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1.
Cell ; 186(10): 2092-2110.e23, 2023 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-37172563

RESUMEN

The third and fourth weeks of gestation in primates are marked by several developmental milestones, including gastrulation and the formation of organ primordia. However, our understanding of this period is limited due to restricted access to in vivo embryos. To address this gap, we developed an embedded 3D culture system that allows for the extended ex utero culture of cynomolgus monkey embryos for up to 25 days post-fertilization. Morphological, histological, and single-cell RNA-sequencing analyses demonstrate that ex utero cultured monkey embryos largely recapitulated key events of in vivo development. With this platform, we were able to delineate lineage trajectories and genetic programs involved in neural induction, lateral plate mesoderm differentiation, yolk sac hematopoiesis, primitive gut, and primordial germ-cell-like cell development in monkeys. Our embedded 3D culture system provides a robust and reproducible platform for growing monkey embryos from blastocysts to early organogenesis and studying primate embryogenesis ex utero.


Asunto(s)
Embrión de Mamíferos , Desarrollo Embrionario , Animales , Macaca fascicularis , Blastocisto , Organogénesis , Primates
2.
Genomics ; 116(3): 110836, 2024 05.
Artículo en Inglés | MEDLINE | ID: mdl-38537809

RESUMEN

The CRISPR/Cas9 system can induce off-target effects in programmed gene editing, but there have been few reports on cleavage detection and their affection in embryo development. To study these events, sgRNAs with different off-target rates were designed and compared after micro-injected into mouse zygotes, and γH2AX was used for DNA cleavage sites analysis by immunostaining and CUT&Tag. Although the low off-target sgRNA were usually selected for production gene editing animals, γH2AX immunofluorescence indicated that there was a relative DSB peak at 15 h after Cas9 system injection, and the number of γH2AX foci at the peak was significantly higher in the low off-target sgRNA-injected group than in the control group. Further, the result of CUT&Tag sequencing analysis showed more double-strand breaks (DSBs) related sequences were detected in low off-target sgRNA-injected group than control and the distribution of DSB related sequences had no chromosome specificity. Gene Ontology (GO) annotation analysis of the DSB related sequences showed that these sequences were mainly concentrated at genes associated with some important biological processes, molecular functions, and cell components. In a conclusion, there are many sgRNA-sequence-independent DSBs in early mouse embryos when the Cas9 system is used for gene editing and the DSB related sequence could be detected and characterized in the genome. These results and method should also be considered in using or optimizing the Cas9 system.


Asunto(s)
Sistemas CRISPR-Cas , Roturas del ADN de Doble Cadena , Embrión de Mamíferos , Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Animales , Ratones , Embrión de Mamíferos/metabolismo , Edición Génica/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , División del ADN , Cigoto/metabolismo , Histonas/metabolismo , Histonas/genética , Femenino
3.
Int J Mol Sci ; 25(3)2024 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-38338922

RESUMEN

Cortical traumatic brain injury (TBI) is a major cause of cognitive impairment accompanied by motor and behavioral deficits, and there is no effective treatment strategy in the clinic. Cell transplantation is a promising therapeutic strategy, and it is necessary to verify the survival and differentiation of cells after transplantation in large animal models like rhesus monkeys. In this study, we transplanted neural stem cells (NSCs) and simultaneously injected basic fibroblast growth factor/epidermal growth factor (bFGF/EGF) into the cortex (visual and sensory cortices) of rhesus monkeys with superficial TBI. The results showed that the transplanted NSCs did not enter the cerebrospinal fluid (CSF) and were confined to the transplantation site for at least one year. The transplanted NSCs differentiated into mature neurons that formed synaptic connections with host neurons, but glial scar formation between the graft and the host tissue did not occur. This study is the first to explore the repairing effect of transplanting NSCs into the superficial cerebral cortex of rhesus monkeys after TBI, and the results show the ability of NSCs to survive long-term and differentiate into neurons, demonstrating the potential of NSC transplantation for cortical TBI.


