Fluorescent Dogs

[Ray Moscow]

(courtesy of Dave Barry's blog):

Scientists Create Fluorescent Puppy (http://news.aol.com/article/glowing-puppy/445317?icid=webmail%7Cwbml-aol%7Cdl3%7Clink3%7Chttp%3A%2F%2Fnews.aol.com%2Far ticle%2Fglowing-puppy%2F445317)

Sometimes you just have to ask, "why?".

[BioBeing]

Sometimes you just have to ask, "why?".

And sometimes, you ask "Why not" :p

[ofro]

As soon as she has a full fur, there won't be much red fluorescence to observe. Mainly just around the mouth.

No need to rush out an buy one. It'll be better to get a GloFish (http://www.glofish.com/video.asp).

[Ray Moscow]

Because we can!

CNN: As world economy collapses, billions face starvation

At least we can create fluorescent dogs, say optimistic scientists

[VoxRat]

Actually, I'm involved in a project to make a fluorescent mouse even as we speak. (Or type, as the case may be).

The serious reason scientists generally have in making such critters, and the reason I have, is that we're using the fluorescent gene as a reporter. We're interested in where (i.e. in which cells in which organs) and when a particular gene is activated - a gene whose product is not easily visualized or locatable. Whatcha do, see, is you take the regulatory sequences for the gene in question, and couple them to the fluorescent gene instead of their usual gene. Then you just examine the animal for which cells, which tissues, fluoresce in response to particular stimuli, or at particular phases in differentiation, and you've learned something valuable about how the gene is controlled. Pretty neat, no? That's why the Nobel Prize in chemistry went to these guys (http://www.sciam.com/article.cfm?id=chemistry-nobel-glows-green) last year.

I don't know if the puppy in this news article had the gene linked to potentially interesting DNA control sequences. The article describes it as a "proof of principle", so I suspect they were just verifying that the approach works in dogs just as well as we've known it works in mice for several years.

[Athier than you]

Dogs like poodles, yorkshire terriers and english bulldogs exist so why not another worthless breed?

[Norrin Radd]

I presume it's named, "Sirius."

[Matty]

what vox said.

GFP, DsREd etc are simply the marker proteins so you can tell that the gene of interest transfected in along with the reporter and assume THAT protein is also being produced. As well as being able to tell about genes switches, triggers promoters, inhibitors etc etc. of course.

A typical example in cell lines we'd use is an over expressed protein that may or may not be implicated in metastasis tagged to a GFP reporter. We can then look at the GFP expressing cells and see if they bear the chemical or morphological hall marks of metastatic cells and infer if there is indeed a role for the protein of interest and if we are lucky, what it is.

I'll be back to gratuiitously spam the thread with pretty fluorescent protein pics in a bit :)

I saw Tsien speak at the last Cytometry conference i went to, he was one of these guys who manage to so enthral a room full of 1000 people that you could have heard a pin drop. Some truly amazing stuff they were doing too. 4d FRET analysis (yeah? Fuck yeah :)) , caged compounds that only become fluorescent when they are in some way modified by the target cell (say, the compound is taken up in hte lysosome and unfolded in particular way - bingo we can see it)

Glad he won that, as a fluorescent imaging specialist, these proteins are kinda important to my job :)

[Matty]

dup.

[Sodong]

4d FRET analysis (yeah? Fuck yeah :)) , caged compounds that only become fluorescent when they are in some way modified by the target cell (say, the compound is taken up in hte lysosome and unfolded in particular way - bingo we can see it)Time resolved FRET? Coupled with FLIM or spectral decon? We don't have a FLIM setup (at least not on-site) but can do the FRET/spectral deconvolution and photo-uncaging on a couple of our instruments. I'm in the midst of designing some experiments with some of his fruits as potentially decent FRET pairs. CFP/YFP kinda bites with diode laser excitation. Anyway, yeah, it's pretty neat. I'd love to hear Roger Tsien speak. Did I read that article correctly? - UV excitation w/ red fluorescence - that's one hell of a stokes shift. How can they be sure they're not just exciting autofluorescence which has that tendency? (ie. excite w/ UV and bleed across the whole spectrum)

