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Thread: Question regarding protein expression and genotyping

  1. #1 Question regarding protein expression and genotyping 
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    Ok, I have a doubt on the field of molecular biology, and I'd be very gratefull if anyone could help me out with it.

    I'm primally interested in neuroscience, but all the sciences overlap sooner or later to some extent. I was reading a very influential article written by Francis Crick in 2003-2004 about a brain structure called the claustrum. One of the techniques widely employed in the neurosciences to study a structure is to selectively destroy that area, and then make behavioral evaluations of the animal performance in certain tasks.

    The problem with the claustrum is the nature of its anatomy and position: it is fairly small and really close to two other regions, the insular cortex and the putamen. So, a lesion to the claustrum normally yields unwanted damage to these other neighbouring areas.

    In the article, Crick suggests: "Molecular biology could help here: if one or more genes were to be specifically and strongly expressed in neurons of the claustrum, it might be possible to silence this population by the judicious use of genetic techniques (...)"

    Ok, so this is my question. What are the techniques used to localize the DNA sequence responsible for the expression of certain proteins in the cells of a brain region?

    If anyone is patient enough, a detailed explanation of the techniques would be awesome, but the simple metion of the name would be enough, and I will google it.


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    Forum Cosmic Wizard spuriousmonkey's Avatar
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    Ok, so this is my question. What are the techniques used to localize the DNA sequence responsible for the expression of certain proteins in the cells of a brain region?

    1. check the literature.


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    Quote Originally Posted by spuriousmonkey
    Ok, so this is my question. What are the techniques used to localize the DNA sequence responsible for the expression of certain proteins in the cells of a brain region?
    1. check the literature.
    Thanks, that's amazing! I wonder why I didn't think of that before. Maybe I don't know what to search for. Only if I made a topic in a forum asking about what terms and technique names I should do a research on...
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    Forum Cosmic Wizard i_feel_tiredsleepy's Avatar
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    Immunohistochemistry

    Oh sorry, didn't read closely, that would just help you find which cells in the brain were expressing a certain protein. For the DNA sequence you just go to a database and look it up...
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    Now THAT was helpful. I just looked up the Wikipedia article, and apparently that's what I was looking for. Many thanks

    The DNA database in question is a database of Drosophila genes, right? I remember I was watching a lecture on the neural basis of language, and the researcher said the FOXP2 gene (that might be linked in some way to language) had it's name from a gene that exists in the fruit fly, which is quite remarkable. Or at least quite remarkable for someone who doesn't really know much about biology, just like me. Anyway, thanks.
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  7. #6  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    Quote Originally Posted by Kadu
    Quote Originally Posted by spuriousmonkey
    Ok, so this is my question. What are the techniques used to localize the DNA sequence responsible for the expression of certain proteins in the cells of a brain region?
    1. check the literature.
    Thanks, that's amazing! I wonder why I didn't think of that before. Maybe I don't know what to search for. Only if I made a topic in a forum asking about what terms and technique names I should do a research on...
    yeah. as a professional I advise you to read the literature. If you are lucky the expression pattern you are looking for is already published. That saves you ten thousands of research money.

    If your structure is an early structure you can try genepaint.org.

    You want a promotor region that is only valid for a certain region in the brain and you want to discover it blindly? good luck. It ain't happening. Because it is very rare that genes are expressed solely in one single small structure.

    What you usually end up with is a set of genes that characterize certain cells.

    And for that people tend to do hard work. Over many many years. Based on previous research of the own group and other groups.

    If you can excise your brain region you can do a microarray. but not the claustrum. Maybe you can facs the cells, but then you need a marker.

    But honestly check the frikking literature.

    it is filled with expression patterns. You just have the check what matches the claustrum.

    But if you are too lazy to the basic work don't go into science, because it is just that kind of stuff you will be doing.

    I checked pubmed. The claustrum is just a layer of tissue in the brain. So, just check the literature. Your work is probably already done.
    [
    quote]What are the techniques used to localize the DNA sequence responsible for the expression of certain proteins in the cells of a brain region? [/quote]

    If you get a gene that is expressed solely in the claustrum you do a promotor analysis.

    Mind you, the promotor region can be different for all genes that you will find expressed in the claustrum

    c'est tout.

    Oh, you want to selectively destroy the claustrum? Well, that isn't going to be easy.

