Document Detail


Generation of recombinant Chinese hamster ovary cell lines by microinjection.
MedLine Citation:
PMID:  16614902     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was cytoplasmic microinjection. Under optimal conditions, 80-90% of the cells were GFP-positive 1 day after microinjection into the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell lines.
Authors:
Madiha Derouazi; Rachel Flaction; Philippe Girard; Maria de Jesus; Martin Jordan; Florian M Wurm
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biotechnology letters     Volume:  28     ISSN:  0141-5492     ISO Abbreviation:  Biotechnol. Lett.     Publication Date:  2006 Mar 
Date Detail:
Created Date:  2006-04-14     Completed Date:  2006-07-28     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8008051     Medline TA:  Biotechnol Lett     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  373-82     Citation Subset:  IM    
Affiliation:
Laboratory of Cellular Biotechnology, Institute of Biological Engineering and Biotechnology, Ecole Polytechnique Fédérale de Lausanne, Lausanne, CH-1015, Switzerland.
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MeSH Terms
Descriptor/Qualifier:
Animals
CHO Cells
Cell Line* / metabolism
Cricetinae
Cricetulus
Gene Transfer Techniques*
Genes, Reporter
Green Fluorescent Proteins / biosynthesis*,  genetics,  metabolism
Microinjections / methods*
Plasmids / genetics
Recombinant Proteins / biosynthesis,  genetics
Transfection
Chemical
Reg. No./Substance:
0/GFP10 Protein; 0/Recombinant Proteins; 147336-22-9/Green Fluorescent Proteins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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