Asunto(s)
Lesiones Traumáticas del Encéfalo , Células-Madre Neurales , Animales , Macaca mulatta , Neuronas/metabolismo , Células-Madre Neurales/metabolismo , Lesiones Traumáticas del Encéfalo/terapia , Lesiones Traumáticas del Encéfalo/metabolismo , Diferenciación Celular , Corteza Cerebral , Trasplante de Células Madre/métodos , Células Cultivadas
4.
Hum Reprod ; 32(1): 76-87, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27864358

RESUMEN

STUDY QUESTION: Can the histone deacetylase inhibitor Scriptaid improve the efficiency of the development of round spermatid injection (ROSI)-fertilized embryos in a mouse model? SUMMARY ANSWER: Treatment of ROSI mouse zygotes with Scriptaid increased the expression levels of several development-related genes at the blastocyst stage, resulting in more efficient in vitro development of the blastocyst and an increased birth rate of ROSI-derived embryos. WHAT IS KNOWN ALREADY: The full-term development of embryos derived through ROSI is significantly lower than that following ICSI in humans and other species. STUDY DESIGN, SIZE, DURATION: Oocytes, spermatozoa and round spermatids were collected from BDF1 (C57BL/6 × DBA/2) mice. For in vitro development experiments, mouse ROSI-derived zygotes were treated with Scriptaid at different concentrations (0, 125, 250, 500 and 1000 nM) and for different exposure times (0, 6, 10, 16 or 24 h). Next, blastocysts of the optimal Scriptaid-treated group and the non-treated ROSI group were separately transferred into surrogate ICR mice to compare in vivo development with the ICSI group (control). Each experiment was repeated at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metaphase II (MII) oocytes, spermatozoa and round spermatids were obtained from sexually mature BDF1 female or male mice. The developmental potential of embryos among the three groups (the ICSI, ROSI and optimal Scriptaid-treated ROSI groups) was assessed based on the rates of obtaining zygotes, two-cell stage embryos, four-cell stage embryos, blastocysts and full-term offspring. In addition, the expression levels of development-related genes (Oct4, Nanog, Klf4 and Sox2) were analysed using real-time PCR, and the methylation states of imprinted genes (H19 and Snrpn) in these three groups were detected using methylation-specific PCR (MS-PCR) sequencing following bisulfite treatment. MAIN RESULTS AND THE ROLE OF CHANCE: The in vitro experiments revealed that treating ROSI-derived zygotes with 250 nM Scriptaid for 10 h significantly improved the blastocyst formation rate (59%) compared with the non-treated group (38%) and further increased the birth rates of ROSI-derived embryos from 21% to 40% in vivo. Moreover, in ROSI-derived embryos, the expression of the Oct4, Nanog and Sox2 genes at the blastocyst stage was decreased, but the optimal Scriptaid treatment restored expression to a level similar to their ICSI counterparts. In addition, Scriptaid treatment moderately repaired the abnormal DNA methylation pattern in the imprinting control regions (ICRs) of H19 and Snrpn. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: Because of the ethics regarding the use of human gametes for ROSI studies, the mouse model was used as an approach to explore the effects of Scriptaid on the developmental potential of ROSI-derived embryos. However, to determine whether these findings can be applied to humans, further investigation will be required. WIDER IMPLICATIONS OF THE FINDINGS: Scriptaid treatment provides a new means of improving the efficiency and safety of clinical human ROSI. STUDY FUNDING/COMPETING INTERESTS: The study was financially supported through grants from the National Key Research Program of China (No. 2016YFC1304800); the National Natural Science Foundation of China (Nos: 81170756, 81571486); the Natural Science Foundation of Shanghai (Nos: 15140901700, 15ZR1424900) and the Programme for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. There are no conflicts of interest to declare.


Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Hidroxilaminas/farmacología , Quinolinas/farmacología , Espermátides/efectos de los fármacos , Animales , Transferencia de Embrión , Femenino , Expresión Génica/efectos de los fármacos , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones Endogámicos ICR , Oocitos/efectos de los fármacos , Inyecciones de Esperma Intracitoplasmáticas
5.
Neural Regen Res ; 19(11): 2444-2455, 2024 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-38526281

