000105004 001__ 105004
000105004 005__ 20190316234000.0
000105004 0247_ $$2doi$$a10.1002/bit.10309
000105004 022__ $$a0006-3592
000105004 02470 $$2ISI$$a000177549400002
000105004 037__ $$aARTICLE
000105004 245__ $$aBalancing GFP reporter plasmid quantity in large-scale transient transfections for recombinant anti-human Rhesus-D IgG1 synthesis
000105004 260__ $$c2002
000105004 269__ $$a2002
000105004 336__ $$aJournal Articles
000105004 500__ $$aLaboratory of Physical Chemistry of Polymers and Membranes, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland.
000105004 500__ $$aComparative Study
000105004 500__ $$aJournal Article
000105004 500__ $$aUnited States
000105004 520__ $$aUsing transient expression, high amounts (>20 mg/mL) of secreted anti-human Rhesus-D IgG1 were produced in a suspension-adapted HEK293 EBNA cell line (Meissner et al., Biotechnol Bioeng 75: 197-203, 2001). Time of harvest was 3 days after transfection. For the estimation of transfection efficiencies, we routinely co-transfected EGFP reporter DNA. At higher reporter plasmid concentrations, >2% of total transfecting plasmid DNA, a substantial reduction of recombinant antibody synthesis, was observed. This phenomenon was investigated in detail by co-expressing various green fluorescent protein (GFP) reporter constructs, which were targeted at different subcellular locations. Enhanced and humanized GFPs targeted to either the endoplasmic reticulum, the cytosol, or the nucleus reduced recombinant antibody production by 30 to 40% when present at higher concentrations in the transfection solution. The most severe effects were observed when the co-transfected EGFP was targeted to the endoplasmic reticulum, leading to a reduction of up to 80% in the presence of only 5% of reporter DNA. Interestingly, one nuclear-targeted GFP variant that was not codon optimized for expression in human cell lines could be added, to up to almost half of the total amount of transfecting DNA, without adverse effect on antibody production. Although the minimum amount of this reporter DNA needed for fluorescence reading was 10 times higher than for the other variants, it provided a much broader quantity range within which the transfection process could be studied without being negatively affected.
000105004 6531_ $$aAnimals
000105004 6531_ $$aCell Line
000105004 6531_ $$aGene Expression Regulation
000105004 6531_ $$a*Genes
000105004 6531_ $$aReporter
000105004 6531_ $$aGreen Fluorescent Proteins
000105004 6531_ $$aHumans
000105004 6531_ $$aImmunoglobulin G/*biosynthesis/*genetics
000105004 6531_ $$aKidney/cytology/embryology
000105004 6531_ $$aLuminescent Proteins/*genetics
000105004 6531_ $$aMacaca mulatta/genetics
000105004 6531_ $$a*Plasmids
000105004 6531_ $$aQuality Control
000105004 6531_ $$aRecombinant Proteins/biosynthesis/genetics
000105004 6531_ $$aTransfection/*methods
000105004 700__ $$0241004$$aPick, H. M.$$g110882
000105004 700__ $$aMeissner, P.
000105004 700__ $$aPreuss, A. K.
000105004 700__ $$0241802$$aTromba, P.$$g106578
000105004 700__ $$0240645$$aVogel, H.$$g106666
000105004 700__ $$0240398$$aWurm, F. M.$$g107554
000105004 773__ $$j79$$k6$$q595-601$$tBiotechnology and bioengineering
000105004 8564_ $$uhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12209806 $$zURL
000105004 909C0 $$0252009$$pLBTC$$xU10180
000105004 909CO $$ooai:infoscience.epfl.ch:105004$$particle$$qGLOBAL_SET
000105004 937__ $$aLBTC-ARTICLE-2002-006
000105004 970__ $$a20/LBTC
000105004 973__ $$aEPFL$$rREVIEWED$$sPUBLISHED
000105004 980__ $$aARTICLE