**2. Materials and Methods**

### *2.1. Generation of Dhddsflx*/*flx CreRPE Mice*

A construct containing lacZ flanked by FLP-FRT and *Dhdds* exon 3 flanked by loxP sites from the Knockout Mouse Project (KOMP, UC Davis, Davis, CA, USA) was linearized and introduced into mouse ES cells (C57Bl/6J background) at the Roswell Park Cancer Institute (RPCI) Gene Targeting and Transgenic Facility (Bu ffalo, NY, USA) using standard technology. To confirm the correctly targeted cells, polymerase chain reaction (PCR) was performed with the primers listed below (see *PCR Genotyping*, below). The lacZ cassette was excised with FLP-FRT recombinase, and excision was confirmed by PCR. Mouse lines that carried the *Dhdds* loxP conditional knockout allele were crossed to generate homozygotes and the latter were also crossed to a mouse line (on a C57Bl/6J background) carrying a homozygous transgene expressing Cre recombinase under the control of the RPE-specific VMD2 (vitelliform macular degeneration 2) promoter [20,21]. RPE-specific expression of Cre in the VMD2 promoter-driven mouse line was confirmed by crossing those mice with a ZsGreen reporter mouse line (B6.Cg-*Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze*/J, The Jackson Laboratory, Bar Harbor, ME, USA) and examining the retinas by confocal fluorescence microscopy. Genotypes of o ffspring were confirmed by PCR with *Dhdds-* and Cre RPE-specific primer pairs. All mice used in this study were treated following the ARVO *Statement on the Use of Animals in Ophthalmic and Vision Research* and the policies of the University of Alabama at Birmingham (UAB) Institutional Animal Care and Use Committee (IACUC). This project was approved for animal use on April 2019 by the UAB IACUC and requires updated approvals each year (protocol number IACUC-21270). All animals were maintained on a standard 12/12-h light/dark cycle, fed standard rodent chow, provided water *ad libitum*, and housed in plastic cages with standard rodent bedding.
