Hi Daniel,

I'm not entirely sure what you mean when you say 'culture effect on phenotypes'. A recent paper from the Field lab may answer your question (Ali & Field, 2013, Experimental Parasitology 134: 249-55); they show that ectopic expression from a rDNA spacer locus declines at high cell density, ie above 3x10^6/ml. Otherwise, I've never noticed any variation in phenotype within the normal density range, ie between 10^4 and 10^6/ml. Obviously, this will depend on the protein that you're working on, and as I said in my previous answer reducing the tet concentration will allow you to reduce the severity of any negative phenotype that you're seeing (particularly in the range of 1-10ng/ml). You should also bear in mind that tetracycline is light sensitive and has a limited half life (~3 d), so the degree of induction will decline over time, if you're not replenishing the medium/drug. Finally, prolonged induction is also not recommended, as the cassette can be lost/mutated if its expression has a detrimental effect on the cells - meaning that phenotypes can appear to change over time (this is particularly common with RNAi cell lines, where revertants are often seen). Hope this goes some way to answering your question.

Daniel- are you working in a prokaryotic or eukaryotic gene expression system?

Daniel- prokaryotic system

I would recommend testing several doses in the range of 20-200ng/mL. Optimal dose may depend on the properties of your reporter gene, and be advised, more is not always better (responses can be biphasic).

Thanks Daniel. That is basically my worry. This is a gene of unknown function. At 1 ng there seem to be varied activity. However, in trypanosomes of which l am working on, as much as 5 ug has been used in some instances. I will definately try out several concentration but l guess one need to know when the effect is due to the tetracycline as a drug and not the gene on is looking at.

Perhaps you can try a control plasmid in parallel with a simple, well-tolerated, reporter gene (GFP or LacZ). This would allow you to measure whether your target gene has any effect distinct from the control case (direct effects of tetracycline, unrelated to your transgene, would be present in both).

Great idea. I do have transfects with GFP from the same construct. Was mindful of the effect of a large reporter such as GFP on my target proteins. I also wanted to look at localization. I will try Tet induction in both. Might be interesting although one might not rule out entirely the possible effect of GFP on the protein especially if it is in a complex

Daniel- I haven't worked in trypanosomes but had some colleagues who worked with T. brucei in Paul Englund's lab. It is a small community and I'm sure they could give you better advice than I. Look up Mark Drew in Internal Medicine at Ohio State University or Soo Hee Lee at Yale University.

Thanks Daniel. Really appreciate it.

Hi Daniel,

I've found that tet-inducible expression in T brucei is usually maximal at 10 to 20 ng/ml, however most people use 1 ug/ml as standard. If you're dealing with toxic expression (significant loss of function RNAi or deleterious over-expression) you can dial the expression over a reasonable dynamic range by using 1 to 10 ng/ml. You also need to be aware that integration of expression cassettes at different rDNA spacer loci leads to different expression levels, especially in bloodstream form parasites (see Alsford et al, 2005, Mol Biochem Parasitology, for details on position effect in T brucei). This will obviously influence the amount of tet you need for each cell line. Hope this helps.

Thanks Sam. Good you commented. I am actually using your pRPa construct. I do see an effect with 1 ug/ml. Just wondering if playing with the Tet concentration will results in different phenotypes.

Happy to help Daniel. 1 ug/ml should always result in expression of the cassette (tag or RNAi; in fact; this concentration is a massive excess), assuming that the cassette is functional (no point mutations, frameshifts, etc), and correctly integrated. Whether or not you see an effect will depend on the sequence being expressed. For example, to deconvolve the consequences of knocking down an essential protein it may be necessary to use a range of tet concentrations. You should also recognise that if you're over expressing a tagged protein, it's not guaranteed that you'll be able to localise it - a short half life, dispersed localisation and failure to complex correctly (etc) can all lead to localisation failure. It's not uncommon to only be able detect a tagged protein by western blotting. In this case, you can get some idea of sub-cellular localisation by doing hypotonic lysis to analyse the soluble and membrane fractions, and ultimately you can do pull downs to look for interacting partner proteins. Probably a bit off-topic towards the end there...

Should've said, glad to hear pRPa (and the 2T1 cells) has made it to the Cape! Which version are you using? Hope it's working well.

The pRPa Tag. Yes it is working well. Although l will like to try out a variations of Tet concentrations to determine the optimal expression levels.

