Conditional functional Vps34 and p85α knockout mice

All animal studies were conducted according to NIH guidelines and approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine. WT C57BL/6J mice were purchased from Jackson lab (Bar Harbor, ME). Mice used for tissue samples were euthanized by CO₂ inhalation prior to dissection.

Vps34 floxed mice²¹ which have lox P sites flanking exons 17 and 18 (the ATP binding domain) were a kind gift from Dr. Fan Wang (Duke University). The C57BL6/129 mixed background Vps34 floxed mice were back-crossed with WT C57BL/6J for at least 6 generations, then bred with transgenic mice containing the opsin promoter controlled iCre-75²³ in a C57BL/6 background (kindly gifted by Dr. Ching-Kang Jason Chen, Baylor College of Medicine) to generate rod-specific conditional functional Vps34 knockout mice (Vps34^∆rod). All the experimental Vps34 knockout mice were maintained with heterozygous iCre-75. Vps34 knockout mice that were also heterozygous for the human rhodopsin-GFP allele at the mouse rhodopsin locus²⁷ (Vps34^∆rod-rho^+/hRhoG(H)) were generated by crossing Vps34^∆rod mice with human rhodopsin-GFP knock-in mice containing a loxH site in the 5′ non-coding region to reduce the human rhodopsin-GFP expression by 80%²⁷. The Vps34 knockout with heterozygous GFP-LC3 (Vps34^∆rod-GFP-LC3) was generated by crossing Vps34^∆rod mice with C57BL/6 background GFP-LC3 mice (BRC No. 00806: GFP-LC3#53, RIKEN Bio Resource Center, Ibaraki, Japan). In order to make a conditional knockout of class I PI-3 kinase in rods, the BALB/c background p85α floxed mice¹ (kindly provided by Dr. Raju V. S. Rajala, University of Oklahoma Health Sciences Center, Oklahoma City, OK) were crossed with wild type C57BL/6J for 8–9 generations, then bred with iCre-75 mice to generate rod-specific p85α knockout mice (p85α^∆rod). Mice with mosaic Vps34 rod knockout (Vsp34^p∆rod) were generated by crossing Vps34 floxed mice with transgenic mice expressing opsin-promoter-controlled Cre in ~50% of rods¹ (kindly provided by Dr. Raju V. S. Rajala, University of Oklahoma Health Sciences Center, Oklahoma City, OK).

Clipped tails and/or retinas were used for genotyping as described²¹. PCR primers L1 5′-AACTGGATCTGGGCCTATG-3′ and A2 5′-GAAGCCTGGAACGAGAAGAG-3′ were used for PCR analysis of the deleted Vps34 allele in the retina, which yields a 672 bp PCR product in Vps34 knockout mice (Supplementary Fig. S2). The absence of rd1 and rd8 alleles was confirmed by PCR and DNA sequencing as described^31,32.

Unless indicated otherwise, mice were maintained in a 12-12 hour light-dark cycle. The life-time dark- or light-adapted mice were raised in light-proof cages or in 4,500 LUX fluorescent illumination until the mice were dissected.