FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1551148190> ?p ?o ?g. }

• W1551148190 abstract "DNA uptake by polyethylenimine (PEI)-mediated transfection was investigated in Chinese hamster ovary (CHO DG44) cells. Rapid DNA uptake was observed with the maximum occurring during the first 45-60 min after addition of PEI-DNA complexes to cells. With longer incubation times, the aggregation of PEI-DNA particles reduced the rate of DNA uptake. At early times after transfection, the kinetics of DNA uptake were constant, suggesting that the limiting factor to PEI transfection was the rate of endocytosis. The most efficient transfection with PEI was observed at a density of 2 x 106 cells/ml and at a PEI:DNA ratio of 2:1 (w/w). DNA uptake at higher PEI:DNA ratios was also efficient, but the toxicity of PEI decreased the recombinant protein yield. The optimal PEI:DNA ratio was found to be related to the number of cells in the culture. Once in the cell, PEI:DNA particles must be disassembled to allow transcription of the recombinant gene. Here their disassembly was investigated using a simple in vitro assay. The particles were formed with either a linear (25 kDa or JetPEITM) or a branched PEI (2, 25, or 750 kDa) and then mixed with varying amounts of a competitor (RNA, DNA, or heparin). The amount of plasmid DNA released from particles was determined by agarose gel electrophoresis or spectroscopy. The stability of the particles to changes in pH, osmolarity, and the PEI:DNA ratio was also determined. Either the presence of heparin or high salt concentrations (1-2 M) yielded complete particle disassembly for all PEIs tested. The addition of RNA to particles formed with linear PEIs or branched 2 kDa PEI resulted in the rapid release of all the plasmid DNA, even at an RNA concentration of 50 µg/ml. However, the presence of RNA at concentrations up to 400 µg/ml induced only partial disassembly of particles formed with branched PEIs of 25 or 750 kDa. In the presence of competitor DNA, slow particle disassembly was observed with particles made with linear 25 kDa PEI, JetPEI, and branched 2 kDa PEI, but DNA release was not seen for particles made with branched PEIs of higher molecular weight. The presence of BSA only resulted in partial disassembly of PEI-DNA particles, and DNA release was not observed after the exposure of particles to acidic pH as low as 3. For all the PEIs tested, their stability increased as the PEI:DNA ratio increased. It was concluded from these studies that branched PEIs have a higher affinity for DNA than linear PEIs and that the intracellular disassembly of PEI-DNA particles may involve interactions between PEI and cellular RNA. In the second part of the work, RNA interference (RNAi) was used to enhance transient gene expression by knockdown of two different mRNAs, the E1A mRNA in human embryonic kidney 293 EBNA (HEK293E) cells and the ube2i mRNA in CHO DG44 cells and HEK293 cells. HEK293 cells are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNAi. The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1α (EF-1α) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to 3-fold. E1A mRNA depletion also resulted in a 2-fold increase in transient expression of a recombinant IgG in suspension cultures when the IgG light and heavy chain genes were driven by the EF-1α promoter. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells. Efficient RNAi-mediated depletion of ube2i mRNA in CHO DG44 cells was accomplished by co-expression of 2 shRNAs targeting two distinct sides in the ube2i mRNA, whereas knockdown with only one of the shRNAs at a time was insufficient for reducing ube2i mRNA levels. The reduction of the ube2i mRNA level in CHO DG44 cells was shown to enhance transient GFP expression 2-fold and IgG expression 2.5-fold. For these experiments, the GFP gene was under the control of the CMV IE promoter and the IgG light and heavy chain genes were under the control of the human EF-1α promoter. Thus, the enhancement of transient gene expression resulting from ube2i mRNA depletion was not promoter-dependent. These results demonstrated that sumoylation has a negative effect on transient gene expression in CHO DG44 and HEK293E cells." @default.
• W1551148190 created "2016-06-24" @default.
• W1551148190 creator A5073401481 @default.
• W1551148190 date "2006-01-01" @default.