RESUMEN

Parkinson's disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Many studies have been performed based on the supplementation of lost dopaminergic neurons to treat Parkinson's disease. The initial strategy for cell replacement therapy used human fetal ventral midbrain and human embryonic stem cells to treat Parkinson's disease, which could substantially alleviate the symptoms of Parkinson's disease in clinical practice. However, ethical issues and tumor formation were limitations of its clinical application. Induced pluripotent stem cells can be acquired without sacrificing human embryos, which eliminates the huge ethical barriers of human stem cell therapy. Another widely considered neuronal regeneration strategy is to directly reprogram fibroblasts and astrocytes into neurons, without the need for intermediate proliferation states, thus avoiding issues of immune rejection and tumor formation. Both induced pluripotent stem cells and direct reprogramming of lineage cells have shown promising results in the treatment of Parkinson's disease. However, there are also ethical concerns and the risk of tumor formation that need to be addressed. This review highlights the current application status of cell reprogramming in the treatment of Parkinson's disease, focusing on the use of induced pluripotent stem cells in cell replacement therapy, including preclinical animal models and progress in clinical research. The review also discusses the advancements in direct reprogramming of lineage cells in the treatment of Parkinson's disease, as well as the controversy surrounding in vivo reprogramming. These findings suggest that cell reprogramming may hold great promise as a potential strategy for treating Parkinson's disease.

6.
Zool Res ; 45(2): 233-241, 2024 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-38287904

RESUMEN

Neural tube defects (NTDs) are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure. Although folate supplementation has been shown to mitigate the incidence of NTDs, some cases, often attributable to genetic factors, remain unpreventable. The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation; at present, however, the underlying mechanism remains unclear. Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate. To determine the role of SHROOM3 in early developmental morphogenesis, we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase. Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei. These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins, namely fibrous actin (F-actin), myosin II, and phospho-myosin light chain (PMLC), to the apical side of the neuroepithelial cells. Notably, these defects were not rescued by folate supplementation. RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis. In summary, we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.


Asunto(s)
Proteínas del Citoesqueleto , Defectos del Tubo Neural , Animales , Proteínas del Citoesqueleto/metabolismo , Tubo Neural/metabolismo , Macaca fascicularis , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismo , Defectos del Tubo Neural/veterinaria , Células Neuroepiteliales/metabolismo , Ácido Fólico/metabolismo , Organoides , Citoesqueleto
7.
Reprod Fertil Dev ; 25(4): 609-16, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23594385

RESUMEN

The goal of this study was to investigate the effect of cryopreservation on oocytes at different times after intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. The study was performed in mouse oocytes fertilised by ICSI, or in artificially-activated oocytes, which were cryopreserved immediately, one hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilisation-activation, embryonic development of oocytes-zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of 0-, 1- and 5-h cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the 0-, 1- and 5-h groups of oocytes frozen after ICSI, while the rates of two-PN development of activated oocytes were different between the 1-h and 5-h groups. Despite these initial differences, there was no difference in the rate of blastocyst formation from two-PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially-activated oocytes cryopreserved at 5h, many oocytes from the 0- and 1-h cryopreservation groups developed to zygotes with abnormal ploidy; this suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that spermatozoa can maintain normal fertilisation capacity in frozen ICSI oocytes and the procedure of freeze-thawing did not affect the later development of zygotes.


Asunto(s)
Núcleo Celular/fisiología , Criopreservación/métodos , Desarrollo Embrionario/fisiología , Microtúbulos/fisiología , Oocitos/fisiología , Animales , Aberraciones Cromosómicas , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Oocitos/citología , Partenogénesis/fisiología , Propilenglicol , Inyecciones de Esperma Intracitoplasmáticas/métodos , Análisis de Supervivencia , Factores de Tiempo
8.
J Assist Reprod Genet ; 30(12): 1605-10, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24114632

RESUMEN

PURPOSE: We investigated the effect of electrical stimulation on rabbit oocyte activation using intracytoplasmic sperm injection (ICSI) to determine whether viable offspring can be produced from deceased rabbit sperm using ICSI. METHODS: Sperm were collected from a heterozygote GFP male rabbit 5 h after sacrifice and cryopreserved in liquid nitrogen. Mature oocytes were fertilized using ICSI. A series of electrical pulse procedures were used to activate oocytes before and/or after ICSI. Following ICSI, zygotes were cultured in B2 medium for 4 days or transferred into the oviducts of recipient rabbits at the 2- or 4-cell stage. RESULTS: The blastocyst formation rate was significantly greater in oocytes that received one or two pulses prior to ICSI compared to controls and other electrically stimulated groups. In the single pulse before ICSI group, 23 % of the blastocysts expressed GFP, which was significantly greater than all other groups. However, those that received treatment before and after, or just following ICSI, showed a significant decrease in embryo survival. Finally, embryos from the single pulse before ICSI group were transferred into recipient female rabbits and a full-term kit was successfully delivered. CONCLUSIONS: One pulse of electrical stimulation prior to sperm injection was an effective method to activate rabbit oocytes for fertilization. Sperm collected from a deceased rabbit is able to produce viable embryos through ISCI that are capable of normal fetal and kit development.


