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1.
Trends Biotechnol ; 27(11): 612-4, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19765844

ABSTRACT

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argue that the importance of technical operations is undeniable, offering possible solutions by drawing on lessons from outdated biotherapeutics production methods and highlighting advances in the field.


Subject(s)
Biomedical Technology/methods , Technology, Pharmaceutical/methods , Animals , Biomedical Technology/economics , Biomedical Technology/legislation & jurisprudence , Quality Control , Research Design/legislation & jurisprudence , Technology, Pharmaceutical/economics , Technology, Pharmaceutical/legislation & jurisprudence
2.
Mol Biotechnol ; 39(2): 105-11, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18327555

ABSTRACT

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.


Subject(s)
Gene Targeting/methods , Genetic Engineering/methods , Oligonucleotide Array Sequence Analysis/methods , Apoptosis , Cell Adhesion , Cell Proliferation , Cell Survival , Culture Media, Serum-Free , Fermentation , Gene Expression Regulation , Genetic Enhancement , HeLa Cells , Humans , Industrial Microbiology , Recombinant Proteins/biosynthesis
3.
Biotechnol Bioeng ; 99(6): 1443-52, 2008 Apr 15.
Article in English | MEDLINE | ID: mdl-18023050

ABSTRACT

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.


Subject(s)
Early Growth Response Protein 1/metabolism , Genetic Enhancement/methods , Intercellular Signaling Peptides and Proteins/metabolism , Kidney/cytology , Kidney/physiology , Protein Engineering/methods , Adaptation, Physiological/physiology , Cell Line , Cell Proliferation , Cell Survival , Culture Media, Serum-Free , Early Growth Response Protein 1/genetics , Humans , Intercellular Signaling Peptides and Proteins/genetics
4.
BMC Biotechnol ; 7: 71, 2007 Oct 18.
Article in English | MEDLINE | ID: mdl-17945021

ABSTRACT

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.


Subject(s)
Cell Proliferation , Cyclin-Dependent Kinases/metabolism , Animals , CHO Cells , Cell Adhesion/physiology , Cell Count , Cell Survival/physiology , Contact Inhibition , Cricetinae , Cricetulus , Cyclin-Dependent Kinases/genetics , Electron Transport Complex IV/biosynthesis , Electron Transport Complex IV/metabolism , Female , Gene Expression Profiling , HeLa Cells , Humans , Interphase/physiology , Oligonucleotide Array Sequence Analysis/standards , Phenotype , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/metabolism , Up-Regulation
5.
Metab Eng ; 9(3): 241-51, 2007 May.
Article in English | MEDLINE | ID: mdl-17240181

ABSTRACT

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.


Subject(s)
Cell Movement/physiology , Gene Expression Regulation/physiology , Cell Adhesion/physiology , Gene Expression Profiling , Gene Expression Regulation/drug effects , HeLa Cells , Humans , Laminin/biosynthesis , Laminin/genetics , Oligonucleotide Array Sequence Analysis , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Sialyltransferases/biosynthesis , Sialyltransferases/genetics
6.
Microb Cell Fact ; 6: 4, 2007 Jan 25.
Article in English | MEDLINE | ID: mdl-17254338

ABSTRACT

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.

7.
Genomics ; 89(2): 300-5, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17125967

ABSTRACT

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.


Subject(s)
Carbon/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Oligonucleotide Array Sequence Analysis/statistics & numerical data , Algorithms , Citric Acid Cycle/genetics , Escherichia coli/classification , Fatty Acids/metabolism , Gluconeogenesis/genetics , Glucose/metabolism , Glycolysis/genetics , Glyoxylates/metabolism , Models, Biological , Models, Genetic , Models, Statistical , Pentose Phosphate Pathway/genetics , Species Specificity
8.
Biotechnol Lett ; 28(13): 951-8, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16786267

ABSTRACT

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.


Subject(s)
Cell Culture Techniques/instrumentation , Chromatography, Ion Exchange/instrumentation , Malaria Vaccines/isolation & purification , Malaria Vaccines/metabolism , Pichia/physiology , Protein Engineering/methods , Protozoan Proteins/isolation & purification , Protozoan Proteins/metabolism , Chromatography, Ion Exchange/methods , Equipment Design , Equipment Failure Analysis , Malaria Vaccines/genetics , Protozoan Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
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