RESUMEN
Research and development connect technology and innovation to product design. However, the term is often used to refer to only a subset of the necessary disciplines to the exclusion of technical operations. Here, we argue that the importance of technical operations is undeniable, offering possible solutions by drawing on lessons from outdated biotherapeutics production methods and highlighting advances in the field.
Asunto(s)
Tecnología Biomédica/métodos , Tecnología Farmacéutica/métodos , Animales , Tecnología Biomédica/economía , Tecnología Biomédica/legislación & jurisprudencia , Control de Calidad , Proyectos de Investigación/legislación & jurisprudencia , Tecnología Farmacéutica/economía , Tecnología Farmacéutica/legislación & jurisprudenciaRESUMEN
Recent studies have demonstrated the utility of DNA microarray technology in engineering cellular properties. For instance, cellular adhesion, the necessity of cells to attach to a surface in order to to proliferate, was examined by comparing two distinct HeLa cell lines. Two genes, one encoding a type II membrane glycosylating sialyltransferase (siat7e) and the other encoding a secreted glycoprotein (lama4), were found to influence adhesion. The expression of siat7e correlated with reduced adhesion, whereas expression of lama4 correlated with increased adhesion, as shown by various assays. In a separate example, a gene encoding a mitochondrial assembly protein (cox15) and a gene encoding a kinase (cdkl3), were found to influence cellular growth. Enhanced expression of either gene resulted in slightly higher specific growth rates and higher maximum cell densities for HeLa, HEK-293, and CHO cell lines. Another investigated property was the adaptation of HEK-293 cells to serum-free media. The genes egr1 and gas6, both with anti-apoptotic properties, were identified as potentially improving adaptability by impacting viability at low serum levels. In trying to control apoptosis, researchers found that by altering the expression levels of four genes faim, fadd, alg-2, and requiem, apoptotic response could be altered. In the present work, these and related studies in microorganisms (prokaryote and eukaryote) are examined in greater detail focusing on the approach of using DNA microarrays to direct cellular behavior by targeting select genes.
Asunto(s)
Marcación de Gen/métodos , Ingeniería Genética/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Apoptosis , Adhesión Celular , Proliferación Celular , Supervivencia Celular , Medio de Cultivo Libre de Suero , Fermentación , Regulación de la Expresión Génica , Mejoramiento Genético , Células HeLa , Humanos , Microbiología Industrial , Proteínas Recombinantes/biosíntesisRESUMEN
Animal-derived serum is an essential media supplement for mammalian cells in cell culture. For a number of reasons including cost, regulatory concerns, lot inconsistency, potential contamination with adventitious agents, and down-stream processing it is desirable to eliminate the use of serum. Existing protocols designed to adapt cells to serum-free media (SFM) are time-consuming and provide little insight into how the cells adapt. To better understand the physiological responses associated with serum withdrawal and to expedite the adaptation process, a Human Embryonic Kidney-293 (HEK-293) cell line was propagated in 10% fetal bovine serum (FBS) and was progressively adapted to SFM and analyzed at specific serum levels by oligonucleotide microarrays. Of the differentially expressed genes two, early growth response 1 (egr1) and growth arrest specific 6 (gas6), were selected for further analysis based on their level of differential expression, overall expression patterns, and proposed functionalities. HEK-293 cells, propagated in 10% FBS were transfected with egr1 or gas6 and then adapted to SFM. Results indicated that higher expression of either gene moderately enhanced the ability of both cell lines to adapt to SFM. Egr1 appeared to have a greater impact on adaptability than gas6. Results also indicated that specific protein production was unaltered when the expression of egr1 was increased. Flow cytometric analysis revealed increased expression of egr1 was associated with an increase in the percentage of cells in the G2/M phases. These results indicate that enhanced expression of egr1 or gas6 facilitate adaptation to SFM by improving growth and viability.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Mejoramiento Genético/métodos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Riñón/citología , Riñón/fisiología , Ingeniería de Proteínas/métodos , Adaptación Fisiológica/fisiología , Línea Celular , Proliferación Celular , Supervivencia Celular , Medio de Cultivo Libre de Suero , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genéticaRESUMEN
BACKGROUND: Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays. RESULTS: Two genes, cyclin-dependent kinase like 3 (cdkl3) and cytochrome c oxidase subunit (cox15), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15. Moreover, flow cytometric analysis indicated that cells with an insert of cdkl3 were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of cox15 was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than cdkl3. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either cdkl3 or cox15. CONCLUSION: These results are consistent with previous studies on the functionalities of cdkl3 and cox15. For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to cox15 expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.