Asunto(s)
Estimulación Eléctrica , Fertilización In Vitro/métodos , Oocitos/fisiología , Inyecciones de Esperma Intracitoplasmáticas/métodos , Animales , Blastocisto/citología , Criopreservación , Transferencia de Embrión/métodos , Desarrollo Embrionario/fisiología , Femenino , Masculino , Conejos , Espermatozoides/fisiología
9.
Sheng Wu Gong Cheng Xue Bao ; 39(7): 3003-3014, 2023 Jul 25.
Artículo en Zh | MEDLINE | ID: mdl-37584144

RESUMEN

The generation of a tau-V337M point mutation mouse model using gene editing technology can provide an animal model with fast disease progression and more severe symptoms, which facilitate the study of pathogenesis and treatment of Alzheimer's disease (AD). In this study, single guide RNAs (sgRNA) and single-stranded oligonucleotides (ssODN) were designed and synthesized in vitro. The mixture of sgRNA, Cas9 protein and ssODN was microinjected into the zygotes of C57BL/6J mice. After DNA cutting and recombination, the site homologous to human 337 valine (GTG) in exon 11 was mutated into methionine (ATG). In order to improve the efficiency of recombination, a Rad51 protein was added. The female mice mated with the nonvasectomy male mice were used as the surrogates. Subsequently, the 2-cell stage gene edited embryos were transferred into the unilateral oviduct, and the F0 tau-V337M mutation mice were obtained. Higher mutation efficiency could be obtained by adding Rad51 protein. The F0 tau-V337M point mutation mice can pass the mutation on to the F1 generation mice. In conclusion, this study successfully established the first tau-V337M mutation mouse by using Cas9, ssODN and Rad51. These results provide a new method for developing AD mice model which can be used in further research on the pathogenesis and treatment of AD.


Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , Animales , Masculino , Femenino , Ratones , Humanos , Sistemas CRISPR-Cas/genética , Recombinasa Rad51/genética , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Recombinación Genética
10.
Front Vet Sci ; 10: 1106016, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36876010

RESUMEN

Introduction: Polycystic kidney disease (PKD) is a common autosomal dominant or recessive genetic disease, often accompanied by polycystic liver disease (PLD). Many cases of PKD in animals have been reported. However, little is known about the genes that cause PKD in animals. Methods: In this study, we evaluated the clinical phenotypes of PKD in two spontaneously aged cynomolgus monkeys and explored the genetic etiology using whole-genome sequencing (WGS). Ultrasonic and histological consequences were further investigated in PKD- and PLD-affected monkeys. Results: The results indicated that the kidneys of the two monkeys had varying degrees of cystic changes, and the renal cortex was thinned and accompanied by fluid accumulation. As for hepatopathy, inflammatory cell infiltration, cystic effusion, steatosis of hepatocytes, and pseudo-lobular were found. Based on WGS results, the variants of PKD1:(XM_015442355: c.1144G>C p. E382Q) and GANAB: (NM_001285075.1: c.2708T>C/p. V903A) are predicted to be likely pathogenic heterozygous mutations in PKD- and PLD-affected monkeys. Discussion: Our study suggests that the cynomolgus monkey PKD and PLD phenotypes are very similar to those in humans, and are probably caused by pathogenic genes homologous to humans. The results indicate that cynomolgus monkeys can be used as the most appropriate animal model for human PKD pathogenesis research and therapeutic drug screening.

11.
J Cell Biochem ; 113(8): 2652-60, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22434705

RESUMEN

Myostatin, a secreted growth factor highly expressed in skeletal muscle, negatively regulates skeletal muscle growth and differentiation. Recently, myostatin is emerged as a potential target for anti-atrophy and anti-fibrotic therapies. Therefore, to investigate the regulation of myostatin in sheep adult fibroblasts, we used the RNA interference mediated by lentiviral vector to gene silence myostatin. Simultaneously, we also had constructed the sheep myostatin overexpression vector to further explore the function of myostatin in fibroblasts. The results here demonstrated that the lentiviral vector could significantly reduce myostatin gene both at mRNA and protein level by 71% and 67%, respectively (P < 0.01). Inhibition of myostatin also resulted in a remarkable increase of activin receptor 2B (ACV2B), p21, PPARγ, leptin, C/EBPß, and MEF2A expression, and a decrease of Akt1, CDK2, MEF2C, and Myf5 expression. Ectopic myostatin mRNA and protein were also present in the fibroblasts transfection. Furthermore, we observed that overexpression of myostatin contributed to an increase of Akt1, CDK2, Myf5 and PPARγ, and a decrease of p21, C/EBPα and leptin at the transcript level. These results suggested that myostatin positively regulated Akt1, CDK2, Myf5, leptin, and C/EBPα, but negatively regulated p21 mRNA expression in adult fibroblasts, and it also expanded our understanding of the regulation mechanism of myostatin. Moreover, the lentiviral system inactivated myostatin gene in fibroblasts would be used to generate transgenic sheep and to ameliorate muscle fibrosis and atrophy by gene therapy in the future.


