Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 10(1): 1234, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874552

RESUMO

Determining cell lineage and function is critical to understanding human physiology and pathology. Although advances in lineage tracing methods provide new insight into cell fate, defining cellular diversity at the mammalian level remains a challenge. Here, we develop a genome editing strategy using a cytidine deaminase fused with nickase Cas9 (nCas9) to specifically target endogenous interspersed repeat regions in mammalian cells. The resulting mutation patterns serve as a genetic barcode, which is induced by targeted mutagenesis with single-guide RNA (sgRNA), leveraging substitution events, and subsequent read out by a single primer pair. By analyzing interspersed mutation signatures, we show the accurate reconstruction of cell lineage using both bulk cell and single-cell data. We envision that our genetic barcode system will enable fine-resolution mapping of organismal development in healthy and diseased mammalian states.


Assuntos
Linhagem da Célula/genética , Código de Barras de DNA Taxonômico/métodos , Edição de Genes/métodos , Elementos Nucleotídeos Longos e Dispersos/genética , Proteína 9 Associada à CRISPR/genética , Diferenciação Celular/genética , Citidina Desaminase/genética , Células HEK293 , Células HeLa , Humanos , Mutagênese , RNA Guia/genética , Análise de Célula Única/métodos , Imagem com Lapso de Tempo
2.
Cell Reprogram ; 21(1): 26-36, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30735078

RESUMO

The potential of induced pluripotent stem (iPS) cells, which have self-renewal ability and can differentiate into three germ layers, led us to hypothesize that iPS cells in pigs can be useful and suitable source for producing transgenic pigs. In this study, we generated iPS-like cells using doxycycline-inducible piggyBac (PB) expression vectors encoding porcine 4 transcription factors. After transfection, transfected cells were cultured until the formation of outgrowing colonies taking least of 7-10 days. The iPS-like cells demonstrated pluripotent characteristics such as self-renewal, high proliferation, expression of pluripotent markers, and aggregation ability. The embryo development through somatic cell nuclear transfer (SCNT), cleavage rate, and blastocyst formation rate did not show any significant differences. However, the total cell number of blastocysts was significantly increased with the established cell line. In conclusion, the iPS-like cell line, generated from porcine transcriptional factors using the PB transposon system, demonstrated pluripotency with the capacity for unlimited self-renewal, and could be used as donor cells to produce cloned embryos by SCNT. These cells will be suitable for gene modification and would contribute to the stability or safety of pig models in biomedical research.


Assuntos
Blastocisto/citologia , Técnicas de Cultura de Células , Clonagem de Organismos , Regulação da Expressão Gênica no Desenvolvimento , Suínos/embriologia , Animais , Animais Geneticamente Modificados , Blastocisto/fisiologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Células Cultivadas , Desenvolvimento Embrionário , Fibroblastos , Técnicas de Transferência Nuclear/veterinária , Células-Tronco Pluripotentes/citologia , Transfecção
3.
Chem Commun (Camb) ; 54(68): 9410-9413, 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30059088

RESUMO

We prepared tetrahedral DNAzymes (TDzs) to overcome potential limitations such as insufficient serum stability and poor cellular uptake of single-stranded DNAzymes (ssDzs). TDzs showed enhanced serum stability and higher cellular uptake efficiency compared to those of ssDzs, providing significantly improved intracellular gene-silencing activity to down-regulate the target mRNA level.


Assuntos
DNA Catalítico/genética , Inativação Gênica , Animais , Bovinos , DNA Catalítico/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Regulação para Baixo , Fibroblastos/metabolismo , Cinética , Camundongos , Células NIH 3T3 , Nanoestruturas/química , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
BMC Genomics ; 19(1): 387, 2018 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-29792157

RESUMO

BACKGROUND: Transposon-mediated, non-viral gene delivery is a powerful tool for generating stable cell lines and transgenic animals. However, as multi-copy insertion is the preferred integration pattern, there is the potential for uncontrolled changes in endogenous gene expression and detrimental effects in cells or animals. Our group has previously reported on the generation of several transgenic cattle by using microinjection of the Sleeping Beauty (SB) and PiggyBac (PB) transposons and seeks to explore the long-term effects of this technology on cattle. RESULTS: Transgenic cattle, one female (SNU-SB-1) and one male (SNU-PB-1), reached over 36 months of age with no significant health issues and normal blood parameters. The detection of transgene integration and fluorescent signal in oocytes and sperm suggested the capacity for germline transmission in both of the founder animals. After natural breeding, the founder transgenic cow delivered a male calf and secreted milk containing fluorescent transgenic proteins. The calf expressed green fluorescent protein in primary cells from ear skin, with no significant change in overall genomic stability and blood parameters. Three sites of transgene integration were identified by next-generation sequencing of the calf's genome. CONCLUSIONS: Overall, these data demonstrate that transposon-mediated transgenesis can be applied to cattle without being detrimental to their long-term genomic stability or general health. We further suggest that this technology may be usefully applied in other fields, such as the generation of transgenic animal models.


