Optimizing the generation of stable neuronal cell lines via pre-transfection restriction enzyme digestion of plasmid DNA
Stuchbury, Grant, and Münch, Gerald (2010) Optimizing the generation of stable neuronal cell lines via pre-transfection restriction enzyme digestion of plasmid DNA. Cytotechnology, 62 (3). pp. 189-194.
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Transfection of mammalian cell lines is a widely used technique that requires significant optimization, including transfection method or product used, DNA vector, cell density, media composition and incubation time. Generation and isolation of stable transfectants from the large pool of untransfected or only transiently transfected cells can be laborious and time-consuming. Transfection of DNA is usually performed with a non-linearized plasmid, since it is assumed that cutting the plasmid beforehand leads to a lower efficiency of transfection or the degradation of linearized DNA by cytosolic nucleases. However, the transfected circular plasmid will be linearized by a random cut within the cell and it might be possible that sensitive parts of the plasmid such as the resistance gene or the gene of interest are destroyed upon linearization. On the other hand, linearizing a plasmid before transfection by a single, defined cut with a selected restriction enzyme in a non-coding area of the gene has the advantage of ensuring the integrity of all necessary gene elements of the plasmid. In this study, we have compared these different methods in order to increase both transient and stable transfection efficiency in mammalian cells. We report that linearization of plasmid DNA prior to transfection can increase both the efficiency of stable clone generation and target gene expression, but is dependant on the site of linearization within the vector.
|Item Type:||Article (Refereed Research - C1)|
|Keywords:||transfection - enzymatic restriction - linearization - stable clones - neuron - EGFP|
|FoR Codes:||11 MEDICAL AND HEALTH SCIENCES > 1109 Neurosciences > 110999 Neurosciences not elsewhere classified @ 100%|
|SEO Codes:||92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920199 Clinical Health (Organs, Diseases and Abnormal Conditions) not elsewhere classified @ 100%|
|Deposited On:||30 May 2011 16:22|
|Last Modified:||18 Oct 2013 01:18|
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