRNAs , Técnicas de Transferência Nuclear , Animais , Blastocisto , Clonagem de Organismos , Desenvolvimento Embrionário/genética , Masculino , Coelhos , Espermatozoides
18.
Mol Reprod Dev ; 88(3): 228-237, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33650239

RESUMO

Ectopic expression of Xist on the putative active X chromosome is a primary cause of the low developmental efficiency of cloned mouse and pig embryos. Suppression of abnormal Xist expression via gene knockout or RNA interference (RNAi) can significantly enhance the developmental competence of cloned mouse and pig embryos. RLIM is a Xist expression activator, whereas REX1 is an Xist transcription inhibitor, as RLIM triggers Xist expression by mediating the proteasomal degradation of REX1 to induce imprinted and random X chromosome inactivation in mice. This study aimed to test whether the knockdown of RLIM and overexpression of REX1 can repress aberrant Xist expression and improve the developmental ability of cloned male pig embryos. Results showed that injection of anti-RLIM small interfering RNA significantly decreased Xist messenger RNA abundance, increased REX1 protein level, and enhanced the preimplantation development of cloned male porcine embryos. These positive effects were not observed in cloned male pig embryos injected with REX1 expression plasmid, which might be due to the low expression efficiency of injected REX1 plasmid and/or the short half-life of expressed REX1 protein. The findings from this study indicated that RLIM participated in the ectopic activation of Xist expression in cloned pig embryos by targeting REX1 degradation. Furthermore, this study provided a new method to improve cloned pig embryo development by the inhibition of Xist expression via RNAi of RLIM.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Longo não Codificante/genética , Ubiquitina-Proteína Ligases/genética , Animais , Clonagem de Organismos , Técnicas de Silenciamento de Genes , Masculino , Técnicas de Transferência Nuclear , RNA Longo não Codificante/metabolismo , Suínos , Ubiquitina-Proteína Ligases/metabolismo
19.
Theriogenology ; 165: 18-27, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33611171

RESUMO

Many studies have reported that interspecies somatic cell nuclear transfer (iSCNT) is considered the prominent method in preserving endangered animals. However, the development rate of iSCNT embryos is low, and there are limited studies on the molecular mechanism of the iSCNT process. This study evaluated the developmental potential of interspecies lycaon (Lycaon pictus)-dog embryos and assessed the mitochondrial content and metabolism of the produced cloned lycaon-dog fetus. Of 678 collected oocytes, 516 were subjected to nuclear transfer, and 419 reconstructed embryos with male lycaon fibroblasts were transferred into 27 surrogates. Of 720 oocytes, 568 were subjected to nuclear transfer and 469 reconstructed embryos with female lycaon fibroblasts were transferred into 31 surrogates. Two recipients who received female reconstructed embryos were identified as pregnant at 30 days. However, fetal retardation with no cardiac activity was observed at 46 days. Microsatellite analysis confirmed that the cloned lycaon-dog fetus was genetically identical to the lycaon donor cell, whereas mitochondrial sequencing analysis revealed that oocyte donor dogs transmitted their mtDNA. We assessed the oxygen consumption rate and mitochondrial content of the aborted lycaon-dog fetus to shed some light on the aborted fetus's cellular metabolism. The oxygen consumption rates in the lycaon-dog fetal fibroblasts were lower than those in adult dog, lycaon and cloned dog fetal fibroblasts. Furthermore, lycaon-dog fetal fibroblasts showed decreased proportions of live and active mitochondria compared with other groups. Overall, we hypothesized that nuclear-mitochondrial incompatibility affects pyruvate metabolism and that these processes cause intrauterine fetal death.


Assuntos
Clonagem de Organismos , Técnicas de Transferência Nuclear , Animais , Clonagem de Organismos/veterinária , Cães , Desenvolvimento Embrionário , Feminino , Feto , Fibroblastos/metabolismo , Masculino , Mitocôndrias , Técnicas de Transferência Nuclear/veterinária , Oócitos/metabolismo , Gravidez
20.
Int J Mol Sci ; 22(3)2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33573215

RESUMO

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from hFUT2×hGLA bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence. The ACFCs derived from non-transgenic (nTG) pigs were served as the negative (TSA-) and positive (TSA+) control groups. For both hFUT2×hGLA and nTG samples, the expression levels of α1,2-FT and α-Gal A proteins in TSA+ cells were more than twofold higher in comparison to TSA- cells. Moreover, a much lower expression of the Galα1→3Gal epitopes was shown in TSA- hFUT2×hGLA cells as compared to the TSA- nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated hFUT2×hGLA and nTG ACFCs were significantly higher than those noticed for their TSA-untreated counterparts. Summing up, ex vivo protection of effectively selected bi-transgenic ACFC lines, in which TSA-dependent epigenetic transformation triggered the enhancements in reprogrammability and subsequent expression of hFUT2 and hGLA transgenes and their corresponding transcripts, allows for cryopreservation of nuclear donor cells, nuclear-transferred female gametes, and resultant porcine cloned embryos. The latter can be used as a cryogenically conserved genetic resource of biological materials suitable for generation of bi-transgenic cloned offspring in pigs that is targeted at biomedical research in the field of cell/tissue xenotransplantation.


Assuntos
Epigênese Genética/efeitos dos fármacos , Epitopos/metabolismo , Rejeição de Enxerto/prevenção & controle , Ácidos Hidroxâmicos/farmacologia , Transplante Heterólogo/efeitos adversos , Animais , Animais Geneticamente Modificados , Linhagem Celular , Clonagem de Organismos/métodos , Criopreservação , Embrião de Mamíferos , Epitopos/genética , Epitopos/imunologia , Fibroblastos , Fucosiltransferases/genética , Fucosiltransferases/imunologia , Fucosiltransferases/metabolismo , Técnicas de Inativação de Genes , Rejeição de Enxerto/imunologia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pele/citologia , Suínos , Transplante Heterólogo/métodos , alfa-Galactosidase/genética , alfa-Galactosidase/imunologia , alfa-Galactosidase/metabolismo
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