Asunto(s)
Proliferación Celular , Quinasas Ciclina-Dependientes/metabolismo , Animales , Células CHO , Adhesión Celular/fisiología , Recuento de Células , Supervivencia Celular/fisiología , Inhibición de Contacto , Cricetinae , Cricetulus , Quinasas Ciclina-Dependientes/genética , Complejo IV de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Perfilación de la Expresión Génica , Células HeLa , Humanos , Interfase/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Fenotipo , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Regulación hacia ArribaRESUMEN
The ability to modify cellular properties such as adhesion is of interest in the design and performance of biotechnology-related processes. The current study was undertaken in order to evaluate the effectiveness of modulating cellular adhesion in HeLa cells from a genomics perspective. Using DNA microarrays, differences in gene expression between two phenotypically distinct, anchorage-dependent and anchorage-independent, HeLa cell lines were identified. With the aid of several statistical methods and an extensive literature search, two genes were selected as potential targets for further study: siat7e and lama4. Subsequently, experiments were carried out to investigate the effects of siat7e and lama4 separately, on adhesion in HeLa cells by altering their expression in vivo. Decreasing the expression of siat7e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells using short interfering RNA (siRNA) resulted in greater aggregation (i.e. clumping) and morphological changes as compared to untreated anchorage-independent HeLa cells. Similar effects were seen in anchorage-independent HeLa cells when the expression of lama4 which encodes laminin alpha4, a member of the laminin family of glycoproteins, was enhanced as compared to untreated anchorage-independent HeLa cells. Using a shear flow chamber, an attachment assay was developed; illustrating either increased expression of siat7e or decreased expression of lama4 in anchorage-dependent HeLa cells reduced cellular adhesion. Collectively, the results of this study are consistent with the roles siat7e and lama4 play in adhesion processes in vivo and indicate modifying the expression of either gene can influence adhesion in HeLa cells. The strategy of applying bioinformatics techniques to characterize and manipulate phenotypic behaviors is a powerful tool for altering the properties of various cell lines for desired biotechnology objectives.
Asunto(s)
Movimiento Celular/fisiología , Regulación de la Expresión Génica/fisiología , Adhesión Celular/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Laminina/biosíntesis , Laminina/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Sialiltransferasas/biosíntesis , Sialiltransferasas/genéticaRESUMEN
With advances in robotics, computational capabilities, and the fabrication of high quality glass slides coinciding with increased genomic information being available on public databases, microarray technology is increasingly being used in laboratories around the world. In fact, fields as varied as: toxicology, evolutionary biology, drug development and production, disease characterization, diagnostics development, cellular physiology and stress responses, and forensics have benefiting from its use. However, for many researchers not familiar with microarrays, current articles and reviews often address neither the fundamental principles behind the technology nor the proper designing of experiments. Although, microarray technology is relatively simple, conceptually, its practice does require careful planning and detailed understanding of the limitations inherently present. Without these considerations, it can be exceedingly difficult to ascertain valuable information from microarray data. Therefore, this text aims to outline key features in microarray technology, paying particular attention to current applications as outlined in recent publications, experimental design, statistical methods, and potential uses. Furthermore, this review is not meant to be comprehensive, but rather substantive; highlighting important concepts and detailing steps necessary to conduct and interpret microarray experiments. Collectively, the information included in this text will highlight the versatility of microarray technology and provide a glimpse of what the future may hold.
RESUMEN
Escherichia coli K (JM109) and E. coli B (BL21) are strains used routinely for recombinant protein production. These two strains grow and respond differently to environmental factors such as glucose and oxygen concentration. The differences have been attributed to differential expression of individual genes that constitute certain metabolic pathways that are part of the central carbon metabolism. By implementing a semiparametric algorithm, which is based on a density ratio model, it was possible to compare and quantify the expression patterns of groups of genes involved in several central carbon metabolic pathways. The groups comprising the glyoxylate shunt, TCA cycle, fatty acid, and gluconeogenesis and anaplerotic pathways were expressed differently between the two strains, whereas no differences were apparent for the groups comprising either glycolysis or the pentose phosphate pathway. These results further characterized differences between the two E. coli strains and illustrated the potency of the semiparametric algorithm.
Asunto(s)
Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Algoritmos , Ciclo del Ácido Cítrico/genética , Escherichia coli/clasificación , Ácidos Grasos/metabolismo , Gluconeogénesis/genética , Glucosa/metabolismo , Glucólisis/genética , Glioxilatos/metabolismo , Modelos Biológicos , Modelos Genéticos , Modelos Estadísticos , Vía de Pentosa Fosfato/genética , Especificidad de la EspecieRESUMEN
An experimental malaria transmission blocking vaccine antigen, Pfs25H, expressed and secreted from Pichia pastoris was recovered and purified using a screenless expanded bed column equipped with a rotating fluid distribution system. This column was able to accommodate feed stock, containing 30% biomass, at a flow rate of 300-400 cm/h without affecting column stability. This capability is three times higher than the capability of the expanded bed column currently in use, which is equipped with a perforated plate fluid distribution system; this design could accommodate biomass concentrations of only up to 10%. The screen-less design did not affect the binding capacity, purification level or process yield and, therefore, shorten the process. Purified Pfs25H of 6.4 g were recovered from 37 l of Pichia pastoris culture in one step.