We are transfecting mutations and matched WT genes. We'll be using antibiotic selection and have generated kill curves to determine the optimal concentration of the selectable marker in the cells we're using, but I'm wondering if there's an additional confirmatory step we can do? We'll be extracting and sequencing DNA from the transfects which will confirm the mutation but not the WT. We are considering doing a western blot as if the WT gene is transfected, the protein product should be overexpressed relative to the parental (untransfected) cells.
Has anyone here transfected WT cells and if so, how did you confirm it? Thanks.
i suggest you KD the endogenous protein with a siNA targetting either 5or 3 'UTR and transfect your exogenous wt and construct lacking the 5/3UTR ..with the appropriate ctl..this is what is usually performed..;alternatively, if your gene is not essential..build a KO cell lines with the cas9 system .. it take roughly a month this allows to probe the effect of your mutant ..
Hi naomi,we also try to generate the stable transfectant in our lab.what we use to confirm our transfectant is that we use GFP( also encoded by the plasmids) as an indicator, so during the screen procedure, we can monitor the GFP positive cells, and get the monoclonal GFP positive transfectant,and then WB will be used to check the expression level of several clones.If you don't wish to have a GFP tag to your N- or C-terminus of your protein,you can put a IRES element between your protein and GFP coding sequences,or, alternatively you can insert another promoter like EF1 promoter(if your plasmid bear the CMV promoter) upstream the GFP coding sequence.
You can also measure mRNA levels by RT-qPCR and than confirms the expression with Western Blot.
What are you transfecting into?? Do you aim for integration or it is to be expressed from plasmid??? Have you looked into recodonising your WT gene?
if the WT in plasmid you can genotype the clones for plasmid sequence part. or generally, any exogen DNA that comes from transfection.
You could also tag your proteins of interest with a Flag epitope tag (or other). When you run your western, use an antibody against the tag instead of your protein of interest. Additionally, if your protein is of moderate size (as opposed to being a large protein) you'll see two bands when using antibodies to your protein of interest, one for endogenous and a higher migrating band for your tagged-ectopic protein. The only caveat to be aware of is that sometimes tags can interfere with a proteinss interactions with other proteins.
WT is not expressed in a cell line certainly, depending on the protein. So even endogen protein level is not an issue considering protein expression, but it is not the case for genomic experiments. But it is need a check for sure.
Best would be to establish a KO of the gene in question using the CRISPR technology. Use the KO cells for your study. It will give you an added advantage of a better comparison of results against the untransfected cells. Remember the protein level and response curve is not linear. Otherwise go for fusion proteins. Just tag your protein with any of the tags available (HA, V5 or FLAG) and use antibody against the tag to differentiate endogenous and the over expressed proteins.
Thanks
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