Hi Lorenzo,

I would recommend you target maybe a glycosylase at the top of the BER pathway, as there is a fair bit of evidence, that lower levels of OGG1 for example increase the propensity for the acquisition of point mutations as damaged guanine bases are more likely left incorporated into the strand and can cause mispairs. That's my specific area of knowledge, although other BER/NER components may also work adequately. I'm not entirely sure how you are going to accurately model tobacco carcinogens for a number of reasons: the main one that springs to mind is because there are so many diverse compounds. I have no knowledge on how similar-an effect you will get with shRNA and any given carcinogen - even if you target a single DNA repair gene to get an effect - you will start a cascade of other interactions modulated by that protein, which may have nothing to do with whichever carcinogen you are trying to model. Sounds difficult. Best of luck with it.

References with regards to OGG1:

http://www.ncbi.nlm.nih.gov/pubmed/?term=Kasai+1997+OGG1

http://www.ncbi.nlm.nih.gov/pubmed/?term=Sunaga+2001+OGG1

Try similar approach as in in vitro NER assay which we've done in following paper:

Impaired nucleotide excision repair pathway as a possible factor in pathogenesis of head and neck cancer. Sliwinski T, Markiewicz L, Rusin P, Kabzinski J, Dziki L, Milonski J, Olszewski J, Blaszczyk J, Szemraj J, Majsterek I. Mutat Res. 2011 Nov 1;716(1-2):51-8. doi: 10.1016/j.mrfmmm.2011.08.002. Epub 2011 Aug 19.

PMID: 21875606 [PubMed - indexed for MEDLINE]

You can access this paper on my profile. Briefly it utilizes CPD (cyclobutane pyrimidine dimers) as one of many damage caused by tobacco leading to carcinogens.

Best of luck

Tobacco carcinogens (in which you have to include oxidative stress due to local inflammation, producing G-T transversions) are in fact a huge mix of compounds leading to many kinds of DNA damages: point mutations, deletions, insertions, double strand breaks...I suppose that what you want to enhance is the sensitivity towards the more specific G-T transversion. In my opinion, though it may be possible to do this (targeting OGG1 for example), I doubt that it could lead to a model really mimicking the tobacco carcinogens damages in vivo. Best regards and good luck to you anyway!

As the others mentioned before: The spectrum of DNA damages originating from cigarette smoke is very broad. So mimicking the effect of tobacco smoke by impairing a repair pathway is basically impossible since there is no specific pathway for tobacco related DNA damages.

The question (as always) is what do you want to test?! If you want to increase the frequency of a specific mutation type you'll have to target the according mechanism that results in that particular mutation. This however, is probably also not really possible, since most mutations either arise from an increase in non-repaired damages or from the repair/bypass mechanism itself. Either way the mutation type is mostly dependent on the damage and not so much on the pathway. So your best bet is to actually use smoke condensates to induce mutations.

But if you really want to increase the mutation rate by altering the pathways here are some guidelines/informations:

NER is the most commonly used pathway for DNA repair and therefore very unspecific. Impairing NER will result in a very significant decrease in survival and DNA damages need to be bypassed and can't be repaired efficiently. This means damages in the genome will likely accumulate significantly over time and you will have a very high rate of mutations (but probably even higher lethality).

Impairing BER will give you much smaller frequency of mutations but is pretty specific to oxidative damage and desamination. Both which are not really related to smoke induced lesions, which are mostly adducts.

Impairing HR will increase the amount of trans lesion synthesis which results in a higher mutation rate due to error prone DNA synthesis. I don't really know how important this is for mouse though (I work in Yeast, where you will get a moderate to very high increase in mutation rates dependent on which component of HR you knock out).

Knocking out Mismatch repair will also give raise to a higher mutation rate but these are not related to DNA damages but replication errors.

Anyways. Again the question is what do you want to test with that model. Just increasing mutation rates can be done in different ways but you'll have to choose what will help test your hypothesis.

In any case I would suggest taking a look at Friedberg et al.: DNA repair and mutagenesis. This book is more or less the bible for working on DNA repair and lesion bypass. Otherwise look for reviews on NER, BER, HR, TLS and Mismatch Repair to get more information specifically suited to your needs.

Hope that helps a bit.

Thanks again for the insightful comments.

Indeed I realize it won't be possible to mimic tobacco carcinogens by simply knocking down/out a DNA repair component. I just wanted to use a genetic trick to switch the tumor genetic from being copy-number driven to mutation driven and ideally (very ideally) introduce a similar type of dna damage as tobacco carcinogens.

In my model, constant, fairly unspecific mutational burden shaping the tumor genome is desirable, because that is indeed what smokers experience. I guess the selective pressure will do the rest. The impaired DNA repair will occur only in infected cells.

In this sense, the genetic trick (e.g. an shOGG1- cre virus) is way simpler than exposing mice to carcinogens for prolonged time (dose/schedules are difficult to optimize and effects quite variable). It also solves the problem of systemic effects that I would encounter using a full KO mouse; I cannot afford the mouse to develop many unrelated tumors, and if one day I want to administer chemotherapy to already long-treated mice it might get confusing.

Finally, I think an shOGG1 will do. Ogg1 KO seems to be cause lung cancer in the long run and it's good to know that such genetic deficiency cannot be easily bypassed by other components of the same pathway.

Chose your cigarette brand carefully. They are not all the same with respect to reactive species.