Abstract:
BACKGROUND:Chinese hamster ovary (CHO) cells have become the host of choice for the production of recombinant proteins, due to their capacity for correct protein folding, assembly, and posttranslational modifications. The most widely used system for recombinant proteins is the gene amplification procedure that uses the CHO-Dhfr expression system. However, CHO cells are known to have a very unstable karyotype. This is due to chromosome rearrangements that can arise from translocations and homologous recombination, especially when cells with the CHO-Dhfr expression system are treated with methotrexate hydrate. The present method used in the industry for testing clones for their long-term stability of recombinant protein production is empirical, and it involves their cultivation over extended periods of time prior to the selection of the most suitable clone for further bioprocess development. The aim of the present study was the identification of marker genes that can predict stable expression of recombinant genes in particular clones early in the development stage. RESULTS:The transcriptome profiles of CHO clones with stable and unstable recombinant protein production were investigated over 10-weeks of cultivation, using a DNA microarray. We identified 14 genes that were differentially expressed between the stable and unstable clones already at 2 weeks from the beginning of the cultivation. Their expression was validated by reverse-transcription quantitative real-time PCR (RT-qPCR). Furthermore, the k-nearest neighbour algorithm approach shows that the combination of the gene expression patterns of only five of these 14 genes is sufficient to predict stable recombinant protein production in clones in the early phases of cell-line development. CONCLUSIONS:The exact molecular mechanisms that cause unstable recombinant protein production are not fully understood. However, the expression profiles of some genes in clones with stable and unstable recombinant protein production allow prediction of such instability early in the cell-line development stage. We have thus developed a proof-of-concept for a novel approach to eliminate unstable clones in the CHO-Dhfr expression system, which saves time and labour-intensive work in cell-line development.
journal_name
BMC Biotechnoljournal_title
BMC biotechnologyauthors
Jamnikar U,Nikolic P,Belic A,Blas M,Gaser D,Francky A,Laux H,Blejec A,Baebler S,Gruden Kdoi
10.1186/s12896-015-0218-9subject
Has Abstractpub_date
2015-10-23 00:00:00pages
98issn
1472-6750pii
10.1186/s12896-015-0218-9journal_volume
15pub_type
杂志文章abstract:BACKGROUND:New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-va...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-11-6
更新日期:2011-01-14 00:00:00
abstract:BACKGROUND:The use of highly reproducible and spatiallyhomogeneous thin film matrices permits automated microscopy and quantitative determination of the response of hundreds of cells in a population. Using thin films of extracellular matrix proteins, we have quantified, on a cell-by-cell basis, phenotypic parameters of...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-6-14
更新日期:2006-03-06 00:00:00
abstract:BACKGROUND:Recombinant factor VIII (FVIII), used for haemophilia A therapy, is one of the most challenging among the therapeutic proteins produced in heterologous expression systems. Deletion variant of FVIII, in which the entire domain B is replaced by a short linker peptide, was approved for medical use. Efficacy and...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-017-0353-6
更新日期:2017-03-24 00:00:00
abstract:BACKGROUND:Analogues of vitamin D3 are extensively used in the treatment of various illnesses, such as osteoporosis, inflammatory skin diseases, and cancer. Functional testing of new vitamin D3 analogues and formulations for improved systemic and topical administration is supported by sensitive screening methods that a...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-11-33
更新日期:2011-04-07 00:00:00
abstract:BACKGROUND:The photorespiratory nitrogen cycle in C₃ plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO₂. Because of the loss of CO₂ and high energy co...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-11-111
更新日期:2011-11-21 00:00:00
abstract:BACKGROUND:To deal with the increasingly severe energy crisis and environmental consequences, biofuels and biochemicals generated from renewable resources could serve as a promising alternative for replacing petroleum as a source of fuel and chemicals, among which isoprene (2-methyl-1,3-butadiene) in particular is of g...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-016-0236-2
更新日期:2016-01-20 00:00:00
abstract:BACKGROUND:Protein cross-coupling reactions demand high yields, especially if the products are intended for bioanalytics, like enzyme-linked immunosorbent assays. Amongst other factors, the coupling yield depends on the concentration of the proteins being used for coupling. Protein supercharging of enzymes can increase...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-014-0088-6
更新日期:2014-10-18 00:00:00
abstract:BACKGROUND:Waste animal fat is a promising feedstock to replace vegetable oil that widely used in commercial biodiesel process, however the high content of free fatty acid in waste fat makes it unfeasible to be processed with commercial base-catalytic process. Enzymatic process is preferable to convert waste fat into b...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-017-0390-1
更新日期:2017-09-09 00:00:00
abstract:BACKGROUND:Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce la...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-12-78
更新日期:2012-10-30 00:00:00
abstract:BACKGROUND:There is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. One of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. The foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw mat...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-13-94
更新日期:2013-10-31 00:00:00
abstract:BACKGROUND:Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV va...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-7-12
更新日期:2007-02-26 00:00:00
abstract:BACKGROUND:Human TGF-β3 has been used in many studies to induce genes coding for typical cartilage matrix components and accelerate chondrogenic differentiation, making it the standard constituent in most cultivation media used for the assessment of chondrogenesis associated with various stem cell types on carrier matr...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-020-00641-y
更新日期:2020-08-27 00:00:00
abstract:BACKGROUND:Analysis of protein-protein interactions (PPIs) is a valuable approach for the characterization of huge networks of protein complexes or proteins of unknown function. Co-immunoprecipitation (coIP) using affinity resins coupled to protein A/G is the most widely used method for PPI detection. However, this tra...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-10-78
更新日期:2010-10-28 00:00:00
abstract:BACKGROUND:The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity. RESULT...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-019-0590-y
更新日期:2019-12-09 00:00:00
abstract:BACKGROUND:Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. In vivo, the virus infects a subpopulation of tissue macrophages. In vitro, PRRSV only replicates in primary pig macrophages and African green monkey kidney derived cells, such as Marc-145....