Hi Sam do you notice a culture effect on phenotypes you have generated in trypanosomes in continuous culture, what and how did you overcome that to get consistent results.

According to my experience 1 microgram/ ml Tetracycline is a optimum conc. for induction. Sometimes we need to check different concentrations of Tetracyclin to find out a optimum concentraion for our construct.

Thanks Munender. Do you happen to notice phenotypic differences from the effect of your constructs from transfection when using different concentrations of Tetracycline? Say does different Tetracycline concentrations give you different growth phenotypes

When I used Tetracyclin below 1microgram/ml conc... the induction was less efficient and they took more than 12 days to get induced for my constructs.

Thanks Munender. Do you happen to notice phenotypic differences from the effect of your constructs from transfection when using different concentrations of Tetracycline? Say does different Tetracycline concentrations give you different growth phenotypes

Hi Daniel,

I'm not entirely sure what you mean when you say 'culture effect on phenotypes'. A recent paper from the Field lab may answer your question (Ali & Field, 2013, Experimental Parasitology 134: 249-55); they show that ectopic expression from a rDNA spacer locus declines at high cell density, ie above 3x10^6/ml. Otherwise, I've never noticed any variation in phenotype within the normal density range, ie between 10^4 and 10^6/ml. Obviously, this will depend on the protein that you're working on, and as I said in my previous answer reducing the tet concentration will allow you to reduce the severity of any negative phenotype that you're seeing (particularly in the range of 1-10ng/ml). You should also bear in mind that tetracycline is light sensitive and has a limited half life (~3 d), so the degree of induction will decline over time, if you're not replenishing the medium/drug. Finally, prolonged induction is also not recommended, as the cassette can be lost/mutated if its expression has a detrimental effect on the cells - meaning that phenotypes can appear to change over time (this is particularly common with RNAi cell lines, where revertants are often seen). Hope this goes some way to answering your question.

Thanks very much Sam. This has been most helpful.

Sam-in our lab, we have noticed even for 427 cells, that below 5 and above 30 passages tends to affect drug sensitivity and even more so for adapted strains. Have you experienced such trends? That is what l mean by culture effect. Sorry l needed to be sure you understood me. Thanks

Hi Daniel,

I've seen some variations but I don't think they correspond to what you describe.

As I mentioned in a previous answer, RNAi against an 'essential' target can begin to 'fail' if induction is maintained for too long (typically more than 72 h with a stem-loop), due to the appearance of revertents that have lost the RNAi cassette. Otherwise, drug resistance profiles appear to be stable; at least I've not had cause to think otherwise. Can you give any more details about the cell lines and culture conditions where you see these effects?

For instance, cells including 427 seems to have a shift to more resistance in drug assays after prolonged passages >30 in media compared with same cells brought from stabilates and taken through say 10 passages. Even growth curves seems to be affected . Is that not culture media adaptation? l believe it might not be restricted to trypanosomes as the same has been observed in Leishmania cells and probably other cells in flasks. These are in normal HMI-9 and other culture media. You do think that might not be the case?

I've never tested whether or not the resistance of recombinant 2T1/T. brucei to the selective drugs (ie. phleo and hyg [on the integrated pRPa construct]) changes through time following thawing, as for these experiments I just want to ensure selection of recombinant T. brucei. Once I have what I want I reduce the drug concentration by at least two-fold to a 'maintenance' level.

How do you know that the cells are becoming more resistant? Is it just that growth is improving over time? Could what you're seeing be simply due to slow recovery after thawing?

Bloodstream-form T. brucei grow significantly slower for at least the first 24 h after thawing; this is especially the case if the glycerol stock is transferred straight into medium without any effort to dilute out the glycerol by washing (typically one centrifigation step in 10 ml complete medium, then resuspend the cells in clean drug free medium). This effect can be further exacerbated by immediate addition of the drugs, so the cells should be left in culture for at least a few hours before adding the drugs at the necessary maintenance concentration.

Thanks Sam. That is good information especially using a two-fold reduction in antibiotics and not the normal selection concentration. Will definitely apply that.

One more question? In making a double KO in 2T1 , after obtaining the single KO with antibiotic replacement, do you still add the antibiotic for the first cassette when you are selecting for the second replacement? And at what concentration should that be if you do. Thanks again

Definitely, though 'maintenance' concentration is fine. If you omit the first drug, it's likely that the second KO cassette will recombine with the first one. Obviously, even following this approach there's no guarantee that double knockouts will be generated! Good luck with it.