Glad he won that, as a fluorescent imaging specialist, these proteins are kinda important to my job :) Yep, mine too. :)

[Matty]

yeah using spectral decon IIRC, it was a couple years back. Beautiful stuff. almost as soon as the attractant was added to the plate you saw the cell spread out and then this burst of fret activity flowing out along the leading edge as the proteins were reconfigured and it started moving. Not heard much about it since though he did say they were still nailing down the details.

UV excitation w/ red fluorescence - that's one hell of a stokes shift. How can they be sure they're not just exciting autofluorescence which has that tendency? (ie. excite w/ UV and bleed across the whole spectrum)i was wondering that myself. I'd have expected a green excitation, that seem to be the case for almost all of the red range. probably a fuck up on the part of the article researcher. i dont know of any fluorochromes with that kind of spectra

yeah we dont have a flim detector, we have the leica sp2 which would be pricey to upgrade (though i did get to play on some shiny new setups with flim fcs and white lasers when i went down to Leica in Penn last summer). its a fun workhorse though, twin photon hooked up and we are doing quite a bit of 3d stuff at the mo. Just as well, my fucking cell sorter died today so i need some techy distraction.

we are getting a peach of a spinning disc in the next year, the grant just got approved etc we just have to wait for all the beaurocracy to play out and we have real time 3d acquisition at our fingertips. As well as confocal intravital video microscopy which my oppo is extremely excited about.

[Sodong]

yeah using spectral decon IIRC, it was a couple years back. Beautiful stuff. almost as soon as the attractant was added to the plate you saw the cell spread out and then this burst of fret activity flowing out along the leading edge as the proteins were reconfigured and it started moving. Eerily beautiful, isn't it? Not heard much about it since though he did say they were still nailing down the details. Somewhere, buried in this heap of papers I call my desk (there is a desk under there somewhere I'm sure) I have what looks like a fairly decent protocol for FRET/spectral decon. I should see if I can find it. :)

UV excitation w/ red fluorescence - that's one hell of a stokes shift. How can they be sure they're not just exciting autofluorescence which has that tendency? (ie. excite w/ UV and bleed across the whole spectrum)i was wondering that myself. I'd have expected a green excitation, that seem to be the case for almost all of the red range. probably a fuck up on the part of the article researcher. i dont know of any fluorochromes with that kind of spectraNo me either. There are a few chromophores that have dual exitation maxima - experimental drugs iirc, and have broad, flat curves beyond 500 nm. The first excitation peak is usually around 300 though and annoyingly just out of range of any of our uv lasers...but I digress. None of the fluorescent proteins have that kind of red shift that I'm aware of. My first thought was that the article's author was mistaken.

yeah we dont have a flim detectorPricey buggers, we have the leica sp2 which would be pricey to upgrade (though i did get to play on some shiny new setups with flim fcsNever done fcs though we hosted a seminar on it some months ago. and white lasers when i went down to Leica in Penn last summer). its a fun workhorse though, twin photon hooked up and we are doing quite a bit of 3d stuff at the mo. Just as well, my fucking cell sorter died today so i need some techy distraction. Aw. We've got an old Zeiss 510 we'd like to retire soon and are just checking under all the empty boxes to see if there's a spare $500K laying around. It's been our workhorse, for sure.

we are getting a peach of a spinning disc in the next year, the grant just got approved etcOh yeah? Cool. We have a DSU and a Yokogawa with a bum 405. We've been looking at several options for reconfiguring it. It had an intensified ccd on it but we removed it and replaced it with a spare Photometrics HQ we had put away. Having the intensified ccd on a scope in a multi-user facility is just plain frightening, as you might imagine. just have to wait for all the beaurocracy to play outHeh. Yeah. We have the pimpdaddy of all beaurocracies...I mustn't speak the name... and we have real time 3d acquisition at our fingertips. As well as confocal intravital video microscopy which my oppo is extremely excited about.An upright confocal that's 2p ready for intravital is on our wish list. We're gonna get a laser scanning cytometer (iCyte) instead though. I've got to go to MA in a while for training.