• W1551148190 modified "2023-09-23" @default.
• W1551148190 title "The use of RNA interference to increase PEI-mediated transient gene expression in mammalian cells" @default.
• W1551148190 doi "https://doi.org/10.5075/epfl-thesis-3450" @default.
• W1551148190 hasPublicationYear "2006" @default.
• W1551148190 type Work @default.
• W1551148190 sameAs 1551148190 @default.
• W1551148190 citedByCount "0" @default.
• W1551148190 crossrefType "journal-article" @default.
• W1551148190 hasAuthorship W1551148190A5073401481 @default.
• W1551148190 hasConcept C104317684 @default.
• W1551148190 hasConcept C116084860 @default.
• W1551148190 hasConcept C153911025 @default.
• W1551148190 hasConcept C175656101 @default.
• W1551148190 hasConcept C185592680 @default.
• W1551148190 hasConcept C2775925850 @default.
• W1551148190 hasConcept C2778271344 @default.
• W1551148190 hasConcept C40767141 @default.
• W1551148190 hasConcept C54009773 @default.
• W1551148190 hasConcept C54355233 @default.
• W1551148190 hasConcept C552990157 @default.
• W1551148190 hasConcept C55493867 @default.
• W1551148190 hasConcept C81885089 @default.
• W1551148190 hasConcept C86803240 @default.
• W1551148190 hasConceptScore W1551148190C104317684 @default.
• W1551148190 hasConceptScore W1551148190C116084860 @default.
• W1551148190 hasConceptScore W1551148190C153911025 @default.
• W1551148190 hasConceptScore W1551148190C175656101 @default.
• W1551148190 hasConceptScore W1551148190C185592680 @default.
• W1551148190 hasConceptScore W1551148190C2775925850 @default.
• W1551148190 hasConceptScore W1551148190C2778271344 @default.
• W1551148190 hasConceptScore W1551148190C40767141 @default.
• W1551148190 hasConceptScore W1551148190C54009773 @default.
• W1551148190 hasConceptScore W1551148190C54355233 @default.
• W1551148190 hasConceptScore W1551148190C552990157 @default.
• W1551148190 hasConceptScore W1551148190C55493867 @default.
• W1551148190 hasConceptScore W1551148190C81885089 @default.
• W1551148190 hasConceptScore W1551148190C86803240 @default.
• W1551148190 hasLocation W15511481901 @default.
• W1551148190 hasOpenAccess W1551148190 @default.
• W1551148190 hasPrimaryLocation W15511481901 @default.
• W1551148190 hasRelatedWork W1940198378 @default.
• W1551148190 hasRelatedWork W1977057199 @default.
• W1551148190 hasRelatedWork W1980741596 @default.
• W1551148190 hasRelatedWork W1982207984 @default.
• W1551148190 hasRelatedWork W1993616429 @default.
• W1551148190 hasRelatedWork W2000747205 @default.
• W1551148190 hasRelatedWork W2014912523 @default.
• W1551148190 hasRelatedWork W2016742083 @default.
• W1551148190 hasRelatedWork W2074284424 @default.
• W1551148190 hasRelatedWork W2075103893 @default.
• W1551148190 hasRelatedWork W2097943461 @default.
• W1551148190 hasRelatedWork W2112007942 @default.
• W1551148190 hasRelatedWork W2137538430 @default.
• W1551148190 hasRelatedWork W2154023987 @default.
• W1551148190 hasRelatedWork W2154882802 @default.
• W1551148190 hasRelatedWork W2289984170 @default.
• W1551148190 hasRelatedWork W2298008529 @default.
• W1551148190 hasRelatedWork W2467789813 @default.
• W1551148190 hasRelatedWork W3198503509 @default.
• W1551148190 hasRelatedWork W345358815 @default.
• W1551148190 isParatext "false" @default.
• W1551148190 isRetracted "false" @default.
• W1551148190 magId "1551148190" @default.
• W1551148190 workType "article" @default.