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[[Category:Neurobiology]] [[Category:Synaptic Plasticity]] [[Category:Neuroinformatics]] [[Category:Genetics]] [[Category:Neuroscience Methods]] [[Category:Methods]] | |||
Latest revision as of 14:19, 5 August 2015
Choosing A Plasmid Backbone
When choosing what plasmid backbone to use, you have many elements to consider. Here is a guide to Addgene's empty vector backbones. For the most part, we will assume that you want to express a gene; however, we have a section at the end for if you are studying a different genetic element or want to express shRNA.
<html> <section id="host">
Species-Specific Expression
If you want to drive expression of your favorite gene, you will need a plasmid with a promoter that will be functional in your host organism.
<thead> </thead> <tbody> </tbody>| Host | Relevant Promoters | Representative Empty Backbones |
|---|---|---|
| Mammalian | CMV, SV40, EF1a, CAG |
|
| Bacteria | Lac, T7, araBAD |
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| Yeast | GAL4, PGK, ADH1, ADE2, TRP1 |
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| Fly | UAS, MT |
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| Worm | unc-54, variety of worm gene promoters |
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| Insect / Baculovirus | Polyhedrin |
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| Xenopus | SP6 for transcription and injection |
|
<a href="#top">Back to Top</a>
</section>
<section id="tag">
Epitope Tag or Fusion Protein
Tags and fusion proteins are excellent tools for further understanding the function of your favorite gene. For example, fusing your protein to an epitope tag, such as Flag or HA, will allow you to easily identify your protein using an antibody against that epitope. This could allow you to conduct western blots or immunoprecipitations of your favorite gene even if you do not have an antibody against it. Another common scenario is fusing your protein to another protein, such as GFP, which allows you to visualize the cellular localization of your protein.
Just remember that when you are designing your plasmid you should keep your gene "in frame" with the fusion protein. This means that the final product should be translated as a single string of amino acids that preserves the sequence of your gene and of the fusion protein.
<thead> </thead> <tbody> </tbody>| Tag or Fusion Protein | Common uses | Representative Empty Backbones |
|---|---|---|
| Flag | Epitope tag |
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| HA | Epitope Tag |
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| Myc | Epitope Tag |
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| 6xHis | Epitope Tag |
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| GST | Protein Purification |
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| GFP | Localization |
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| Other Fluorescent Proteins | Localization | See full <a href="http://www.addgene.org/fluorescent-proteins/">Fluorescent Protein Guide</a> |
| NLS | Nuclear Localization |
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| Myr | Membrane Localization |
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For more information about tags, including amino acid sequences, see our <a href="http://www.addgene.org/mol_bio_reference/genetic_code/#tags">list of common tags</a>.
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</section>
<section id="selection">
Selectable Markers
Regardless of your delivery method, it's unlikely that all of your cells will take up your plasmid. Thus, many plasmids have markers on them so that you can find or select for only the cells that received the plasmid.
<thead> </thead> <tbody> </tbody>| Selectable Marker | Typical Host Organism | Representative Empty Backbones |
|---|---|---|
| Neomycin (G418) | Mammalian |
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| Puromycin | Mammalian |
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| Hygromycin | Mammalian |
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| Zeocin/Bleo | Mammalian |
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| GFP | Varies |
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| URA3 | Yeast |
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| TRP1 | Yeast |
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| LEU2 | Yeast |
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| HIS3 | Yeast |
|
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</section>
<section id="viral">
Viral Expression and Packaging
Although transient expression is sufficient for some experiments, scientists often want to create stable cell lines in which the plasmid is incorporated into the host DNA. For mammalian cells, you can do this through viral delivery. <a href="http://www.addgene.org/viral-vectors/">Visit our viral vector page for more information.</a> Below are some common delivery methods:
<tbody> <thead> </thead> <tbody> </tbody>| Delivery method | Advantages | Representative Empty Backbones |
|---|---|---|
| Lentiviral | Can transduce both dividing and nondividing cells | Addgene has an extensive collection of plasmids for
packaging and expression. See our dedicated <a href="/viral-vectors/lentivirus/">lentiviral plasmid page</a>. |
| Retroviral | Easy and safe to use |
|
| Adenoviral | High transduction efficiency, but do NOT integrate into host genome. <a href="http://www.addgene.org/viral-vectors/aav/">View our AAV page for more information.</a> |
|
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</section>
<section id="special">
Reporters, shRNA expression, transgenics and genome modification
<thead> </thead> <tbody> </tbody>| Element | Details | Representative Empty Backbones |
|---|---|---|
| Promoter | Measure promoter strength |
|
| shRNA/RNAi | For gene silencing experiments |
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| Transgenic | Expression in organisms |
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| TALENs | Gene targeting |
|
</section> </html>