Abstract
Purpose :
Electroporation of plasmid DNA into mouse retinal progenitor cells (RPCs) has been extensively used to investigate gene function during retinal development. To perform high-throughput genetic screens, it is important to know how many plasmid copies are delivered to individual electroporated RPCs and their progeny. Here we investigate multiplicity of electroporation in the retina based on fluorescent protein expression and high-throughput sequencing of barcoded plasmids.
Methods :
CD1 mouse pups were electroporated on postnatal day 1 (P1) with plasmids containing a CMV-beta-actin (CAG) promoter driving expression of one of three fluorescent proteins, EGFP, tdTomato, or mKate. The plasmids were mixed in equimolar concentrations totaling 10, 100, or 1000 ng/ul. DNA was injected subretinally and electroporated using five 80V pulses. Mice were euthanized at postnatal day 21 (P21), and fixed retinas were cryosectioned and imaged on a Zeiss confocal microscope. In separate experiments, pups were electroporated with a library of plasmids containing BFP tagged with random 18-nucleotide barcodes. Individual electroporated cells were FACS-purified, and barcodes were PCR-amplified to perform high-throughput DNA sequencing.
Results :
All plasmid concentrations resulted in most transfected cells being labeled with all three fluorescent proteins, with random variation in expression levels resulting in a rainbow-like range of hues. Modeling suggests that most cells receive at least 6-10 plasmid copies at the lowest concentration and as many as 1000 copies at high concentrations. Experiments in which barcodes were recovered from individual cells by high-throughput sequencing allowed us to more directly estimate multiplicity of electroporation across a range of plasmid concentrations, further supporting our prior estimates.
Conclusions :
Our results suggest that gene delivery by plasmid electroporation in the retina results in high plasmid copy numbers in each cell. These data support the premise of using high-throughput screening by electroporating libraries of plasmids, but highlights the importance of titrating the concentration of such libraries in order to avoid simultaneously manipulating thousands of genes in each cell.
This is a 2021 ARVO Annual Meeting abstract.