Asunto(s)
Fibroblastos/metabolismo , Miostatina/genética , Animales , Western Blotting , Células Cultivadas , Femenino , Silenciador del Gen , Miostatina/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Ovinos
12.
Sheng Wu Gong Cheng Xue Bao ; 38(6): 2087-2104, 2022 Jun 25.
Artículo en Zh | MEDLINE | ID: mdl-35786464

RESUMEN

CRISPR/Cas9 is a cutting-edge gene-editing technology that has emerged as a promising tool for gene therapy. Nevertheless, the safe and efficient delivery of CRISPR/Cas9 is still an urgent issue in clinical application. Nanoparticles, such as lipid-based nanoparticles, polymer nanoparticles, gold nanoparticles, and biofilm nanoparticles, are expected to bring new opportunities for CRISPR/Cas9-based gene therapy because of their biocompatibility, safety and designability. This review briefly introduced the characteristics of nanoparticles and the development of CRISPR/Cas9 in gene therapy. Moreover, the application of nanoparticles in the delivery of different forms of CRISPR/Cas9 were elaborated. Finally, the challenges and safety of nanoparticle-based gene therapy were discussed.


Asunto(s)
Sistemas CRISPR-Cas , Nanopartículas del Metal , Sistemas CRISPR-Cas/genética , Edición Génica , Terapia Genética , Oro
13.
Front Aging Neurosci ; 14: 1039780, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36389074

RESUMEN

Parkinson's disease (PD) is the second most common neurodegenerative disease. Studies have shown that abnormal accumulation of α-synuclein (α-Syn) in the substantia nigra is a specific pathological characteristic of PD. Abnormal accumulation of α-Syn in PD induces the activation of microglia. Microglia, which are immune cells in the central nervous system, are involved in the function and regulation of inflammation in PD by autophagy. The role of microglial autophagy in the pathophysiology of PD has become a hot-pot issue. This review outlines the pathways of microglial autophagy, and explores the key factor of microglial autophagy in the mechanism of PD and the possibility of microglial autophagy as a potential therapeutic target for PD.

14.
Cell Biosci ; 12(1): 5, 2022 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-34983631

RESUMEN

BACKGROUND: During male meiosis, the Y chromosome can form perfect pairing with the X chromosome. However, it is unclear whether mammalian Female germline stem cells (FGSCs) without a Y chromosome can transdifferentiate into functional haploid spermatid-like cells (SLCs). RESULTS: We found that spermatogenesis was restarted by transplanting FGSCs into Kitw/wv mutant testes. Complete meiosis and formation of SLCs was induced in vitro by testicular cells of Kitw/wv mutant mice, cytokines and retinoic acid. Healthy offspring were produced by sperm and SLCs derived from the in vivo and in vitro transdifferentiation of FGSCs, respectively. Furthermore, high-throughput chromosome conformation capture sequencing(Hi-C-seq) and "bivalent" (H3K4me3-H3K27me3) micro chromatin immunoprecipitation sequencing (µChIP-seq) experiments showed that stimulated by retinoic acid gene 8 (STRA8)/protamine 1 (PRM1)-positive transdifferentiated germ cells (tGCs) and male germ cells (mGCs) display similar chromatin dynamics and chromatin condensation during in vitro spermatogenesis. CONCLUSION: This study demonstrates that sperm can be produced from FGSCs without a Y chromosome. This suggests a strategy for dairy cattle breeding to produce only female offspring with a high-quality genetic background.