[Matty]

ooh. lsm is a tool and a half. nice. i'd quite like one of those and one of those imaging flow cytometers to cover all bases, in an ideal and very rich world of course :)

and fwiw the FCS strikes me as a module you are going to shell out for if that is specifically what your lab needs. i doubt we'd use it too much in a core facility. Dunno what yours is like but ours is maybe 75% pretty standard CF images, then the rest is some 3d modeling a, bit of fret and frap from time to time, and the odd spurt of live cell stuff tracking and wound injury that type of thing.

Pissed off about the cell sorter, looks like the same circuit board blew that we replaced about 14 months back for 12 fucking grand. gah. Time for some applications for readies for a new sorter methinks. The newly upgraded moflo looks sweet not that i have much call for 24-36 colours but still it would be nice to say i can......:)


eta we did it agin eh., killed the fluorescence thread with jargon :D

[Sodong]

ooh. lsm is a tool and a half. nice. i'd quite like one of those and one of those imaging flow cytometers to cover all bases, in an ideal and very rich world of course :) Yeah, it's pretty neat, though for some reason they can only provide it with three laser lines. I'll gloat more when we finally get it installed :D

and fwiw the FCS strikes me as a module you are going to shell out for if that is specifically what your lab needs. i doubt we'd use it too much in a core facility.I thought it had pretty limited application for our facility too. Dunno what yours is like but ours is maybe 75% pretty standard CF images, then the rest is some 3d modeling a, bit of fret and frap from time to time, and the odd spurt of live cell stuff tracking and wound injury that type of thing.Very similar though we do quite a bit of live cell work and just got some of our space equipped for doing our own cell culture. We also have some in vivo fluorescence, bioluminescence and microdissection equipment as well as whole slide brightfield scanning. Check out Genie (http://www.aperio.com/newsevents/nov_13_07.asp)

Pissed off about the cell sorter, looks like the same circuit board blew that we replaced about 14 months back for 12 fucking grand. gah.Opted out of the service contract? Time for some applications for readies for a new sorter methinks. The newly upgraded moflo looks sweet not that i have much call for 24-36 colours but still it would be nice to say i can......:)We don't do much of that. It's done in one/some of the other cores.


eta we did it agin eh., killed the fluorescence thread with jargon :DProlly. You never know though, someone might pipe up with a "what the fuck are you talking about" question and I'm sure either of us would be happy to deconstruct all the acronyms...I guess I shouldn't hold my breath while I wait though ;) I need some sleep now anyway.

[Matty]

Opted out of the service contract?
always. we get the odd nasty bill but myself and my colleague are reasonably hand at fixing and rigging (well i'm okay, hes fucking awesome) . Reckon that over the whole instrument base we save about 50-75K a year doing it ourselves. Not that that stops the bosses shitting bricks when you drop a 12grand bill on their desk :)

Panned out this am too, just been on the phone to tech services for a while trying stuff via (to me) incomprehensible dos menus and some underlying controll software etc, and got it going again. Apparently the two things that look identical to a point are the blown board and a dos instrument control file corruption . luckily this time is was the cheaper and quicker one one. woot.

it still needs to be replaced at some point in the next couple years. Its old enough to run out of tech support at the end of 2012, and there has been a serious burst forward in laser and capillary technology let alone processing power since this one was brand new too.

rolly. You never know though, someone might pipe up with a "what the fuck are you talking about" question and I'm sure either of us would be happy to deconstruct all the acronyms...always.

