Hi All,
We have problem getting good transfection of PX459 by Lipofectamine 3000 into A549 cells (I am not 100% sure, just that we don't get any editing with all 4 gRNA we designed based on SURVEYOR assay results). Do you all use calcium phosphate methods to tranfect cells?
Thank you for sharing your experiences!
Lyra
Transfecting 4 different sequences at once can be risky, and it's likely that you'll need controls to verify that each one was successfully transfected. For a549, lipofection works (https://altogen.com/product/a549-transfection-reagent-lung-carcinoma-ccl-185/) so if you're having problems with transfection efficiency try an a549-targeted transfection reagent and that should boost your results.
Hello
Try with Lipofectamine LTX reagent (Invitrogene,Cat#15388-100)
http://www.farnhamlab.com/protocols/Farnham%20Lab_Puro%20CRISPR%20Protocol_201 60115.pdf
http://research-repository.uwa.edu.au/files/9740124/Li_Ruohan_2016.pdf
Hi,
You can chech if you can use a transfection kit for your cell line of interest , to achieve higher efficinecy. For example, for U2OS cells you can use the Amaxa® Cell Line Nucleofector® Kit V for U2OS cells (Lonza). Additionallly, you can use a GFP plasmid as a positive control for your transfection & puromycin for selection of the transfected clones.
Good luck,
Ester
Hi Lyra,
Adding a GFP gene to your plasmid would help a lot, as mentioned above. You could, if you want, test a couple of different reagents and different amounts of plasmid. But in principle you can then already simply sort for high GFP expressors via FACS and then do a limiting dilution and genotype. Or if you are knocking out a protein for which you could obtain an antibody, then you could even do a subsequent sorting step with the high expressors, to obtain single clones that lack your protein of interest.
Any lipid based transfection method should work fine and provided your Puro selection (switch to puro 16-24 hours after transfection and select for 48 hours) is enriching for your transfectants and your guides are optimal you shouldn't be having any trouble. We do not use Surveyor and find it much easier/cost-effective to perform a quick western on some of the surviving cell pool, if it is working you should see at least 50% knockdown as compared to your control. The cell pool with the greatest decrease in target expression should be progressed to single cell cloning.
We have a number of projects that involve transfection of various materials to such cells. A549 cells are particularly sensitive to lipid based transfection vectors. We have devised our own peptide based materials which have recently been adapted to improve delivery to the nucleus (latest paper - http://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.6b00338). I would be happy to provide these materials as we are very interested to know if they can be effective in the CRISPR system you are using.
Take a look at MTI-GlobalStem's DNA-In CRISPR Transfection Reagent.
It is optimized for large plasmid delivery of CRISPR/Cas9 vectors into a range of cell types.
https://www.mti-globalstem.com/DNAIn-CRISPR-Cas9?search=crispr
Transfecting 4 different sequences at once can be risky, and it's likely that you'll need controls to verify that each one was successfully transfected. For a549, lipofection works (https://altogen.com/product/a549-transfection-reagent-lung-carcinoma-ccl-185/) so if you're having problems with transfection efficiency try an a549-targeted transfection reagent and that should boost your results.
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