I'm planning on generating conditional knockout cell lines of apparently essential genes to test the functionality of some fusion proteins in bloodstream form T. brucei. The idea is to knockout the endogenous copies while ectopically expressing a rescue version - with or without the fusion (using pLEW100v5 construct) - under the control of Tetracycline in the "Single Marker" cell line, or one transfected with the SMOx construct from Steve Kelly.
Unfortunately, the expression levels of my genes of interest are highly critical, so that over-expression is lethal and even generating a single allele knockout is very difficult. To overcome this, I was planning on repeating the knockout transfections while inducing expression of the 'rescue' copy at different levels by varying the tetracycline concentration - hopefully I'll hit upon a concentration that results in the right amount of expression to complement the knockout of each individual allele.
My question is (Finally!): can anyone recommend a sensible range of Tetracycline concentrations that will allow me to vary the amount of expression? Does anyone know whether expression level is linearly related to Tetracycline concentration?
Any advice would be greatly appreciated!
Hi Matt,
I've done this sort of thing when trying to show that RNAi knockdown of an 'essential' gene can impact drug EC50, e.g. CATL and suramin. It can be done, unfortunately the dynamic range of tetracycline isn't great. I've found that you get a reasonable range of expression levels between 1 ng and 10 ng/ml tet; beyond this, the expression level quickly flattens off (at least by western blot). I tested this about 9 years ago with SM cells transfected with a pLEW100-related construct, and the result can be found on Dave Horn's lab web site:
http://www.lifesci.dundee.ac.uk/groups/david-horn//inducible-protein-expression-bloodstream-form-t-brucei
Therefore, if I'm dealing with a particularly 'lethal' RNAi knockdown, I titrate the tet concentrations across the following range: 1, 2.5, 5 and 10 ng/ml. The short half life (24 h) of tet may complicate it, if you use this approach over the long-term - when doing conditional knockouts, I've always induced in 1 ug/ml tet (massive excess) and replenished the tet on day 3 of a transfection (obviously, not what you want!).
Hope this helps,
Sam.
Hi Matt,
Something else to bear in mind are position and density effects.
The plasmid you're planning to use targets random rDNA spacer loci, and these support a range of expression levels. This might be advantageous, as you'll be able to select a 'low level' expresser, which may make expression 'tuning' easier.
Recently, a paper out of Mark Field's lab (http://www.ncbi.nlm.nih.gov/pubmed/23538029) showed that protein expression from these loci is sensitive to cell density (BSF cells), so you'll need to be scrupulous about maintaining your cultures below 2x10^6/ml, if you want to maintain the correct expression level.
You should also consider incorporating the native UTRs into your expression construct.
Sam.
Hi Sam,
Many thanks for the very fast and detailed responses.
I remembered the Tet titrations you did for the independent RNAi validation of the drug resistance phenotypes from the RIT-seq experiments, but wasn't sure if the Tet sensitivity range would be the same for protein expression constructs versus RNAi.
From what you say, it looks like the Tet dosing regimen will have to be optimised separately for every single cell line... But at least I now have a sensible range to start from!
Thanks again,
Matt
Hi Matt,
Just a though in regards to what Sam mentioned above about the short half life of tetracycline - doxycycline is more stable and should help reduce the variability over time in your titration experiment.
Hi Sam,
Thanks for the doxycycline tip. Sounds like it may be a more consistent inducer for longer term inductions than Tet. - Do you (or anyone else) happen to know if the kinetics of action of doxycycline are similar to tet? ie: does it have roughly the same dynamic range of induction as tet of 1 - 10 ng per ml that Sam Alsford demonstrated?
Matt
Hi Matthew,
We use it at the same concentration as tetracycline (1ug per ml), but i've never done a comparative titration. From what I read they exhibit similar dose response and induction characteristics, but doxycycline has a 48 hour half life, compared with 24 hours for tetracycline.
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