Nucleofection of whole murine retinas

Abstract

The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.

Mutant dominant-negative rhodopsin∆I256 causes protein aggregates degraded via ERAD and prevents normal rhodopsin from proper membrane trafficking

“…To investigate this, we transfected Rho ΔI256 -EGFP into retinal explants from C57BL/6J (wild-type, WT) mice, which naturally produce RHO WT that localizes to the rod outer segments (ROS). To determine whether RHO ΔI256 affects endogenous RHO WT localization, we used reverse magnetofection, a technique to deliver Rho ΔI256 -EGFP into the C57BL/6J retina (Gomez-Tourino et al, 2013;Sen et al, 2021a;Bassetto et al, 2021). We labeled total RHO with a specific red fluorescing dye conjugated antibody to differentiate between EGFP-tagged exogenous RHO (displayed fluorescence in both red and green) and endogenous RHO (exhibited only red fluorescence).…”

“…The 26S proteasome consists of one 20S protein subunit and two 19S regulatory cap subunits. The 20S core region is responsible for substrate degradation (Gomez-Tourino et al, 2013). To analyze proteasomal rhodopsin degradation, we used a specific antibody against the proteasome 20S subunit beta 5 (PSMB5) to visualize this subunit in HEK293 and COS-7 cells transfected with plasmids encoding Rho and to observe its colocalization with RHO ΔI256 aggregates.…”