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.
