Enrichment strategies for siRNA-transfected cells in an untransfected background

Gene silencing experiments in difficult-to-transfect cells are often hampered by the presence of a background of untransfected cells. We present proof-of-concept data from two different strategies for enrichment of siRNA-transfected cells. In the first approach, a heterologous surface antigen is exp...

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Published inJournal of biotechnology Vol. 130; no. 3; pp. 209 - 212
Main Authors Narz, Frank, Hübner, Silke, Magyar, Silvia, Bielke, Wolfgang, Weber, Martin, Dennig, Jörg
Format Journal Article
LanguageEnglish
Published Lausanne Elsevier B.V 30.06.2007
Amsterdam Elsevier
New York, NY
Subjects
Biological and medical sciences
Biotechnology
CD4 Antigens - metabolism
Cell Line, Tumor
Cell separation
Drug Resistance - genetics
Fundamental and applied biological sciences. Psychology
Humans
RNA, Small Interfering - genetics
RNA, Small Interfering - metabolism
siRNA
Transfection
Transfection - methods
MAPK1
siRNA
Transfection
Cell separation
GAPDH
Small Interference RNA
Separation
RNA interference
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Abstract Gene silencing experiments in difficult-to-transfect cells are often hampered by the presence of a background of untransfected cells. We present proof-of-concept data from two different strategies for enrichment of siRNA-transfected cells. In the first approach, a heterologous surface antigen is expressed from a plasmid that is co-transfected with an siRNA targeting an endogenous mRNA. The surface antigen is then used for enrichment of successfully transfected cells using antibody-coated magnetic particles. In the second strategy, a eukaryotic antibiotic resistance gene is expressed from a co-transfected plasmid. Addition of the corresponding antibiotic 24 h after transfection results in killing of untransfected cells, which can be washed away. Elimination of untransfected cells will allow more accurate interpretation of the effects of gene silencing.
AbstractList Gene silencing experiments in difficult-to-transfect cells are often hampered by the presence of a background of untransfected cells. We present proof-of-concept data from two different strategies for enrichment of siRNA-transfected cells. In the first approach, a heterologous surface antigen is expressed from a plasmid that is co-transfected with an siRNA targeting an endogenous mRNA. The surface antigen is then used for enrichment of successfully transfected cells using antibody-coated magnetic particles. In the second strategy, a eukaryotic antibiotic resistance gene is expressed from a co-transfected plasmid. Addition of the corresponding antibiotic 24h after transfection results in killing of untransfected cells, which can be washed away. Elimination of untransfected cells will allow more accurate interpretation of the effects of gene silencing.
Gene silencing experiments in difficult-to-transfect cells are often hampered by the presence of a background of untransfected cells. We present proof-of-concept data from two different strategies for enrichment of siRNA-transfected cells. In the first approach, a heterologous surface antigen is expressed from a plasmid that is co-transfected with an siRNA targeting an endogenous mRNA. The surface antigen is then used for enrichment of successfully transfected cells using antibody-coated magnetic particles. In the second strategy, a eukaryotic antibiotic resistance gene is expressed from a co-transfected plasmid. Addition of the corresponding antibiotic 24 h after transfection results in killing of untransfected cells, which can be washed away. Elimination of untransfected cells will allow more accurate interpretation of the effects of gene silencing.
Author Magyar, Silvia
Weber, Martin
Narz, Frank
Bielke, Wolfgang
Hübner, Silke
Dennig, Jörg
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10.1002/ijc.2910190505
10.2144/99264st04
10.1038/35078107
10.1016/0076-6879(92)16035-I
10.1016/j.jbiotec.2006.12.015
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Keywords MAPK1
siRNA
Transfection
Cell separation
GAPDH
Small Interference RNA
Separation
RNA interference
Language English
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Lozzio (10.1016/j.jbiotec.2007.04.018_bib5) 1973; 50
De la Luna (10.1016/j.jbiotec.2007.04.018_bib1) 1992; 216
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Snippet Gene silencing experiments in difficult-to-transfect cells are often hampered by the presence of a background of untransfected cells. We present...
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SubjectTerms Biological and medical sciences
Biotechnology
CD4 Antigens - metabolism
Cell Line, Tumor
Cell separation
Drug Resistance - genetics
Fundamental and applied biological sciences. Psychology
Humans
RNA, Small Interfering - genetics
RNA, Small Interfering - metabolism
siRNA
Transfection
Transfection - methods
Title Enrichment strategies for siRNA-transfected cells in an untransfected background
URI https://dx.doi.org/10.1016/j.jbiotec.2007.04.018
https://www.ncbi.nlm.nih.gov/pubmed/17555840
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