Assuntos
Técnicas de Transferência de Genes , Saúde , Óvulo/metabolismo , Espermatozoides/metabolismo , Transposases/genética , Animais , Animais Geneticamente Modificados , Bovinos , Feminino , Masculino , Transgenes/genética , Sequenciamento Completo do Genoma
5.
Artigo em Inglês | MEDLINE | ID: mdl-29423215

RESUMO

The production of transgenic farm animals (e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos (zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure. However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer (SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies (e.g., ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies will accelerate our understanding of genetic traits in bovine and will be readily adapted for bio-medical applications in cattle.

7.
Stem Cells Transl Med ; 6(3): 1040-1051, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28186692

RESUMO

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) exhibit potency for the regeneration of infarcted hearts. Vascular endothelial growth factor (VEGF) is capable of inducing angiogenesis and can boost stem cell-based therapeutic effects. However, high levels of VEGF can cause abnormal blood vessel growth and hemangiomas. Thus, a controllable system to induce therapeutic levels of VEGF is required for cell therapy. We generated an inducible VEGF-secreting stem cell (VEGF/hUCB-MSC) that controls the expression of VEGF and tested the therapeutic efficacy in rat myocardial infarction (MI) model to apply functional stem cells to MI. To introduce the inducible VEGF gene cassette into a safe harbor site of the hUCB-MSC chromosome, the transcription activator-like effector nucleases system was used. After confirming the integration of the cassette into the locus, VEGF secretion in physiological concentration from VEGF/hUCB-MSCs after doxycycline (Dox) induction was proved in conditioned media. VEGF secretion was detected in mice implanted with VEGF/hUCB-MSCs grown via a cell sheet system. Vessel formation was induced in mice transplanted with Matrigel containing VEGF/hUCB-MSCs treated with Dox. Moreover, seeding of the VEGF/hUCB-MSCs onto the cardiac patch significantly improved the left ventricle ejection fraction and fractional shortening in a rat MI model upon VEGF induction. Induced VEGF/hUCB-MSC patches significantly decreased the MI size and fibrosis and increased muscle thickness, suggesting improved survival of cardiomyocytes and protection from MI damage. These results suggest that our inducible VEGF-secreting stem cell system is an effective therapeutic approach for the treatment of MI. Stem Cells Translational Medicine 2017;6:1040-1051.


Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Infarto do Miocárdio/terapia , Animais , Feminino , Humanos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular
8.
Mol Ther ; 24(9): 1644-54, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27434585

RESUMO

Mesenchymal stem cells (MSCs) promote therapeutic angiogenesis to cure serious vascular disorders. However, their survival period and cytokine-secretory capacity are limited. Although hepatocyte growth factor (HGF) can accelerate the rate of angiogenesis, recombinant HGF is limited because of its very short half-life (<3-5 minutes). Thus, continuous treatment with HGF is required to obtain an effective therapeutic response. To overcome these limitations, we produced genome-edited MSCs that secreted HGF upon drug-specific induction. The inducible HGF expression cassette was integrated into a safe harbor site in an MSC chromosome using the TALEN system, resulting in the production of TetOn-HGF/human umbilical cord blood-derived (hUCB)-MSCs. Functional assessment of the TetOn-HGF/hUCB-MSCs showed that they had enhanced mobility upon the induction of HGF expression. Moreover, long-term exposure by doxycycline (Dox)-treated TetOn-HGF/hUCB-MSCs enhanced the anti-apoptotic responses of genome-edited MSCs subjected to oxidative stress and improved the tube-formation ability. Furthermore, TetOn-HGF/hUCB-MSCs encapsulated by arginine-glycine-aspartic acid (RGD)-alginate microgel induced to express HGF improved in vivo angiogenesis in a mouse hindlimb ischemia model. This study showed that the inducible HGF-expressing hUCB-MSCs are competent to continuously express and secrete HGF in a controlled manner. Thus, the MSCs that express HGF in an inducible manner are a useful therapeutic modality for the treatment of vascular diseases requiring angiogenesis.

