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Green to red photoconversion of GFP for protein tracking in vivo.

Sattarzadeh A, Saberianfar R, Zipfel WR, Menassa R, Hanson MR - Sci Rep (2015)

Bottom Line: A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light.However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms.We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Cornell University, Department of Molecular Biology and Genetics, Ithaca, NY, 14853 USA.

ABSTRACT
A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

No MeSH data available.


Related in: MedlinePlus

GFP variants used for photoconversion.(a) Alignment of the coding region of the GFP variants. The alignment was created using GeneDoc (http://www.nrbsc.org/gfx/genedoc/). (b) Schematic representation of GFP transgenes used in photoconversion experiments. Pr1b-SP, secretory signal peptide from the tobacco pathogenesis-related protein 1; EGFP, enhanced green fluorescent protein; L, linker (GGGS)3; KDEL, endoplasmic reticulum retrieval signal peptide; HttQ103, exon 1 of human HttQ103 gene; Cytosolic mGFP4-T in tobacco cell culture and cytosolic S65T-GFP in Drosophila gut cells. In cytosolic mGFP4-T, V (valine) was replaced by A (alanine), and Q (glutamine) was replaced by R (arginine) relative to EGFP. In cytosolic S65T-GFP, L (leucine) was replaced by F (phenylalanine) relative to EGFP.
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f1: GFP variants used for photoconversion.(a) Alignment of the coding region of the GFP variants. The alignment was created using GeneDoc (http://www.nrbsc.org/gfx/genedoc/). (b) Schematic representation of GFP transgenes used in photoconversion experiments. Pr1b-SP, secretory signal peptide from the tobacco pathogenesis-related protein 1; EGFP, enhanced green fluorescent protein; L, linker (GGGS)3; KDEL, endoplasmic reticulum retrieval signal peptide; HttQ103, exon 1 of human HttQ103 gene; Cytosolic mGFP4-T in tobacco cell culture and cytosolic S65T-GFP in Drosophila gut cells. In cytosolic mGFP4-T, V (valine) was replaced by A (alanine), and Q (glutamine) was replaced by R (arginine) relative to EGFP. In cytosolic S65T-GFP, L (leucine) was replaced by F (phenylalanine) relative to EGFP.

Mentions: While performing photobleaching studies on plant cells labeled with GFP, we made the unexpected observation that EGFP, mGFP4-T, and GFP modified by an S65T mutation can be photoconverted by 405 nm light in vivo. The sequences of the variant GFPs we have analyzed are shown in Fig. 1a.


Green to red photoconversion of GFP for protein tracking in vivo.

Sattarzadeh A, Saberianfar R, Zipfel WR, Menassa R, Hanson MR - Sci Rep (2015)

GFP variants used for photoconversion.(a) Alignment of the coding region of the GFP variants. The alignment was created using GeneDoc (http://www.nrbsc.org/gfx/genedoc/). (b) Schematic representation of GFP transgenes used in photoconversion experiments. Pr1b-SP, secretory signal peptide from the tobacco pathogenesis-related protein 1; EGFP, enhanced green fluorescent protein; L, linker (GGGS)3; KDEL, endoplasmic reticulum retrieval signal peptide; HttQ103, exon 1 of human HttQ103 gene; Cytosolic mGFP4-T in tobacco cell culture and cytosolic S65T-GFP in Drosophila gut cells. In cytosolic mGFP4-T, V (valine) was replaced by A (alanine), and Q (glutamine) was replaced by R (arginine) relative to EGFP. In cytosolic S65T-GFP, L (leucine) was replaced by F (phenylalanine) relative to EGFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493561&req=5

f1: GFP variants used for photoconversion.(a) Alignment of the coding region of the GFP variants. The alignment was created using GeneDoc (http://www.nrbsc.org/gfx/genedoc/). (b) Schematic representation of GFP transgenes used in photoconversion experiments. Pr1b-SP, secretory signal peptide from the tobacco pathogenesis-related protein 1; EGFP, enhanced green fluorescent protein; L, linker (GGGS)3; KDEL, endoplasmic reticulum retrieval signal peptide; HttQ103, exon 1 of human HttQ103 gene; Cytosolic mGFP4-T in tobacco cell culture and cytosolic S65T-GFP in Drosophila gut cells. In cytosolic mGFP4-T, V (valine) was replaced by A (alanine), and Q (glutamine) was replaced by R (arginine) relative to EGFP. In cytosolic S65T-GFP, L (leucine) was replaced by F (phenylalanine) relative to EGFP.
Mentions: While performing photobleaching studies on plant cells labeled with GFP, we made the unexpected observation that EGFP, mGFP4-T, and GFP modified by an S65T mutation can be photoconverted by 405 nm light in vivo. The sequences of the variant GFPs we have analyzed are shown in Fig. 1a.

Bottom Line: A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light.However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms.We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Cornell University, Department of Molecular Biology and Genetics, Ithaca, NY, 14853 USA.

ABSTRACT
A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

No MeSH data available.


Related in: MedlinePlus