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BACKGROUND: The faithful determination of the concentration and viability of yeast cells is important for biological research as well as industry. To this end, it is important to develop an automated cell counting algorithm that can provide not only fast but also accurate and precise measurement of yeast cells. RESULTS: With the proposed method, we measured the precision of yeast cell measurements by using 0%, 25%, 50%, 75% and 100% viability samples. As a result, the actual viability measured with the proposed yeast cell counting algorithm is significantly correlated to the theoretical viability (R2 = 0.9991). Furthermore, we evaluated the performance of our algorithm in various computing platforms. The results showed that the proposed algorithm could be feasible to use with low-end computing platforms without loss of its performance. CONCLUSIONS: Our yeast cell counting algorithm can rapidly provide the total number and the viability of yeast cells with exceptional accuracy and precision. Therefore, we believe that our method can become beneficial for a wide variety of academic field and industries such as biotechnology, pharmaceutical and alcohol production.
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The need for efficient high-throughput gene delivery system for mammalian cells is rapidly increasing with the growing request for functional genomics studies and drug discoveries in various physiologically relevant systems. However, plasmid-based gene delivery has limitations in transfection efficiency and available cell types. Viral vectors have great advantages over plasmid-based vectors, but construction of recombinant viruses remains to be a big hurdle for high-throughput applications. Here we demonstrate a rapid and simple high-throughput system for constructing recombinant adenoviruses which have been used as efficient gene delivery tools in mammalian systems in vitro and in vivo. By combining Gateway-based site-specific recombination with Terminal protein-coupled adenovirus vector, the adenovirus high-throughput system (AdHTS) generates multiple recombinant adenoviruses in 96-well plates simultaneously without the need for additional cloning or recombination in bacteria or mammalian cells. The AdHTS allows rapid and robust cloning and expression of genes in mammalian cells by removing shuttle vector construction, bacterial transformation, or selection and by minimizing effort in plaque isolation. By shortening the time required to convert whole cDNA library into desired viral vector constructs, the AdHTS would greatly facilitate functional genomics and proteomics studies in various mammalian systems.
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Adenoviridae/genética , Clonación Molecular/métodos , Vectores Genéticos , Ensayos Analíticos de Alto Rendimiento/métodos , Recombinación Genética , Biotecnología/métodos , ADN Viral/genética , Células HEK293 , HumanosRESUMEN
Low transfection and expression of target genes has been a problem in gene delivery for clinical therapy. In this study, we present a method for enhancing the transfection efficiency of target genes by electroporation using carrier genes. To evaluate the transfection efficiency, we transfected HeLa cells with luciferase genes (pGL3-control) and then measured the luciferase activity. In our experiments, the prokaryotic expression vector pCR2.1 was used as a carrier gene in the electro-mediated gene delivery. The result shows that the luciferase gene can be effectively transferred into the cell membrane with the aid of carrier genes. In the presence of carrier genes, luciferase activities increased two- to three-fold compared with that in the absence of carrier genes. We also investigated the effect of the weight ratio of luciferase genes to carrier genes on transfection efficiency and found no significant relationship between them. Consequently, we believe that carrier genes are potentially beneficial for promoting transfection and expression of target genes in biological applications.