9.
Sci Rep ; 6: 27185, 2016 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-27324781

RESUMO

Here, we efficiently generated transgenic cattle using two transposon systems (Sleeping Beauty and Piggybac) and their genomes were analyzed by next-generation sequencing (NGS). Blastocysts derived from microinjection of DNA transposons were selected and transferred into recipient cows. Nine transgenic cattle have been generated and grown-up to date without any health issues except two. Some of them expressed strong fluorescence and the transgene in the oocytes from a superovulating one were detected by PCR and sequencing. To investigate genomic variants by the transgene transposition, whole genomic DNA were analyzed by NGS. We found that preferred transposable integration (TA or TTAA) was identified in their genome. Even though multi-copies (i.e. fifteen) were confirmed, there was no significant difference in genome instabilities. In conclusion, we demonstrated that transgenic cattle using the DNA transposon system could be efficiently generated, and all those animals could be a valuable resource for agriculture and veterinary science.


Assuntos
Animais Geneticamente Modificados/genética , Elementos de DNA Transponíveis/genética , Transgenes/genética , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Blastocisto/metabolismo , Bovinos , Feminino , Instabilidade Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Transposases/genética
10.
J Vet Sci ; 17(3): 261-8, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27030199

RESUMO

Animal models, particularly pigs, have come to play an important role in translational biomedical research. There have been many pig models with genetically modifications via somatic cell nuclear transfer (SCNT). However, because most transgenic pigs have been produced by random integration to date, the necessity for more exact gene-mutated models using recombinase based conditional gene expression like mice has been raised. Currently, advanced genome-editing technologies enable us to generate specific gene-deleted and -inserted pig models. In the future, the development of pig models with gene editing technologies could be a valuable resource for biomedical research.


Assuntos
Animais Geneticamente Modificados/genética , Técnicas de Transferência de Genes/veterinária , Modelos Animais , Sus scrofa/genética , Animais
11.
Theriogenology ; 86(2): 596-603, 2016 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-27020877

RESUMO

In animal reproduction technologies, the in vitro embryo culture system has advanced over the past few decades. However, in vitro cultured embryos still have reduced functional and physiological abilities compared with those from in vivo conditions, and many factors of oviduct and uterine environments have not yet been revealed. Here, we demonstrated the in vitro culture of domestic goat (Capra hircus) embryos using two types of culture media, modified synthetic oviductal fluid (mSOF) and a two-step chemically defined medium (DI/II). To obtain parthenogenetic goat embryos, oocytes were matured in vitro in tissue culture media-199 supplemented with 10% fetal bovine serum for 22 to 24 hours, and activated with 5 µM, Ca(2+) ionomycin for 4 minutes, followed by 1.9 mM, 6-dimethylaminopurine treatment for 4 hours. After 2 days of embryo culture in different culture media, there were no significant differences in cleavage rates (96.6% vs. 95.4% in mSOF vs. DI/II, respectively). However, the DI/II group showed improved development competence to blastocysts (64.6% vs. 82.3% in mSOF vs. DI/II, respectively) and the total cell number of blastocysts (144.3 ± 9.2 vs. 264.4 ± 15.2 in mSOF vs. DI/II, respectively) at Day 7. After the cryopreservation of early-stage blastocysts at Day 6 via the conventional slow-freezing procedure, the surviving embryos were analyzed. The re-expansion rate after freezing and thawing was significantly higher in DI/II (39.66% vs. 67.69% in mSOF vs. DI/II, respectively), but there were no statistical differences in total cell numbers (142.3 ± 12.1 vs. 172.1 ± 11.6 in mSOF vs. DI/II, respectively), apoptotic index (4.9 ± 0.8% vs. 3.8 ± 0.7 in mSOF vs. DI/II, respectively), and the gene expression levels (BAX, GLUT1, MnSOD, and OCT4) among the re-expanded blastocysts. Overall, our data reported that the defined in vitro culture media for goat embryos were established with high efficiency, which will be very useful for goat embryo production.