15.
Mol Biol Rep ; 38(5): 3161-71, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20140706

RESUMEN

Ovis aries is one of the most important agricultural livestock for meat production, and also is an ideal model organism for biological and comparative genomics studies. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about O. aries miRNAs. In this study, combining a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library, we identified 31 miRNAs belong to 24 families in sheep, 2 of which were novel miRNAs which had never been previously identified in any species. Especially, we cloned 12 miRNAs from the sheep skeletal muscle, which were good candidate miRNAs to be studied about the miRNA-dependant regulated process of muscle development, and we identified four pairs of miRNA/miRNA and one pair of miRNA-3p/miRNA-5p from sheep EST sequences. Expression analysis indicated that some miRNAs were expressed in a specific tissue, and the pair of miRNA-3p/miRNA-5p and one pair of miRNA/miRNA had a similar relative expression pattern in some tissues, respectively. Further, we predicted 120 potential target genes of 31 oar-miRNAs on the 3'UTR of O. aries genes. Gene ontology analysis showed that most of these genes took part in the cellular process and metabolic process. Our results enriched the O. aries miRNA database and provided useful information for investigating biological functions of miRNAs and miRNA in sheep.


Asunto(s)
Biología Computacional/métodos , MicroARNs/genética , Ovinos/genética , Animales , Secuencia de Bases , Biblioteca de Genes , MicroARNs/clasificación , Datos de Secuencia Molecular , Músculo Esquelético/fisiología , Filogenia , Ovinos/anatomía & histología , Distribución Tisular
16.
J Clin Invest ; 131(4)2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33301426

RESUMEN

How particular bone marrow niche factors contribute to the leukemogenic activities of leukemia-initiating cells (LICs) remains largely unknown. Here, we showed that ATP levels were markedly increased in the bone marrow niches of mice with acute myeloid leukemia (AML), and LICs preferentially localized to the endosteal niche with relatively high ATP levels, as indicated by a sensitive ATP indicator. ATP could efficiently induce the influx of ions into LICs in an MLL-AF9-induced murine AML model via the ligand-gated ion channel P2X7. P2x7 deletion led to notably impaired homing and self-renewal capacities of LICs and contributed to an approximately 5-fold decrease in the number of functional LICs but had no effect on normal hematopoiesis. ATP/P2X7 signaling enhanced the calcium flux-mediated phosphorylation of CREB, which further transactivated phosphoglycerate dehydrogenase (Phgdh) expression to maintain serine metabolism and LIC fates. P2X7 knockdown resulted in a markedly extended survival of recipients transplanted with either human AML cell lines or primary leukemia cells. Blockade of ATP/P2X7 signaling could efficiently inhibit leukemogenesis. Here, we provide a perspective for understanding how ATP/P2X7 signaling sustains LIC activities, which may benefit the development of specific strategies for targeting LICs or other types of cancer stem cells.


Asunto(s)
Adenosina Trifosfato/metabolismo , Médula Ósea/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales , Células Madre Neoplásicas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Microambiente Tumoral , Adenosina Trifosfato/genética , Animales , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Receptores Purinérgicos P2X7/genética , Transducción de Señal/genética
17.
Theriogenology ; 157: 314-320, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32827989

RESUMEN

Successful fertilization by intracytoplasmic sperm injection (ICSI) is possible as long as the sperm genome is intact, even in the context of defective sperm or sustained adverse treatment. However, there are few reports on rescuing gene-modified mouse lines after accidental death. To investigate whether sperm from a dead transgenic mouse can fertilize an oocyte and enable embryo development into a pup, Nestin-GFP transgenic male mice were sacrificed, and sperm was collected 14 h, 24 h, and 48 h after death. The collected sperm was injected into oocytes from hybrid B6D2F1 or inbred C57BL/6 N mice. The results showed that the sperm in the three groups activated oocytes from B6D2F1 and supported embryo development to the blastocyst stage. For ICSI embryos derived from B6D2F1 mice, the cleavage and blastocyst rates were significantly lower in the three experimental groups than in the control group (0 h) (P < 0.05), and the birth rate in the 24 h and 48 h groups was significantly lower than that in the 14 h and control groups (0 h). For C57BL/6N-derived ICSI embryos, the cleavage rates were significantly lower at 24 h and 48 h than at 14 h and 0 h (control group), and the birth rate in the three experimental groups was significantly lower than that in the control group (0 h). The F0 mice derived from B6D2F1 and C57BL/6 N oocytes had normal reproductive ability, and F1 mice were successfully obtained. The characteristics of the GFP gene were preserved and inherited. The histone H2AX phosphorylation assay showed that the proportion of focus-negative embryos was markedly and significantly lower in the 14 h, 24 h, and 48 h groups than in the control group (0 h). The proportion of focus-negative embryos was significantly lower at 48 h than at 14 h or 24 h. The number of foci was significantly higher in the three experimental groups than in the control group (0 h), indicating that sperm DNA sustained more damage after death and that few sperm had an intact genome. In summary, sperm obtained from mice 14 h, 24 h, and 48 h after death is capable of activating an oocyte and supporting complete embryo development into a pup. This study provides an effective way to rescue accidently died mouse strains.


