RESUMO
The relative proportion of kynurenine aminotransferase (KAT) I-IV activities in the brain is similar between humans and rats. Moreover, KAT II is considered to be the main enzyme for kynurenic acid production in the brain. Taken together, human KAT II knock-in (hKAT II KI) rats will become a valuable tool for the evaluation of KAT II targeted drugs as a human mimetic model. Although we initially tried the approach by conventional gene-targeting via embryonic stem cells (ESCs) to generate them, we had to give up the production because of no recombinant ESCs. Accordingly, we developed a method to improve the efficiency of homologous recombination (HR) in ESCs by the combination with the CRISPR/Cas system. Co-electroporation of Cas9 plasmid, single guide RNA plasmid and hKAT II KI vector increased the number of drug-resistant colonies and greatly enhanced the HR efficiency from 0 to 36 %. All the clones which we obtained showed the same sequence as designed. These recombinant clones resulted in chimeras that transmitted the hKAT II KI allele to their offspring. hKAT II KI rats showed no reduction of KATs mRNA expression and the amount of kynurenic acid was similar between the hKAT II KI rats and the wild type in their brains. These results indicate that the methodology presented in this report can overcome the problem encountered in conventional gene-targeting that prevented production of humanized rats.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Embrionárias/enzimologia , Marcação de Genes , Transaminases/genética , Animais , Sequência de Bases , Southern Blotting , Células Cultivadas , Células-Tronco Embrionárias/citologia , Feminino , Recombinação Homóloga , Humanos , Ácido Cinurênico/metabolismo , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , Ratos , Ratos Long-Evans , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Transaminases/metabolismoRESUMO
Gene-targeting technology using mouse embryonic stem (ES) cells has become the "gold standard" for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, 2003) harboring 3 mutated genes (APPswe, TauP301L, and PS1M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F1 mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.
Assuntos
Doença de Alzheimer/genética , Células-Tronco Embrionárias/fisiologia , Marcação de Genes/métodos , Camundongos Transgênicos , Peptídeos beta-Amiloides/genética , Animais , Blastocisto/fisiologia , Linhagem Celular , Modelos Animais de Doenças , Células Germinativas , Glicosilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Ratos , Tetraploidia , Proteínas tau/genéticaRESUMO
One of the remarkable achievements in knockout (KO) rat production reported during the period 2008-2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.
Assuntos
Animais Geneticamente Modificados , Células-Tronco Embrionárias , Técnicas de Inativação de Genes , Receptor PAR-2/genética , Animais , Blastocisto/citologia , Técnicas de Cultura de Células , Marcação de Genes , Recombinação Homóloga , Miócitos Cardíacos/citologia , RatosRESUMO
The overnight preservation of bovine ovaries would be highly useful in the subsequent harvest of viable oocytes for reproductive study. The present study aimed to optimize conditions for overnight preservation of bovine ovaries by examining the effects of temperature, solution and supplementation. In Experiment 1, the rate of development to the blastocyst stage of oocytes derived from ovaries preserved at 15°C was higher than that at either 5 or 25°C (p < 0.05). In Experiment 2, the rate of development to the blastocyst stage of oocytes derived from ovaries preserved in University of Wisconsin solution was higher than when PBS or saline was used (p < 0.05). In Experiment 3, oocytes preserved in saline supplemented with 0.3 mM glutathione (GSH) exhibited an increase in the rate of blastocyst formation compared with oocytes supplemented with 0 or 3 mM GSH (p < 0.05). In Experiment 4, supplementation with 10 µM epigallocatechin gallate during ovary preservation increased the rate of blastocyst formation (p < 0.05). The blastocysts derived from ovaries stored in saline supplemented with GSH at 15°C for 24 h were shown to develop into normal offsprings following transfer to recipient heifers. Our studies indicate that bovine IVM/IVF embryos derived from ovaries preserved in saline supplemented with an antioxidant at 15°C for 24 h can successfully develop to the blastocyst stage and result in offspring.
Assuntos
Antioxidantes/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Soluções para Preservação de Órgãos/química , Preservação de Tecido/métodos , Animais , Bovinos , Feminino , Fertilização in vitro , Ovário , Gravidez , Temperatura , Técnicas de Cultura de TecidosRESUMO
Taking advantage of the phenomenon that animal cells adhering to a culture substrate are temporarily immobilized by light irradiation, we established a technique to manipulate the cells adhering to a culture substrate under microscopic observation. Using this technique, we demonstrated a separation of cells adhering to a culture substrate and fabrication of an elaborately patterned co-culture system.
Assuntos
Adesão Celular , Separação Celular/métodos , Luz , Animais , Células CHO , Técnicas de Cocultura , Cricetinae , CricetulusRESUMO
Cell micropatterning, a method to place cells at arbitrary regions, is becoming an essential tool to conduct cell biology and tissue engineering. Conventional cell patterning techniques usually allow only single patterning with single cell type on the same culture surface. However, biomedical research today requires even sophisticated fabrication methods that require spatiotemporal control of multiple cell arrangements. Here we introduce in situ cell micropatterning system which enables stepwise cell patterning using a photoresponsive cell culture surface (PRCS) whose cell adhesiveness could be altered by the UV irradiation. To demonstrate an application to tissue engineering, a liver-mimic tissue array was fabricated and liver-specific gene expressions were quantified with real time PCR. Patterned co-culture systems composed of HepG2 spheroids with Balb/3T3 were fabricated, and the optimum spheroid diameter, which yielded the highest cellular functions, was determined to be 150 microm. After 20 days of patterned co-culture of HepG2 spheroids and Balb/3T3, CYP3A4 expression increased 50-fold higher than conventionally cultured HepG2; CYP3A4 expression was 20% higher than randomly co-cultured HepG2 and Balb/3T3. Thus the combination of PRCS and the photomask-free irradiation apparatus showed the versatility of experimental setups and proved to be a powerful tool for biomedical studies.
Assuntos
Adesão Celular/efeitos da radiação , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual/métodos , Animais , Linhagem Celular , Técnicas de Cocultura , Citocromo P-450 CYP3A/biossíntese , Fibroblastos , Perfilação da Expressão Gênica , Hepatócitos , Humanos , Camundongos , Raios UltravioletaRESUMO
In order to meet the diversifying demand for the cell manipulation in the rapid progress of cell engineering, we developed a novel technique to capture the living cells on a culture substrate by irradiating light in a multiple manner. In clear contrast to the conventional cell patterning using the previously patterned substrate, the cell-retaining area can be defined even after cell seeding, and the captured cells can continue to grow freely beyond the defined area afterwards. After the light irradiation in arbitrary micropattern by using a newly developed apparatus and the process to remove non-captured cells including EDTA treatment, the highly contrasted cell patterns were formed with the precision of single cell size. Moreover, it was determined that the cell capturing arose just after light irradiation and diminished gradually in a time scale of 10h. It was confirmed that the cells maintained their viability well after the manipulation process including photo-induced cell capturing.