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Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. [ 1 ] [ 2 ] It may also refer to other methods and cell types, although other terms are often preferred: " transformation " is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including ...
If stable, long-term gene expression is desired, stable transfection of cells is more useful. However, since successful integration of a DNA vector into the chromosome is a rare occurrence, this process is more difficult and time-consuming, and is reserved for large-scale protein production, gene therapies, and long-term pharmacology studies.
A typical optical transfection protocol is as follows: [11] 1) Build an optical tweezers system with a high NA objective 2) Culture cells to 50-60% confluency 3) Expose cells to at least 10 μg/mL of plasmid DNA 4) Dose the plasma membrane of each cell with 10-40 ms of focussed laser, at a power of <100 mW at focus 5) Observe transient transfection 24-96h later 6) Add selective medium if the ...
The 293T cell line was created in Michele Calos's lab at Stanford by stable transfection of the HEK 293 cell line with a plasmid encoding a temperature-sensitive mutant of the SV40 large T antigen; it was originally referred to as 293/tsA1609neo. [9]
Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. [3] This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. [4]
Professor Kingston's advancements within the field of biotechnology extend into developing eukaryotic cell transfection protocols, for he also developed two methods of calcium phosphate transfection for transient and stable transfections. [16] These two methods use a precipitate to introduce plasmid DNA into monolayer cell cultures. [16]
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