difficulty with paGFP

[Aryeh Weiss]

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This is a not-really-confocal question, but I think it is well within
the list's expertise.

We have been trying to create  a stable line of B16 melanoma cells which
will express photoactivatable GFP, and can be lit up whenever we like.
So we transfected with what we think is a suitable plasmid
(11910 pPAGFP-C1 from Addgene), and sure enough, we have  cells that
survive the selection marker (Neo) while the controls die.
So we think we have our stable cell line.

Problem is -- none of them light up. They dont light up in a widefield
scope,  and they dont light up in the confocal, when zapped with 405nm
excitation (we tried different exposures, from 100ms to many seconds).

So I must be missing something obvious, because this is not a new
technology. If someone can point me in the right direction, that would
be wonderful.

--aryeh
--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

[Sarang Kulkarni]

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Screen your stable cell lines by PCR to confirm if the gfp gene is still there.

Sarang Kulkarni

Sent from my iPhone

On 2013-06-28, at 3:02 AM, "Aryeh Weiss" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> This is a not-really-confocal question, but I think it is well within the list's expertise.
>
> We have been trying to create  a stable line of B16 melanoma cells which will express photoactivatable GFP, and can be lit up whenever we like.
> So we transfected with what we think is a suitable plasmid
> (11910 pPAGFP-C1 from Addgene), and sure enough, we have  cells that survive the selection marker (Neo) while the controls die.
> So we think we have our stable cell line.
>
> Problem is -- none of them light up. They dont light up in a widefield scope,  and they dont light up in the confocal, when zapped with 405nm
> excitation (we tried different exposures, from 100ms to many seconds).
>
> So I must be missing something obvious, because this is not a new technology. If someone can point me in the right direction, that would be wonderful.
>
> --aryeh
> --
> Aryeh Weiss
> Faculty of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
>
> Ph:  972-3-5317638
> FAX: [phone]

[George McNamara]

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If the PCR is successful, still leaves to do RT-PCR to see if the
messenger RNA is still there (and the right size). The promoter may not
work well in your cells.
May be simpler to do immunofluorescence (with red or far-red
fluorophore), with an EGFP cell line as a positive control.

Several months ago I mentioned in a posting Michael Davidson's five
favorite photoactivatable/convertible/etc proteins:

Michael Davidson, who told me: "The best photoswitchable proteins are tdEos, mEos4, PS-CFP2, PA-mCherry1, and Dendra2."

http://lists.umn.edu/cgi-bin/wa?A2=ind1304&L=confocalmicroscopy&P=11619 
<http://lists.umn.edu/cgi-bin/wa?A2=ind1304&L=confocalmicroscopy&P=11619>

Consider using one of those instead. Caveat - Dendra2 is in addgene, but
only in C elegans or no expression plasmids. You can ask Michael
Davidson about availability, and possibly a stable cell line from one of
his publications. mEos4 is from Loren Looger's lab, and is described at
https://www.linkedin.com/pub/maria-gabriela-paez-segala/61/978/b55

    In particular, I have made substantial progress in engineering a
    version of mEos with increased protein stability (mEos2 to mEos4).
    mEos2 was the last fluorescent protein designed by Sean McKinney in
    the Looger lab. It is a photoconvertible fluorescent protein
    (changes fluorescence from green to red by hitting with UV light),
    and basically is the best standard photoconvertible FP (fluorescent
    protein) until date. Our codename for a better mEos2 now is mEos4. I
    first began by working on improving the thermal stability, and
    currently I am specifically working on fixatives and
    auto-fluorescence of some variants of mEos2. With the electron
    microscopy (EM) team, Richard Fetter, Yalin Wang, and Mei Sun, our
    main discovery has been a new protocol for EM that will allow us to
    use CLEM for any fluorophore and fluorescent protein, by decreasing
    the auto-fluorescence of fixatives, the harsh conditions of OsO4,
    and increasing the ultra-structure preservation of cell tissue.

Hopefully mEos4 will be published soon - you could email Loren Looger at
HHMI Janelia Farm
http://www.hhmi.org/research/groupleaders/looger_bio.html

Or, consider buying rsEGFP2 or Dreiklang from Abberior
http://www.abberior.com/products/productlist/cat/fluorescent-proteins/prod/abberior-rsegfp/


best wishes,

George


On 6/28/2013 5:48 AM, Sarang Kulkarni wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Screen your stable cell lines by PCR to confirm if the gfp gene is still there.
>
> Sarang Kulkarni
>
> Sent from my iPhone
>
> On 2013-06-28, at 3:02 AM, "Aryeh Weiss"<[hidden email]>  wrote:
>
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> This is a not-really-confocal question, but I think it is well within the list's expertise.
>>
>> We have been trying to create  a stable line of B16 melanoma cells which will express photoactivatable GFP, and can be lit up whenever we like.
>> So we transfected with what we think is a suitable plasmid
>> (11910 pPAGFP-C1 from Addgene), and sure enough, we have  cells that survive the selection marker (Neo) while the controls die.
>> So we think we have our stable cell line.
>>
>> Problem is -- none of them light up. They dont light up in a widefield scope,  and they dont light up in the confocal, when zapped with 405nm
>> excitation (we tried different exposures, from 100ms to many seconds).
>>
>> So I must be missing something obvious, because this is not a new technology. If someone can point me in the right direction, that would be wonderful.
>>
>> --aryeh
>> --
>> Aryeh Weiss
>> Faculty of Engineering
>> Bar Ilan University
>> Ramat Gan 52900 Israel
>>
>> Ph:  972-3-5317638
>> FAX: [phone]
>>      
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054

