Cells by design: a mini-review of targeting cell engineering using DNA microarrays.
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.
- SourceAvailable from: Leila Kokabee[Show abstract] [Hide abstract]
ABSTRACT: Cell line development is the most critical and also the most time consuming step in the production of recombinant therapeutic proteins. In this regard a variety of vector and cell engineering strategies have been developed for generating high producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA) producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on CERT and XBP1s genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, or XBP1s were overexpressed in a recombinant t-PA producing CHO cell line. Overexpression of CERT S132A increased specific productivity of t-PA producing CHO cells up to 35%. In contrast, heterologous expression of XBP1s did not affect t-PA expression rate. Our results suggest that CERT S132A based secretion engineering could be an effective strategy for enhancing recombinant t-PA production in CHO cells.Journal of Microbiology and Biotechnology 05/2013; · 1.32 Impact Factor
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ABSTRACT: Increase in both productivity and product yields in biopharmaceutical process development with recombinant protein producing mammalian cells can be mainly attributed to the advancements in cell line development, media, and process optimization. Only recently, genome-scale technologies enable a system-level analysis to elucidate the complex biomolecular basis of protein production in mammalian cells promising an increased process understanding and the deduction of knowledge-based approaches for further process optimization. Here, the use of gene expression profiling for the analysis of a low titer (LT) and high titer (HT) fed batch process using the same IgG producing CHO cell line was investigated. We found that gene expression (i) significantly differed in HT versus LT process conditions due to differences in applied chemically defined, serum-free media, (ii) changed over the time course of the fed batch processes, and that (iii) both metabolic pathways and 14 biological functions such as cellular growth or cell death were affected. Furthermore, detailed analysis of metabolism in a standard process format revealed the potential use of transcriptomics for rational media design as is shown for the case of lipid metabolism where the product titer could be increased by about 20% based on a lipid modified basal medium. The results demonstrate that gene expression profiling can be an important tool for mammalian biopharmaceutical process analysis and optimization.Biotechnology and Bioengineering 02/2010; 105(2):431-8. DOI:10.1002/bit.22549 · 4.16 Impact Factor
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ABSTRACT: Experimental data about the modulation of adhesion and proliferation of anchorage-dependent HeLa cells with monochromatic or quasimonochromatic radiation in red to near-infrared region are presented. Cell adhesion and proliferation can be increased by irradiation with light of certain wavelengths (maxima in action spectrum are 619, 675, 740, 760, and 820 nm) or decreased when the activity of photoacceptor (cytochrome c oxidase in mitochondrial respiratory chain) is inhibited by chemicals before the irradiation. This modality allows controlling the number of attached and/or proliferating cells. Possible biotechnology applications of this method are outlined.International Union of Biochemistry and Molecular Biology Life 01/2011; 63(9):747-53. DOI:10.1002/iub.514 · 2.76 Impact Factor