Assuntos
Blastocisto/fisiologia , Criopreservação/veterinária , Técnicas de Cultura Embrionária/veterinária , Cabras/embriologia , Partenogênese/fisiologia , Animais , Clonagem de Organismos , Meios de Cultura , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia
12.
Prion ; 9(4): 278-91, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26217959

RESUMO

Even though prion (encoded by the PRNP gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing Bmi-1 and hTert genes; 2) investigate the disruption of the PRNP in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with Bmi-1and hTert. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the PRNP gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (CSNK2A1, FAM64A, MPG and PRND) were affected after PRNP disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using Bmi-1 and hTert genes, and used TALENs to knockout the PRNP gene in these immortalized cells. The efficient PRNP KO is expected to be a useful technology to develop our understanding of in vitro prion protein functions in cattle.


Assuntos
Príons/genética , Deleção de Sequência/genética , Animais , Bovinos , Linhagem Celular , Células Cultivadas , Encefalopatia Espongiforme Bovina/genética , Predisposição Genética para Doença/genética , Masculino , Regiões Promotoras Genéticas/genética
13.
PLoS One ; 10(3): e0108874, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25815812

RESUMO

Adipose tissue mesenchymal stem cells (ATMSCs) represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of liver regeneration medicine. ATMSCs overexpressing Oct4 and Sox2 (Oct4/Sox2-ATMSCs) showed enhanced proliferation and multipotency. Hence, we hypothesized that Oct4 and Sox2 can increase "transdifferentiation" of ATMSCs into cells of the hepatic lineage. In this study, we generated Oct4- and Sox2-overexpressing human ATMSCs by liposomal transfection. We confirmed the expression of mesenchymal stem cell surface markers without morphological alterations in both red-fluorescent protein (RFP) (control)- and Oct4/Sox2-ATMSCs by flow cytometry. After induction of differentiation into hepatocyte-like cells, the morphology of ATMSCs changed and they began to appear as round or polygonal epithelioid cells. Hepatic markers were evaluated by reverse transcription-polymerase chain reaction and confirmed by immunofluorescence. The results showed that albumin was strongly expressed in hepatogenic differentiated Oct4/Sox2-ATMSCs, whereas the expression level of α-fetoprotein was lower than that of RFP-ATMSCs. The functionality of hepatocytes was evaluated by periodic acid-Schiff (PAS) staining and urea assays. The number of PAS-positive cells was significantly higher and urea production was significantly higher in Oct4/Sox2-ATMSCs compared to that in RFP-ATMSCs. Taken together, the hepatocyte-like cells derived from Oct4/Sox2-ATMSCs were mature hepatocytes, possibly functional hepatocytes with enhanced capacity to store glycogen and produce urea. In this study, we demonstrated the enhanced transdifferentiation of Oct4- and Sox2-overexpressing ATMSCs into hepatocyte-like cells that have enhanced hepatocyte-specific functions. Therefore, we expect that Oct4/Sox2-ATMSCs may become a very useful source for hepatocyte regeneration or liver cell transplantation.


Assuntos
Tecido Adiposo/citologia , Engenharia Celular , Transdiferenciação Celular/genética , Hepatócitos/citologia , Células-Tronco Mesenquimais/citologia , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Humanos , Imunofenotipagem , Proteínas Luminescentes/genética , Transfecção
14.
Zygote ; 23(6): 916-23, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25424059

RESUMO

Genome-editing technologies are considered to be an important tool for generating gene knockout cattle models. Here, we report highly efficient disruption of a chromosomally integrated eGFP gene in bovine somatic cells using RNA-guided endonucleases, a new class of programmable nucleases developed from a bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. In the present study, we obtained homogenously eGFP-expressing primary fibroblasts from cloned bovine transgenic embryonic tissues and employed them for further analysis. CRISPR/Cas9 plasmids specifically targeting the eGFP gene were transfected into the eGFP fibroblasts by electroporation. After 10 days of culture, more than 40% of the cells had lost eGFP expression in fluorescence activated cell sorting (FACS) analysis. Targeted sequences of the transfected cells were analyzed, and various small indel mutations (6-203 bp deletions) in the target sequence were found. The fibroblasts mutated with the CRISPR/Cas9 system were applied for somatic cell nuclear transfer, and the reconstructed embryos were successfully developed into the blastocyst stage. In conclusion, the CRISPR/Cas9 system was successfully utilized in bovine cells and cloned embryos. This will be a useful technique to develop livestock transgenesis for agricultural science.


Assuntos
Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes/métodos , Proteínas de Fluorescência Verde/genética , Animais , Blastocisto/fisiologia , Bovinos , Células Cultivadas , Endonucleases/genética , Feminino , Fibroblastos , Proteínas de Fluorescência Verde/metabolismo , Mutação , Técnicas de Transferência Nuclear , Gravidez , RNA Guia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA