Adeno-associated virus (AAV)-mediated Cre recombinase expression in melanopsin ganglion cells without leaky expression in rod/cone photoreceptors.

BACKGROUND:Constituting about 5 % of mouse retinal ganglion cells (RGCs), intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin (gene symbol Opn4) and drive such photoresponses as pupil constriction, melatonin suppression, and circadian photoentrainment. Opn4Cre mice with Cre recombinase-expressing ipRGCs have enabled genetic manipulation of ipRGCs; unfortunately, while Cre expression within the inner retina is ipRGC-specific, leaky expression also occurs in some outer retinal photoreceptors, so Cre-induced alterations in the latter cells may confound certain studies of ipRGC function. Methods that express Cre in ipRGCs but not rods or cones are needed. NEW METHOD:We have constructed a recombinant serotype-2 adeno-associated virus, rAAV2-Opn4-Cre, with the improved Cre recombinase (iCre) gene under the control of a ∼3kbp Opn4 promoter sequence, and injected it intravitreally into mice to transduce inner retinal neurons while sparing the outer retina. RESULTS:We introduced rAAV2-Opn4-Cre into Cre reporter mice in which enhanced green fluorescent protein (EGFP) expression indicates Cre expression. Single-cell electrophysiological recordings and intracellular dye fills showed that 84 % of the EGFP+ cells were ipRGCs including M1-M6 types, while 16 % were conventional RGCs. COMPARISON WITH EXISTING METHODS:Whereas Opn4Cre mice express Cre in some rod/cone photoreceptors, intravitreally applied rAAV2-Opn4-Cre induces Cre only in the inner retina, albeit with leaky expression in some conventional RGCs. CONCLUSIONS:rAAV2-Opn4-Cre has overcome a significant limitation of Opn4Cre mice. We recommend usage scenarios where the Cre-expressing conventional RGCs should not pose a problem.