B6.Cg-Gt(ROSA)26Sor^{tm1(CAG-PA-GFP)Rmpl}/J

Stock number: 021071
Product name: R26 PA-GFP::NLS
Research areas: Neurobiology Research, Research Tools

Description

These mice conditionally express nuclear enriched photoactivatable GFP (PA-GFP::NLS) and allow broad and strong fluorescent expression in most tissues of the body, and may be useful to rapidly mark and follow selected population of cells in the living animal.

Detailed description

Mice heterozygous for the R26 PA-GFP::NLS allele are viable and fertile. The R26 PA-GFP::NLS allele contains a CAG promoter, loxP-flanked neomycin-β-galactosidase fusion protein (βgeo) with a STOP codon, and a photoactivatable green fluorescent protein (PA-GFP) fused to a nuclear localization signal (NLS). This construct was inserted into the endogenous Gt(ROSA)26Sor locus. In the absence of Cre, PA-GFP expression is prevented by the βgeo sequence. After removal of the loxP-flanked cassette via cre-mediated recombination, PA-GFP is expressed in all cells where Cre recombinase is expressed. PA-GFP::NLS is enriched in the nucleus, but can be also detected in the cytoplasm of neuronal cells. PA-GFP shows very weak fluorescence at 517nm when excited at 488nm before photoactivation. Upon photoactivation with light at ~400nm, PA-GFP is converted into its fluorescent form. This form shows strong emission at 517nm when excited at 488nm and allows the conditional fluorescence labeling of individual, living cells which can be imaged using conventional GFP filter sets. Photoactivation can also be efficiently achieved using 2-photon excitation at ~750nm and imaged after photoconversion using excitation at ~950nm.

Development

The R26 PA-GFP::NLS targeting vector was designed with (from 5' to 3') the CMV-IE enhancer/chicken beta-actin hybrid promoter (CAG; from the pCAGGS vector), a loxP-flanked neomycin-β-galactosidase fusion protein (βgeo) with a STOP codon, a photoactivatable green fluorescent protein (PA-GFP) fused to a nuclear localization signal (NLS), and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability). This vector was inserted into the Gt(ROSA)26Sor locus and was electroporated into 129P2/OlaHsd;C57BL/6J-derived A9 embryonic stem (ES) cells. Resulting chimeric mice were bred to C57BL6/J mice establish the colony. R26 PA-GFP::NLS mice were bred to C57BL/6J for at least 5 generations. Upon arrival at The Jackson Laboratory, mutant mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-PA-GFP)Rmpl}
Name: targeted mutation 1, Simon Rumpel
MGI accession ID: MGI:5449399
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (C57BL/6 x 129P2/OlaHsd)F1
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: When bred to mice that express Cre recombinase, photoactivatable GFP will be expressed in tissues where promoters driving Cre recombinase are expressed.
Molecular note: The R26 PA-GFP::NLS targeting vector was designed with (from 5' to 3') the CMV-IE enhancer/chicken beta-actin hybrid promoter (CAG; from the pCAGGS vector), a loxP-flanked neomycin-beta-galactosidase fusion protein (betageo) with a STOP codon, a photoactivatable green fluorescent protein (PA-GFP) fused to a nuclear localization signal (NLS), and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability).
General note: After removal of the loxP-flanked cassette via cre-mediated recombination, PA-GFP is expressed in all cells where Cre recombinase is expressed. PA-GFP::NLS is enriched in the nucleus, but can be also detected in the cytoplasm of neuronal cells. PA-GFP shows very weak fluorescence at 517nm when excited at 488nm before photoactivation. Upon photoactivation with light at ~400nm PA-GFP is converted into its fluorescent form. This form shows strong emission at 517nm when excited at 488nm and allows the conditional fluorescence labeling of individual, living cells which can be imaged using conventional GFP filter sets. Photoactivation can also be efficiently achieved using 2-photon excitation at ~750nm and imaged after photoconversion using excitation at ~950nm.