Biopharma Insights This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.
Total time
~1 day (including culture time)
Steps
1
Collect mouse eyes
Enucleate the mouse eyes carefully to preserve retinal tissue integrity for subsequent dissection.
▶ 01:30
2
Dissect the retina
Isolate the retina from the eye cup, ensuring clean separation from the retinal pigment epithelium and other surrounding tissues.
▶ 02:43
3
Prepare for electroporation
Mount the isolated retina on a membrane and position the electrodes for optimal DNA delivery.
▶ 03:58
4
Electroporate the retinal explants
Apply electrical pulses to introduce the DNA construct into the retinal cells.
▶ 04:34
5
Culture electroporated retinas
Transfer the electroporated retinal explants to culture medium and maintain them under appropriate conditions for gene expression.
▶ 05:27
6
Visualize reporter expression in retinal cells
Fix and process the retinal explants to visualize the reporter gene driven by the cis-regulatory element.
▶ 06:23