    I predict roughly that you need about 10-30 years and a budget of 5-100M.
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    Quote Originally Posted by spuriousmonkey
    I checked pubmed. The claustrum is just a layer of tissue in the brain. So, just check the literature. Your work is probably already done.
    The claustrum is a layer of tissue. And? How is this something new? I just said that in my original post.

    The question here is the cognitve function of the claustrum, what kind of information processing it does. The only thing I could find were experiments with lesioned animals in a context of provoked epileptic seizures, and that has nothing to do with cognition.

    And I'm not using research money. I'm not a researcher. I'm just an undergrad interested to know how scientists do it.

    But thanks for the genepaint.org link anyway.
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  9. #8  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    A layer of tissue is impossible to dissect. That is the problem.

    Why don't you go back to dreamland then if you don't want to know about reality.

    ffs
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  10. #9  
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    You should quit being such a major douche. Stop giving half assed answers that do not even yield a proper answer.
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  11. #10  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    I gave a proper answer. Search the literature first. That is what professionals do. You want to hear some answer you want to hear.

    If you can't find the information in the literature you just spend a lot of money.

    One very standard scenario:

    Breed 50-200 mice to the proper stage.
    depends on facilities (DOF): 500-5.000

    Microlaser dissection of claustrum. There aren't really any other options since it is a tissue layer. You could facs if you had markers, but if you had markers you would have stopped at stage one: read the literature. And you would have your proteins.
    DOF: 2.000-10.000

    RNA purification
    DOF: 100-500

    RNA amplification
    DOF: ~100-500

    RNA quality control
    ~DOF 500

    Microarray of claustrum vs random tissue (could be whole brain for instance or your left toe). in triplet.
    4.750 Agilent (outsource that part for sure)

    Analyze results of microarray with software.
    DOF: 1.000-5.000-10.000 (there is free software. Otherwise a software suite can already be 3.000 +)

    Validate microarray analysis, with q-pcr or in situ hybridization.
    DOF, Depends on how many candidates: 5.000-20.000 The sky is the limit.


    pick most promising candidates.
    For free.

    cross fingers.
    For free.

    when finding a suitable candidate construct transgenic mice with floxed allele and reporter function, preferably inducible.
    DOF: ~40.000-60.000


    A year later analyze expression again.
    DOF: ? too many unknowns


    Analyze phenotype of knockout.
    DOF: ? too many unknowns

    When results aren't completely unexpected you are now ready to start real research.
    Costs: priceless.

    Alternatively you can do proteomics, but you can't amplify proteins, and hence you will have to laser microdissect a lot more.



    I didn't really count labour into the costs.

    What i described is a three year project.

    One postdoc: 120.000 - 150.000 gross (3 years)
    One technician 90-120.000 (3 yrs)
    One groupleader: 150.000-?????? (3 yrs)

    And you still don't have any real answers of course after these three years.

    Then the real work starts.

    Who is willing to donate?


    Alternatives:

    Viral work to determine function of candidates in vitro. Nasty business. special facilities required.
    RNAi/morpholinos/miRNAs: many unknowns. How to apply to target tissue?
    High throughput: you need to set up the criteria and system that would make a high throughput system possible. Expensive. Good chance of zero results.
    Random screening with probes: guessing involved. Luck needed.
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  12. #11  
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    Quote Originally Posted by spuriousmonkey
    I didn't really count labour into the costs.

    Come on dude, everyone knows the labor is virtually free since you are using doctoral and postdoctoral students. $20k (USD) is virtually free relative to the knowledge/skills a 3rd year and beyond grad student has.


    Also, be mindful that we all were undergrads once and we all had scientific curiousness that led us into the field of research. Without asking the questions, as Kadu did, the scientific method would never have developed. Further, when you don't have a year of graduate work under your belt, then you don't necessarily know what the techniques might be or the means to search specifically for them. We all know how quickly you can drown in pubmed if you don't have very specific keywords to search for.
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  13. #12  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    although you can get away with 20k if you manage to put a student on a tax free grant, the moment you start talking proper salaries, cheap went out of the window.

    I myself have a 3 year postdoc grant for which is allocated the sum of 160.000 euros.

    That doesn't mean I get that kind of money as a salary. It is just what it costs to pay someone a salary for three years and contribute to overhead costs etc.
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