Asunto(s)
Inyecciones de Esperma Intracitoplasmáticas , Espermatozoides , Animales , Blastocisto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oocitos , Inyecciones de Esperma Intracitoplasmáticas/veterinaria
18.
Hum Gene Ther ; 30(9): 1079-1092, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31144528

RESUMEN

The application of CRISPR/Cas9 has opened a new era in gene therapy, making it possible to correct mutated genomes in vivo. Exon replacement can correct many mutations and has potential clinical value. In this study, we used a lentivirus-delivered transgene to obtain transgenic mice in which Cas9 and green fluorescent protein (GFP) were driven by the hTBG promoter and were specifically expressed in the liver. In Cas9-positive mice, only ∼11.6% of hepatocytes were GFP positive. The newborn Cas9-positive F1 mice were injected via the temporal vein with rAAV carrying a modified homologous replacement sequence for exon 8 of Atp7b and a pair of single-strand guide RNAs targeting the introns surrounding exon 8. When the Cas9-positive hepatocytes were sorted and analyzed by PCR and next-generation deep sequencing with different labels, ∼16.34 ± 4.02% to 19.37 ± 6.50% of the analyzed copies of exon 8 were replaced by the donor template in the genome of GFP-positive hepatocytes, that is, 1.81 ± 0.29% to 2.09 ± 0.54% replacement occurred in all liver genomes. However, when rAAV carrying a modified homologous replacement sequence was injected into the adult spCas9 mice, a double-cut deletion ratio of up to 99%, only about 1.10-1.13% of the exon 8 replacement rate was detected in Cas9-positive hepatocytes. This study is the first to achieve exon replacement via CRISPR/Cas9, which will benefit research on CRISPR/Cas9 technology for gene therapy.


Asunto(s)
Sistemas CRISPR-Cas , ATPasas Transportadoras de Cobre/genética , Exones , Edición Génica , Animales , Línea Celular , Dependovirus/genética , Orden Génico , Marcación de Gen , Genes Reporteros , Ingeniería Genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Hepatocitos/metabolismo , Lentivirus/genética , Ratones , Ratones Transgénicos , Mutación , ARN Guía de Kinetoplastida , Análisis de Secuencia de ADN , Transducción Genética
19.
Sci Rep ; 8(1): 1332, 2018 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-29358698

RESUMEN

CRISPR/Cas9 has recently been developed as an efficient genome engineering tool. The rabbit is a suitable animal model for studies of metabolic diseases. In this study, we generated ATP7B site-directed point mutation rabbits to simulate a major mutation type in Asians (p. Arg778Leu) with Wilson disease (WD) by using the CRISPR/Cas9 system combined with single-strand DNA oligonucleotides (ssODNs). The efficiency of the precision point mutation was 52.94% when zygotes were injected 14 hours after HCG treatment and was significantly higher than that of zygotes injected 19 hours after HCG treatment (14.29%). The rabbits carrying the allele with mutant ATP7B died at approximately three months of age. Additionally, the copper content in the livers of rabbits at the onset of WD increased nine-fold, a level similar to the five-fold increase observed in humans with WD. Thus, the efficiency of precision point mutations increases when RNAs are injected into zygotes at earlier stages, and the ATP7B mutant rabbits are a potential model for human WD disease with applications in pathological analysis, clinical treatment and gene therapy research.


Asunto(s)
ATPasas Transportadoras de Cobre/genética , Modelos Animales de Enfermedad , Degeneración Hepatolenticular/genética , Oligodesoxirribonucleótidos/administración & dosificación , Mutación Puntual , Animales , Sistemas CRISPR-Cas , Cobre/análisis , ADN de Cadena Simple/administración & dosificación , ADN de Cadena Simple/farmacología , Humanos , Hígado/química , Microinyecciones , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/farmacología , Conejos , Cigoto/química , Cigoto/crecimiento & desarrollo
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