B6.Cg-Ptprc^a Tg(UBC-PA-GFP)1Mnz/J

Stock number: 022486
Product name: UBC PA-GFP
Research areas: Neurobiology Research, Research Tools

Description

A photactivatable variant of Aequorea victoria green fluorescent protein was placed under the regulatory control of the human ubiquitin C (UBC) promoter in this transgenic strain. Photoactivation enables rapid, stable fluorescent labeling of living cells with great microanatomical precision.

Detailed description

A photactivatable (PA) T203H variant of Aequorea victoria green fluorescent protein (GFP) was placed under the regulatory control of the widely-expressed human ubiquitin C (UBC) promoter in this transgenic strain. Peak excitation wavelength of PA-GFP shifts to ~495 nm upon one-photon irradiation at ~415 nm or two-photon irradiation at ~720-840 nm.

Cells can be photoactivated within intact lymph nodes with great microanatomical precision (~10 microns in the Z dimension, or close to one cell diameter) by two-photon irradiation at 830 nm, and subsequently identified by two-photon excitation at 940 nm or flow cytometry using a conventional 488 nm laser. After a brief recovery period, the migration of photoactivated naive T and germinal center B cells is indistinguishable from that of control cells. The half-life of photoactivated PA-GFP in naive B cells is estimated to be 30 hours, a figure consistent with previous estimates for the half-life of GFP in living cells.

These mice express high levels in all hematopoietic cell populations. Expression in other tissues has not been evaluated. Mutant mice are viable, fertile, and produce normal size litters. Homozygous mice express approximately twice as much protein as hemizygotes, as determined by flow cytometry. Photoactivation enables rapid, stable fluorescent labeling of living cells with great microanatomical precision.

Development

A transgenic vector carrying photoactivatable green fluorescent protein (PA-GFP, T203H variant) under the transcriptional control of the human ubiquitin C promoter was injected into C57BL/6 embryos. Resultant mice were crossed with a congenic strain on a C57BL/6 background to introduce a Ptprc^a (CD45.1/Ly5.1) allele (see Stock No. 002014). These mice were maintained on a C57BL/6 genetic background by the donating laboratory (see SNP note below).

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Ptprc^a
Name: a variant
MGI accession ID: MGI:4819849
Generation method: Not Applicable
Synonyms: CD45.1; Ly5^a; Ptprc^SJL
Marker symbol: Ptprc
Marker name: protein tyrosine phosphatase, receptor type, C
Marker synonyms: B220; CD45; CD45R; GP180; IMD105; LCA; L-CA; LY5; Ly-5; Lyt-4; RT7; T200
Chromosome: 1
Strain of origin: Not Applicable
Site of expression: Widely expressed on all adaptive and innate immune cells.
Molecular note: Ptprc^a is found in strains SJL/J, STS/A, and DA. Ptprc^b is found in strains C57BL/6, C3H/An, DBA/2, AKR, and many others (J:13367, J:12054, J:12077, J:8603). Twelve nucleotide differences between the a and b alleles have been identified. These base substitutions correspond to five amino-acid changes within the extracellular domain of the encoded protein. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat sequences (J:22485).

Note that the allele designations originally described were reversed in 1987 (J:8603); all publications prior to 1987 show SJL/J, STS/A, and DA as having the b allele and the C57BL/6J group as having the a allele (J:22341).

Allele

Symbol: Tg(UBC-PA-GFP)1Mnz
Name: transgene insertion 1, Michel C Nussenzweig
MGI accession ID: MGI:5476728
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Tg(UBC-PA-GFP)1Mnz
Marker name: transgene insertion 1, Michel C Nussenzweig
Chromosome: UN
Strain of origin: C57BL/6J
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: Fluorescent labeling is expressed in high levels in all hematopoietic cell populations.
Molecular note: This transgene comprises 1.2 kb of 5'-flanking DNA (nucleotides -1225 to -6) of the human ubiquitin C gene, which is expressed in a broad range of cell types, including all hematopoietic cells, driving expression of a photoactivatable green fluorescent protein cDNA, which is followed by both the SV40 and the bovine growth hormone polyadenylation signals. PA-GFP differs from EGFP by the substitution of histidine for threonine at amino acid position 203 (T203H); this modification results in ~100-fold enhanced fluorescence of PA-GFP upon excitation at 488 nm after preliminary photoactivation with intense 413-nm light, greatly increasing the optical contrast.