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CRE-based lineage mapping

  1. Jan 10, 2005 #1

    DocToxyn

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    I am interested in following the fate of cells in which a particular cytosolic receptor/transcription factor has been activated during development. Would something like a CRE recombinase transgenic work for this? Can anyone who worked with this sytem or has working knowledge of it give me a quick and dirty explanation of the CRE technique?
     
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  3. Jan 10, 2005 #2

    Monique

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    The cre/lox method is to introduce mutations when the organism has already matured: you don't have to introduce germline mutations, which can be embryonic lethal.

    The way it works is that you put the cre gene under the control of a (tissue-) specific promotor that you can turn on yourself, or that is turned on in specific tissues only. You also make a construct of your gene of interest with a stop codon just after it's promotor, you surround this stop codon with two lox sites. When cre is not active, your gene will be prematurely terminated and thus not be expressed, when cre is active, the area between the two lox sites will be excised (the part containing the stop codon) and your gene of interest will become active.

    I think it might be more usefull for you to make a lacZ construct that is under the control of the promotor of your cytosolic protein. This way you can visualize the cells that express the protein (they will turn blue if you add gal, since lacZ is produced), and thus follow their fate.

    If you want to introduce a mutant of the protein and your cells dye prematurely before you can study them (which cell system are you using?) the cre/lox system might be an option. I've never used the technique.
     
  4. Jan 10, 2005 #3

    Monique

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    Basically the point of the cre/lox system is that you can induce recombination when and where you want at a specific point in time and with a specific gene.
     
  5. Jan 10, 2005 #4

    Moonbear

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    As Monique suggested, putting a marker under the control of the promoter of interest might be more useful. LacZ is one you can use, which is good if you're going to be studying embryonic expression patterns and need an easy way to look at all the tissues at once. If you have a specific target organ in mind and want to look for cell-specific expression, LacZ can be a bit difficult to work with (hard to do a double-label immunocytochemical identification of the LacZ expressing cells; not necessarily impossible, but it can be difficult), in which case you might want to try something like GFP. GFP can also be useful if you only want to look at expression after a certain point in time/development, because you can use photobleaching to get rid of the signal at your starting point and only examine what is expressed after that time. On the other hand, you can also get antibodies for GFP and easily do immunocytochemical detection which is more sensitive than trying to examine the natural fluorescence of GFP, which can fade with processing. This also gives you the ability to do multiple-label techniques if you need to know more about specific cells expressing your gene of interest.

    So, your choice of marker under the control of the promoter of interest will depend somewhat on what tissue/endpoint you're interested in.
     
  6. Jan 10, 2005 #5

    DocToxyn

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    Moonbear, Monique thanks for the input. Your suggestion for a lacZ reporter gene is well taken, especially considering that I already have that. It is very useful, but only gives me a snapshot of what is going on at a single time of exposure/development. I realize I can set up a timecourse with multiple timed pregnant dams and I'm currently doing that for some endpoints.

    What I was looking for was a way to tag cells at a specific time during development by activating that receptor and then follow them for a longer period of time than the lacZ will most likely allow. I'm interested in what lineage is derived from the cells that express that receptor at the time of exposure and if their function and/or lineage committment is altered. Maybe I can't do this, but I thought getting some info on the CRE system would be the place to start.

    I have thought about a GFP label as well because it would provide us with more options on the immunotox. side of things that also get done in our lab- flow sorting of live cells, etc, but I thought if I was going to create a new transgenic I might try something different.
     
  7. Jan 10, 2005 #6

    Moonbear

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    Oh, I think I see what you're looking for. Have you considered treatment with BrdU at the time of interest? Then you would want to look for the cells containing BrdU as those that were present at the time of exposure. I'm not sure what sort of time course you're looking for. I don't have much experience with using BrdU to know how long it lasts, but at least in theory, it sounds like something that might work for you. I don't know if it gets taken up by all cells though.

    Alternatively, if what you want to do is specifically activate your receptor at a given timepoint, you could put it under control of a conditional promoter, such as one activated by tetracycline, then you just treat with tetracycline when you want your gene expressed (so, it ends up under control of a double promoter, one for your gene of interest to keep it cell specific, and one that is conditional so you can allow it to turn on when you want it to turn on). My understanding is some of those promoters can be leaky (some low level constitutive expression), so this may be a tough undertaking, but if you can get it to work it would be a great tool to have.
     
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