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Electroporación/métodos , Vectores Genéticos/genética , Transfección/métodos , Electroquímica , Células HeLa , Humanos , Luciferasas/genéticaRESUMEN
CLOCK and BMAL1 are bHLH-PAS-containing transcription factors that bind to E-box elements and are indispensable for expression of core circadian clock components such as the Per and Cry genes. A key step in expression is the heterodimerization of CLOCK and BMAL1 and their accumulation in the nucleus with an approximately 24-h periodicity. We show here that nucleocytoplasmic shuttling of BMAL1 is essential for transactivation and for degradation of the CLOCK/BMAL1 heterodimer. Using serial deletions and point mutants, we identified a functional nuclear localization signal and Crm1-dependent nuclear export signals in BMAL1. Transient-transfection experiments revealed that heterodimerization of CLOCK and BMAL1 accelerates their turnover, as well as E-box-dependent clock gene transcription. Moreover, in embryonic mouse fibroblasts, robust transcription of Per2 is tightly associated with massive degradation of the CLOCK/BMAL1 heterodimer. CRY proteins suppressed this process during the transcription-negative phase and led to nuclear accumulation of the CLOCK/BMAL1 heterodimer. Thus, these findings suggest that the decrease of BMAL1 abundance during the circadian cycle reflects robust transcriptional activation of clock genes rather than inhibition of BMAL1 synthesis.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Procesamiento Proteico-Postraduccional , Transactivadores/metabolismo , Activación Transcripcional , Factores de Transcripción ARNTL , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas CLOCK , Núcleo Celular/metabolismo , Células Cultivadas , Ritmo Circadiano/fisiología , Citoplasma/metabolismo , Dimerización , Células HeLa , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Células 3T3 NIH , Señales de Exportación Nuclear , Señales de Localización Nuclear/química , Estructura Terciaria de Proteína , Transporte de Proteínas , Transcripción Genética/genética , Ubiquitina/metabolismoRESUMEN
Anaplastic thyroid carcinomas are a highly aggressive and extremely lethal form of human cancer, but the biological characteristics related to their aggressive nature are not understood. Moreover, Gadd45 family proteins have been implicated in a variety of growth-regulatory mechanisms, including DNA replication and repair, G(2)/M checkpoint control, and apoptosis. In this study we found that Gadd45gamma RNA was present at significantly lower levels in anaplastic cancer cells, compared with normal primary cultured thyrocytes. In addition, the adenovirus-mediated reexpression of Gadd45gamma significantly inhibited the proliferation of anaplastic thyroid carcinoma cells, ARO, FRO, and NPA cells, which was attributed to apoptosis. Furthermore, the adenovirus-mediated delivery of Gadd45gamma gene in anaplastic thyroid cancer resulted in the inhibition of tumor growth in vivo. This in vitro and in vivo activity of the adenovirus-mediated transduction of CR6/Gadd45gamma, on anaplastic thyroid cancer cell growth suppression, was reminiscent of the effects of p53. This study demonstrates that the Gadd45gamma gene has potential use as a candidate gene for gene therapy in anaplastic thyroid cancer.
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Apoptosis/genética , Carcinoma/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Biosíntesis de Proteínas , Neoplasias de la Tiroides/metabolismo , Adenoviridae/genética , Animales , Northern Blotting , Western Blotting , Carcinoma/genética , Genes p53/genética , Terapia Genética , Humanos , Inmunohistoquímica , Indicadores y Reactivos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas/genética , ARN Neoplásico/biosíntesis , Sales de Tetrazolio , Tiazoles , Neoplasias de la Tiroides/genética , Transducción Genética , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteinas GADD45RESUMEN
The Gadd45 family of proteins includes Gadd45alpha, MyD118/Gadd45beta, and CR6/OIG37/Gadd45gamma. These proteins play important roles in maintaining genomic stability and in regulating the cell cycle. This study reports the cloning of a novel protein called CR6-interacting factor 1 (CRIF1) which interacts with Gadd45alpha, MyD118/Gadd45beta, and CR6/OIG37/Gadd45gamma. CRIF1 binds specifically to the Gadd45 family proteins, as determined by an in vitro glutathione S-transferase pull-down assay and an in vivo mammalian cell two-hybrid assay along with coimmunoprecipitation assays. CRIF1 mRNA is highly expressed in the thyroid gland, heart, lymph nodes, trachea, and adrenal tissues. CRIF1 localizes exclusively to the nucleus and colocalizes with Gadd45gamma. Recombinant CRIF1 inhibits the histone H1 kinase activity of immunoprecipitated Cdc2-cyclin B1 and Cdk2-cyclin E, and the inhibitory effects were additive with Gadd45 proteins. Overexpression of CRIF1 increases the percentage of cells in G1, decreases the percentage of cells in S phase, and suppresses growth in NIH3T3 cells. The down-regulation of endogenous CRIF1 by the transfection of the small interfering RNA duplexes resulted in the inactivation of Rb by phosphorylation and decreased the G1 phase cell populations. Expression of CRIF1 is barely detectable in adrenal adenoma and papillary thyroid cancer and much lower than in adjacent normal tissue. The results presented here suggest that CRIF1 is a novel nuclear protein that interacts with Gadd45 and may play a role in negative regulation of cell cycle progression and cell growth.