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-10-48
更新日期:2010-06-29 00:00:00
abstract:BACKGROUND:One of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for con...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-015-0121-4
更新日期:2015-02-13 00:00:00
abstract:BACKGROUND:Microarrays are powerful tools for DNA-based molecular diagnostics and identification of pathogens. Most target a limited range of organisms and are based on only one or a very few genes for specific identification. Such microarrays are limited to organisms for which specific probes are available, and often ...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-10-13
更新日期:2010-02-17 00:00:00
abstract:BACKGROUND:Xylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by h...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-014-0107-7
更新日期:2014-12-16 00:00:00
abstract:BACKGROUND:Carboxyl-terminal protease (CtpA) plays essential functions in posttranslational protein processing in prokaryotic and eukaryotic cells. To date, only a few bacterial ctpA genes have been characterized. Here we cloned and characterized a novel CtpA. The encoding gene, ctpAp (ctpA of Paenibacillus lautus), wa...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-13-89
更新日期:2013-10-25 00:00:00
abstract:BACKGROUND:Ultrasound and microbubbles (USMB) have been shown to enhance the intracellular uptake of molecules, generally thought to occur as a result of sonoporation. The underlying mechanism associated with USMB-enhanced intracellular uptake such as membrane disruption and endocytosis may also be associated with USMB...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-017-0364-3
更新日期:2017-05-18 00:00:00
abstract:BACKGROUND:We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine. RESULTS:Transformation of plants was only achieved with the HPV-1...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-7-56
更新日期:2007-09-12 00:00:00
abstract:BACKGROUND:Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) has been wildly used to generate gene knockout models through inducing indels causing frame-shift. However, there are few studies concerning the post-transcript effects caused by CRISPR-mediated genome editing...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-018-0472-8
更新日期:2018-10-03 00:00:00
abstract:BACKGROUND:Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-11-14
更新日期:2011-02-07 00:00:00
abstract:BACKGROUND:In the last three decades, the Baculovirus expression vector system (BEV) has evolved to one of the most widely used eukaryotic systems for heterologous protein expression including approved vaccines and therapies. Despite the significant improvements introduced during the past years, the BEV system still ha...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-017-0400-3
更新日期:2017-11-16 00:00:00
abstract:BACKGROUND:Copper is an essential chemical element for life as it is a part of prosthetic groups of enzymes including super oxide dismutase and cytochrome c oxidase; however, it is also toxic at high concentrations. Here, we present the trade-off of copper availability and growth inhibition of a common host used for co...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-016-0251-3
更新日期:2016-02-20 00:00:00
abstract:BACKGROUND:Methylated resveratrol analogs show similar biological activities that are comparable with those of the resveratrol. However, the methylated resveratrol analogs exhibit better bioavailability as they are more easily transported into the cell and more resistant to degradation. Although these compounds are wid...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-14-67
更新日期:2014-07-17 00:00:00
abstract:BACKGROUND:The isothermal amplification of RNA in vitro has been used for the study of in vitro evolution of RNA. Although Qβ replicase has been traditionally used as an enzyme for this purpose, we planned to use norovirus replicase (NV3D(pol)) due to its structural simplicity in the scope of in vitro autonomous evolut...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-13-85
更新日期:2013-10-09 00:00:00
abstract:BACKGROUND:Transformed cells of Escherichia coli DH5-alpha with pGFPuv, induced by IPTG (isopropyl-beta-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-p...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-2-7
更新日期:2002-04-24 00:00:00
abstract:BACKGROUND:Thermally stable α-L-rhamnosidase with cleaving terminal α-L-rhamnose activity has great potential in industrial application. Therefore, it is necessary to find a proper method to improve the thermal stability of α-L-rhamnosidase. RESULTS:In this study, addition of sorbitol has been found to increase the th...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-017-0342-9
更新日期:2017-02-27 00:00:00
abstract:BACKGROUND:Bacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ1-dehydrogenase activity towards various 3-oxosteroids and oth...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-021-00668-9
更新日期:2021-01-13 00:00:00