[Aryeh Weiss]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Many thanks for the quick and informative responses to my question about
paGFP.
They provide me with a number of ideas to try.

--aryeh


On 6/28/13 3:27 PM, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If the PCR is successful, still leaves to do RT-PCR to see if the
> messenger RNA is still there (and the right size). The promoter may not
> work well in your cells.
> May be simpler to do immunofluorescence (with red or far-red
> fluorophore), with an EGFP cell line as a positive control.
>
> Several months ago I mentioned in a posting Michael Davidson's five
> favorite photoactivatable/convertible/etc proteins:
>
> Michael Davidson, who told me: "The best photoswitchable proteins are
> tdEos, mEos4, PS-CFP2, PA-mCherry1, and Dendra2."
>
> http://lists.umn.edu/cgi-bin/wa?A2=ind1304&L=confocalmicroscopy&P=11619
> <http://lists.umn.edu/cgi-bin/wa?A2=ind1304&L=confocalmicroscopy&P=11619>
>
> Consider using one of those instead. Caveat - Dendra2 is in addgene, but
> only in C elegans or no expression plasmids. You can ask Michael
> Davidson about availability, and possibly a stable cell line from one of
> his publications. mEos4 is from Loren Looger's lab, and is described at
> https://www.linkedin.com/pub/maria-gabriela-paez-segala/61/978/b55
>
>     In particular, I have made substantial progress in engineering a
>     version of mEos with increased protein stability (mEos2 to mEos4).
>     mEos2 was the last fluorescent protein designed by Sean McKinney in
>     the Looger lab. It is a photoconvertible fluorescent protein
>     (changes fluorescence from green to red by hitting with UV light),
>     and basically is the best standard photoconvertible FP (fluorescent
>     protein) until date. Our codename for a better mEos2 now is mEos4. I
>     first began by working on improving the thermal stability, and
>     currently I am specifically working on fixatives and
>     auto-fluorescence of some variants of mEos2. With the electron
>     microscopy (EM) team, Richard Fetter, Yalin Wang, and Mei Sun, our
>     main discovery has been a new protocol for EM that will allow us to
>     use CLEM for any fluorophore and fluorescent protein, by decreasing
>     the auto-fluorescence of fixatives, the harsh conditions of OsO4,
>     and increasing the ultra-structure preservation of cell tissue.
>
> Hopefully mEos4 will be published soon - you could email Loren Looger at
> HHMI Janelia Farm
> http://www.hhmi.org/research/groupleaders/looger_bio.html
>
> Or, consider buying rsEGFP2 or Dreiklang from Abberior
> http://www.abberior.com/products/productlist/cat/fluorescent-proteins/prod/abberior-rsegfp/
>
>
>
> best wishes,
>
> George
>
>
> On 6/28/2013 5:48 AM, Sarang Kulkarni wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Screen your stable cell lines by PCR to confirm if the gfp gene is
>> still there.
>>
>> Sarang Kulkarni
>>
>> Sent from my iPhone
>>
>> On 2013-06-28, at 3:02 AM, "Aryeh Weiss"<[hidden email]>  wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> This is a not-really-confocal question, but I think it is well within
>>> the list's expertise.
>>>
>>> We have been trying to create  a stable line of B16 melanoma cells
>>> which will express photoactivatable GFP, and can be lit up whenever
>>> we like.
>>> So we transfected with what we think is a suitable plasmid
>>> (11910 pPAGFP-C1 from Addgene), and sure enough, we have  cells that
>>> survive the selection marker (Neo) while the controls die.
>>> So we think we have our stable cell line.
>>>
>>> Problem is -- none of them light up. They dont light up in a
>>> widefield scope,  and they dont light up in the confocal, when zapped
>>> with 405nm
>>> excitation (we tried different exposures, from 100ms to many seconds).
>>>
>>> So I must be missing something obvious, because this is not a new
>>> technology. If someone can point me in the right direction, that
>>> would be wonderful.
>>>
>>> --aryeh
>>> --
>>> Aryeh Weiss
>>> Faculty of Engineering
>>> Bar Ilan University
>>> Ramat Gan 52900 Israel
>>>
>>> Ph:  972-3-5317638
>>> FAX: [phone]
>
>