Hi everyone. Has anyone worked with Raji cell transfection? I recently used AMAXA technology to transfect Raji cells with a CRISPR construct that resulted in a very poor efficiency (~1%). When I tried the same construct in HEK293T cells with Xtreme gene9 I got more than 80% transfected cells plus very high levels of GFP expression that was part of the cassette. Any alternative suggestions for RAJI transfection? I know that Xtreme gene9 probably won't work well on them...
Electroporation is the best route for suspension cell lines, and Raji has some pre-optimized electroporation kits (https://altogen.com/product/raji-electroporation-kit-burkitts-lymphoma-cells-ccl-86/) that can help out with problems in efficiency. One of the major problems will be delivering CRISPR/Cas9 to the nucleus, and usually we expect around 10-15% efficiency if you're trying to replace a gene, so be sure to find optimal routes, like attaching nuclear localization sequences to your Cas9 protein.
Dear Nik,
as far as I know, raji cells are quite difficult to transfect ; this is mainly due to their physiology and their phenotype (issued from suspension cells).
we have recently launched a line of transfection reagents dedicated to CRISPR/Cas9 application.
you can find more information in the link below;
as the transfection reagents are really recent, we have no results on Raji cells but I do think this should be a solution to try.
would you need more information, please do not hesitate to contact me via Researchgate or directly at: [email protected]
best regards,
Cedric
http://www.ozbiosciences.com/content/58-transfection-reagents-for-genome-editing
Dear Nik,
have you tried Amaxa® Cell Line Nucleofector® Kit V, ® Program M-013? I have had about 50 to 60% transfection efficiency.
good luck,
kebria
Hi Kebria,
Thanks for your answer. Both me and a colleague of mine tried it as a first line strategy and got really lousy results (~1,2% transfection efficiency). Now I am trying to find alternative approaches, so any suggestions are greatly appreciated!
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