Nucleofector® Technology and Stable Transfections
The number of stably expression clones resulting from standard transfection experiments is often low, especially for cells which are difficult to transfect. In contrast to lipofection reagents the Nucleofector® technology is well suited even for the generation of stable expressing clones in difficult-to-transfect cell types. Efficient transfection of difficult-to-transfect cell lines enables the application of stable transfectants to address various scientific questions. As an example an application note by Gebreselassie et al. demonstrates the use of Nucleofection® for stably expressing HLA molecules in human lymphoblastoid cells.
Furthermore Nucleofection® was used to establish single-integration clones and clones used for protein production.
Stable Nucleofection® of your specific cell-type
For the transfection of your specific cell-type, we recommend using the respective cell-type specific Nucleofector® Kit together with the Optimized Protocol (for more information on the available Nucleofector® Kits please see our product section). A special protocol optimization for stable transfection is not necessary. Nevertheless, parameters such as ideal from of DNA, culture conditions, seeding densities, G418 concentration as well as clonal analysis still have to be optimized.
For further information on how to generate a stable cell line please download our guideline from this page.
For any additional questions regarding stable transfection with the Nucleofector® Technology please contact our scientific support team.
Please also check our Citation Database for more publications on stable transfection using the Nucleofector® Technology.
Higher integration rates in difficult-to-transfect cell lines using Nucleofection®.
Jurkat cells were transfected using either Nucleofection® (2 µg DNA) or competitor lipofection reagent L (0.7 µg DNA) according to the respective manufacturers instructions. 24 hours after transfection cells were plated on a 96-well plate containing culture medium supplemented with G418 for selection of stably transfected cells. 30 days after plating cells were analyzed for clonal outgrowth (Integration frequency = number of resistant clones per number of living cells seeded). Due to toxic effects lipofection with 10 µg circular DNA could not be performed.
