Site-specific integration of targeted DNA into animal cell genomes

Koch, Katherine S.; Aoki, Takashi; Wang, Y; Atkinson, Allan E.; Gleiberman, Anatoli S.; Glebov, O K; Leffert, Hyam L.

doi:10.1016/s0378-1119(00)00153-0

Cited by 16 publications

(4 citation statements)

References 31 publications

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“…Various methods have been employed during transfection in an attempt to overcome the problems encountered with random insertion of recombinant genes into chromosomes; for example, the use of episomal vectors Hörtnagel et al, 1995). However, one of the most promising methods involves targeted integration of gene(s) into areas that are thought to be relevant for high expression by "sitespecific recombination" (Bode et al, 2000;Feng et al, 1999;Gorman and Bullock 2000;Koch et al, 2000). Sitespecific recombination using homologous recombination commonly employs one of two systems, the Cre/loxP or the Flp/FRT system (Bode et al, 2000), although other systems have been developed (Ringrose et al, 1997).…”

Section: Approaches To Improve Expression Levels and Ensure Stabilitymentioning

confidence: 99%

Stability of protein production from recombinant mammalian cells

Barnes¹,

Bentley²,

Dickson³

2003

Biotech & Bioengineering

198

171

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One of the most important criteria for successful generation of a therapeutic protein from a recombinant cell is to obtain a cell line that maintains stability of production. If this is not achieved it can generate problems for process yields, effective use of time and money, and for regulatory approval of products. However, selection of a cell line that sustains stability of production over the required time period may be difficult to achieve during development of a therapeutic protein. There are several studies in the literature that have reported on the instability of protein production from recombinant cell lines. The causes of instability of production are varied and, in many cases, the exact molecular mechanisms are unknown. The production of proteins by cells is modulated by molecular events at levels ranging from transcription, posttranscriptional processing, translation, posttranslational processing, to secretion. There is potential for regulation of stability of protein production at many or all of these stages. In this study we review published information on stability of protein production for three industrially important cell lines: hybridoma, Chinese hamster ovary (CHO), and nonsecreting (NS0) myeloma cell lines. We highlight the most likely molecular loci at which instability may be engendered and indicate other areas of protein production that may affect stability from mammalian cells. We also outline approaches that could help to overcome the problems associated with unpredictable expression levels and maximized production, and indicate the consequences these might have for stability of production.

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Section: Approaches To Improve Expression Levels and Ensure Stabilitymentioning

confidence: 99%

Stability of protein production from recombinant mammalian cells

Barnes¹,

Bentley²,

Dickson³

2003

Biotech & Bioengineering

198

171

View full text Add to dashboard Cite

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“…Elements such as locus control regions, insulators, S/MARs and ubiquitous chromatin opening elements, which are known to influence the localised structure of chromatin, have been incorporated into vectors (Benton et al, 2002;Festenstein et al, 1996;Kim et al, 2004;Li et al, 1999;Pikaart et al, 1998). In addition, techniques such as targeted integration of the recombinant genes using site-specific recombination and recombinase-mediated cassette exchange have been used to identify potential ''hot spots'' of expression for subsequent targeted recombinant gene insertion to these specific areas (Baer and Bode, 2001;Barnes et al, 2003;Bode et al, 2000;Feng et al, 1999;Gorman and Bullock, 2000;Koch et al, 2000). However, due to the plasticity of the mammalian genome, even techniques such as these cannot guarantee complete success at the level of selection of cloned cell line, and it is still largely unclear how long-term high-level producers can be identified.…”

Section: Introductionmentioning

confidence: 99%

Molecular analysis of successful cell line selection in transfected GS‐NS0 myeloma cells

Barnes

Bentley

Moy

et al. 2006

Biotech & Bioengineering

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The production of recombinant proteins from mammalian cells is now an essential part of biotechnology. However, despite this importance, the detailed characteristics of good producing cell lines remain largely unknown. The industrially important GS-NS0 mammalian expression system is able to produce large amounts of protein from relatively few copies of recombinant genes. This makes GS-NS0 cell lines ideal candidates to study the consequence of recombinant plasmid transfection in mammalian cells. This study investigated the molecular features of a panel of 17 randomly chosen GS-NS0 cell lines engineered to produce a recombinant antibody. The research analysed antibody production via enzyme-linked immunosorbent assay (ELISA), and investigated the molecular features of the transfectants by Northern, Southern and copy number analysis. The cell lines generated produced a range of antibody concentrations. In addition, for transfectants defined as producers of recombinant antibody there was a positive correlation between specific productivity and heavy chain mRNA expression. The use of Northern and Southern analysis allowed determination of the functional integrity of the transfected plasmid. Over 50% of the transfectants studied had molecular defects at the level of mRNA and/or cDNA. Cell lines were identified with suspected defects in the regulatory regions of transfected genes in addition to cell lines which lacked recombinant genes. Also, "false-positive" cell lines were generated which were able to overcome the GS selection pressure without producing any recombinant antibody. This article discusses these findings in relation to vector design.

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“…Especially for in vivo applications, enzymatic recombination has gained strong importance for excision, inversion, integration and exchange of genetic elements. Conditional knock-out mouse model systems employ site-specific recombination as well as transgenic plant systems (6–8). …”

Section: Introductionmentioning

confidence: 99%

An ultrasensitive site-specific DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy

Jahnz

Schwille

2005

Nucleic Acids Research

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Site-specific exchange of genetic information is mediated by DNA recombinases, such as FLP or Cre, and has become a valuable tool in modern molecular biology. The so far low number of suitable recombinating enzymes has driven current research activities towards alteration of catalytic properties, such as thermostability or recognition sequences. However, identification and analysis of new mutants requires sensitive in vitro activity assays, which traditionally are based on gel electrophoresis. Here, we describe the development of a new sensitive DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy (DC-FCCS), which works in homogenous solution and does not require any separation step such as electrophoresis. The assay was validated with unlabeled FLP recombinase and different fluorescently labeled DNA substrates containing the FLP recognition target (FRT). This strategy fulfills all requirements for possible application in high throughput screening and engineering of new site-specific DNA recombinases starting from the FLP-FRT system, and is easily adjustable to other systems like Cre/loxP.

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Site-specific integration of targeted DNA into animal cell genomes

Cited by 16 publications

References 31 publications

Stability of protein production from recombinant mammalian cells

Stability of protein production from recombinant mammalian cells

Molecular analysis of successful cell line selection in transfected GS‐NS0 myeloma cells

An ultrasensitive site-specific DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy

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