Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 5 de 5
Filter
Add more filters










Database
Language
Publication year range
1.
Adv Biochem Eng Biotechnol ; 127: 251-84, 2012.
Article in English | MEDLINE | ID: mdl-22068842

ABSTRACT

Animal cells can be regarded as factories for the production of relevant proteins. The advances described in this chapter towards the development of cell lines with higher productivity capacities, certain metabolic and proliferation properties, reduced apoptosis and other features must be regarded in an integrative perspective. The systematic application of systems biology approaches in combination with a synthetic arsenal for targeted modification of endogenous networks are proposed to lead towards the achievement of a predictable and technologically advanced cell system with high biotechnological impact.


Subject(s)
Biotechnology/methods , Cell Line , Synthetic Biology/methods , Animals , Cell Differentiation/genetics , Humans , Signal Transduction/genetics
2.
Hum Gene Ther ; 21(8): 979-91, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20222806

ABSTRACT

The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line.


Subject(s)
Cell Line/metabolism , Chromosomes , Genetic Loci , Genetic Vectors/metabolism , Retroviridae/genetics , Virus Assembly , Gene Targeting , Genetic Therapy/methods , Promoter Regions, Genetic , Retroviridae/physiology , Transduction, Genetic , Virus Integration
3.
Gene Ther ; 17(2): 272-80, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19865181

ABSTRACT

The clinical application of self-inactivating (SIN) retroviral vectors has been hampered by the lack of reliable and efficient vector production technologies. To enable production of SIN gamma-retroviral vectors from stable producer clones, a new PG13-based packaging cell, known as PG368, was developed. Viral vector expression constructs can be reliably inserted at a predefined genomic locus of PG368 packaging cells by an Flp-recombinase-mediated targeted cassette exchange (RMCE) reaction. A new, carefully designed vector-targeting construct, pEMTAR-1, eliminated the co-packaging of the selectable marker gene used for the identification of successful recombination at the predefined genomic locus and thus, improved the safety of the production system. Selected clones produced vector supernatants at consistent titers. The targeted insertion of therapeutically relevant SIN vectors for chronic granulomatous disease and X-linked severe combined immunodeficiency into PG368 cells results in stable titers within the range necessary for clinical application. The production of retroviral SIN vectors from stable clinical-grade producer cells is feasible and will contribute to the safe production and application of SIN gamma-retroviral vectors for clinical trials.


Subject(s)
DNA Nucleotidyltransferases , Gene Transfer Techniques , Genetic Vectors , Retroviridae/genetics , Cell Line , Feasibility Studies , Gene Targeting , Genetic Therapy/methods , Granulomatous Disease, Chronic/therapy , Humans , Severe Combined Immunodeficiency/therapy
4.
J Biotechnol ; 124(2): 457-68, 2006 Jul 13.
Article in English | MEDLINE | ID: mdl-16529836

ABSTRACT

Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.


Subject(s)
Cell Line/virology , Genetic Vectors/biosynthesis , Genetic Vectors/genetics , Retroviridae/genetics , Transfection/methods , DNA Nucleotidyltransferases/genetics , Gene Expression Regulation/genetics , Genetic Therapy/methods , Humans
5.
Microb Drug Resist ; 11(4): 360-70, 2005.
Article in English | MEDLINE | ID: mdl-16359196

ABSTRACT

In this study, we analyzed the antimicrobial resistance properties and T antigenic types of 511 isolates collected in Lisbon district, Portugal, from throat swabs of healthy subjects (n=341), during 2000-2002 and from diverse infection sites (n=170) of outpatients and inpatients, during 1999-2002. Erythromycin resistance was higher in tonsillitis/pharyngitis (27.4%) and skin infection isolates (21.1%), than in carriage and invasive isolates (

Subject(s)
Anti-Bacterial Agents/pharmacology , Carrier State/microbiology , Drug Resistance, Bacterial , Streptococcus pyogenes/drug effects , Streptococcus pyogenes/isolation & purification , Adolescent , Adult , Agglutination Tests , Bacterial Typing Techniques , Child , Child, Preschool , Colony Count, Microbial , Humans , Infant , Infant, Newborn , Microbial Sensitivity Tests , Polymerase Chain Reaction , Portugal
SELECTION OF CITATIONS
SEARCH DETAIL
...