[Sodong]

<snip> Whatcha do, see, is you take the regulatory sequences for the gene in question, and couple them to the fluorescent gene instead of their usual gene. Then you just examine the animal for which cells, which tissues, fluoresce in response to particular stimuli, or at particular phases in differentiation, and you've learned something valuable about how the gene is controlled. <snip> I just got a look today at the latest and greatest in vivo fluorescence tomography instrument (we have an older version of it). I've yet to be convinced that for the money, (it doesn't come cheap) it's a vast improvement over excision and conventional fluorescence imaging.

[Sodong]

Opted out of the service contract?
always. we get the odd nasty bill but myself and my colleague are reasonably hand at fixing and rigging (well i'm okay, hes fucking awesome) . Reckon that over the whole instrument base we save about 50-75K a year doing it ourselves. Not that that stops the bosses shitting bricks when you drop a 12grand bill on their desk :) Heh. I'll bet.

Panned out this am too, just been on the phone to tech services for a while trying stuff via (to me) incomprehensible dos menus and some underlying controll software etc, and got it going again. Apparently the two things that look identical to a point are the blown board and a dos instrument control file corruption . luckily this time is was the cheaper and quicker one one. woot.

it still needs to be replaced at some point in the next couple years. Its old enough to run out of tech support at the end of 2012, and there has been a serious burst forward in laser and capillary technology let alone processing power since this one was brand new too. We're way too busy to have to mess with that stuff and actually have a bit of clout where service contract pricing is concerned. When the instruments fail people usually want it fixed...well, immediately. If it's too much longer than immediately some have a tendency to go nuclear. :) We fix whatever's not covered in the service contracts. You'd be surprised how much that is. If something like the 2P laser ever went belly-up and we didn't have a contract for it we'd be knee deep.

rolly. You never know though, someone might pipe up with a "what the fuck are you talking about" question and I'm sure either of us would be happy to deconstruct all the acronyms...always.So for those of you who don't already know, who wants to know what FRAP, FRET, FLIP, FLIM, FACS, FCS or other fluorescence techniques are all about? Matty and I know lots of F-words and could titillate you with the details :D ... C'mon, aren't you at least a little curious about them?

[Matty]

We're way too busy to have to mess with that stuff and actually have a bit of clout where service contract pricing is concerned. When the instruments fail people usually want it fixed...well, immediately. Thats actually part of the issue for us too. we had downtime before now waiting for the service call where it was so bloody simple that if they sent the part we'd have it done days earlier.

this probably differs depending on where you are i'd imagine.

the previous time this went down we had to wait for the refurbed circuit board to arrive from Cali (2weeks) and BC wouldnt even schedule a service call until the bloody thing was in our hands, in case it was delayed etc, that then took another 10days. the fix itself took about 20minutes.

Luckily the engineer we have knows we know our way round the machines and even for stuff that is out of our league to some degree but not too much., he'll gladly send the relevent protocols and talk us though it if needs be.

[Sodong]

We're way too busy to have to mess with that stuff and actually have a bit of clout where service contract pricing is concerned. When the instruments fail people usually want it fixed...well, immediately. Thats actually part of the issue for us too. we had downtime before now waiting for the service call where it was so bloody simple that if they sent the part we'd have it done days earlier.

this probably differs depending on where you are i'd imagine.
the previous time this went down we had to wait for the refurbed circuit board to arrive from Cali (2weeks) and BC wouldnt even schedule a service call until the bloody thing was in our hands, in case it was delayed etc, that then took another 10days. the fix itself took about 20minutes.

Luckily the engineer we have knows we know our way round the machines and even for stuff that is out of our league to some degree but not too much., he'll gladly send the relevent protocols and talk us though it if needs be.We're a fairly large and central operation and as I said, we have a bit of clout with the manufacturers so when we call they're usually pretty good at getting the problem fixed. We're fairly pushy about it, especially since we've paid for the contracts. :) Still, there is at least one manufacturer who remains smug about their position in our facility. They seem unaware of their error and tend to take their sweet time getting things done.