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Fluoro-Immune Mice

Antibodies are really useful in research, let's make them better.
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So, antibodies are in use in many many many labs, right here in our lab, there are over 1000 different types. They're incredibly useful. In ideal conditions, they can be used to detect a single variant of a single protein among 1000s. Which is nice.

Now, I want to improve them. Simply genetically modify mice/rats/rabbits to produce all of their antibodies with a fluorescent protein tag, such as CFP/YFP/GFP etc.

Then, simply produce antibodies in the normal way: Inoculate, wait a bit, harvest & purify.

There are several reasons why this is great:

1. Their immune system will be fluorescent, which is fun, but not immediately useful to non-immunologists.

2. Antibody purification and quantification may be more effective when assisted and monitored by fluorometry.

3. They will not require secondary antibodies for Immunofluorescence and fluorescent Western Blot experiments. This a: saves between 30mins and 1hr for every experiment of this type ever performed. b: removes 1 layer of complexity and source of false positives.

4. The resolution of microscopy experiments will be improved: Normally, the fluorophore is on the end of the secondary antibody, which is attached to the primary antibody. Each antibody is about 10nm, so, the fluorophore could be anywhere in a 20nm radius of the protein of interest. This cuts that to 10nm. So you can get a better idea of where things are, and it's useful for super- resolution microscopy<link>, which is becoming all easy- peasy lemon-squeezy

5. Co-localization experiments are enhanced by the possibility of using fluorescence resonance electron transfer (FRET)<link>. Normally, you can use 2 separate antibodies to probe for two independent proteins, and look to see if they're in the same place, sort of. Now, it's very difficult to draw strong conclusions from these data, as a microscope's resolution is a few hundred nm. Many proteins that really don't interact at all can be found within a few hundred nm... so it's vague supportive evidence at best. Looks good though, hence it's persistence in the literature. Now, FRET, only works if the proteins are REALLY close, so the CFP antibody you got from a mouse and the YFP antibody from a Rabbit only produce a positive co-localization in a FRET experiment if they're right next to each other or directly interacting.

6. You can attach some of those whizz-bang photoswitchable proteins, like Dronpa, to antibodies... 'cause, you know they're what the with-it kids are all about these days.

Right, got to go and wash my secondary antibody off....

bs0u0155, Nov 14 2012

Superresolution microscopy http://en.wikipedia...solution_microscopy
[bs0u0155, Nov 14 2012]

FRET http://en.wikipedia...nce_energy_transfer
[bs0u0155, Nov 14 2012]

FRET pairs http://www.plosone....ournal.pone.0001916
[bs0u0155, Jan 17 2013]

Ab-FRET http://www.perkinel...tection%20reagents1
[bs0u0155, Jan 17 2013]

[link]






       The title is somewhat misleading, since GM animals (specifically sheep) are WKTE.   

       "Flourescent tagging of GM-produced antibodies" would be more concise.
8th of 7, Nov 14 2012
  

       point taken, done.
bs0u0155, Nov 15 2012
  

       no takers? I thought this was a pretty good idea...
bs0u0155, Jan 17 2013
  

       I am sure its a great idea, but I don't actually know anything about the field, so it is difficult to form an opinion about whether this is a good idea or not.
Kansan101, Jan 17 2013
  

       ^
FlyingToaster, Jan 17 2013
  

       It's actually a pretty good idea, and it makes sense. (Only one caveat - do two antibodies really get close enough for FRET when they bind adjacently to a target? And are various GFP variants good for FRET? I thought they all had similar excitation spectra, but I'm not at all sure.)   

       This is the sort of thing you could base a startup on, if it's not already patented.
MaxwellBuchanan, Jan 17 2013
  

       //And are various GFP variants good for FRET?//   

       Sure are, can do lots of combinations nowadays CFP- YFP, GFP-RFP <link>. I even suspect that triple-FRET is possible, so you could have CFP-GFP-RFP triplets, and prove that proteins A,B & C are adjacent, rather than go through the tedium of doing A&B, then B&C then A&C.
bs0u0155, Jan 17 2013
  

       In that case, I am prepared to add cinnamon to my bun. Why isn't this already done?   

       Whereabouts would you put the GFP? If you put it on the rear end of the antibody you'd block some of its functions. Can you insert it in a constant region, or would this interfere with something? (I really, really ought to know this.)
MaxwellBuchanan, Jan 17 2013
  

       // rear end of the antibody you'd block some of its functions//   

       I should also know this, but as long as the antibodies get made, I don't really care if the critters are immuno compromised. In fact it could be useful, if they were like nude mice, and had to be kept as such, then the protein you exposed them to would be the only pseudopathogenic protein they'd EVER been exposed to, so your antibodies wouldn't have the "noise" of background immunity.   

       Actually, could you simply mess about with a immune stem-cell line, give a nude mouse a bone marrow transplant and you're done? it wouldn't be heritable, but you could freeze down the appropriate stem cell line for each antibody, then start/stop production in a bank of multi-purpose nude mice at any time. You'd only need ~100,000 or so to cover the proteome, which is about 260 384 well plates. So, one freezer's worth, with a bit of room left for super-fast emergency wine chilling.
bs0u0155, Jan 18 2013
  

       must find a tame immunologist.
bs0u0155, Jan 18 2013
  

       // You'd only need ~100,000 or so to cover the proteome//   

       You'd actually want to create a line of mice with GFP inserted in the antibody C-region genes, then use those mice to raise antibodies against the target protein you're interested in, shirley?   

       In fact, you'd want a few lines with different GFP variants in their C-genes so that you could (for example) raise a green antibody against protein A and a red antibody against protein B. Or did I miss your point?
MaxwellBuchanan, Jan 19 2013
  

       Yes that was the original point exactly. I was just thinking of an alternative which might allow a little more commercial responsiveness. You could synthesize peptides corresponding to a bunch of known proteins, inoculate a whole lot of mice, wait for a response then store the corresponding bone marrow in the freezer, then when a request comes in, you can scale up super quick in nude mice. Thinking a little harder, it's actually easier to go recombinant at that point.
bs0u0155, Jan 19 2013
  

       Ah, right, sorry. Problem is, you'll be making a lot of lines, most of which will never be ordered. Moreoveralso, how many of your peptides would evoke responses that worked against the full proteins?
MaxwellBuchanan, Jan 19 2013
  

       yup, which is why the original idea's best.
bs0u0155, Jan 19 2013
  
      
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