Generation of recombinant Chinese hamster ovary cell lines by microinjection.
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.
Derouazi
Madiha
M
Laboratory of Cellular Biotechnology, Institute of Biological Engineering and Biotechnology, Ecole Polytechnique Fédérale de Lausanne, Lausanne, CH-1015, Switzerland.
Flaction
Rachel
R
Girard
Philippe
P
de Jesus
Maria
M
Jordan
Martin
M
Wurm
Florian M
FM
D000818:Animals; D016466:CHO Cells; D002460:Cell Line / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008845:Microinjections / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
C485191:GFP10 Protein; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins