Cln3Δex7/8 knock-in mice have been previously described [12]. Littermate pups from heterozygote crosses were used for primary culture establishment, by previously described methods that yield cerebellar granule neuron-enriched cultures [26]. Postnatal day 4 (P4) cerebella were dissected in Hank's Balanced Salt Solution (HBSS, Sigma), supplemented with 35 mM glucose. Tail biopsies were also collected for genomic DNA isolation and genotypic analysis. Trypsin/EDTA (10 mg/ml, Sigma) and DNase I (100 μg/ml, Sigma), suspended in HBSS, helped dissociate cerebella for primary culture plating onto 0.01% poly-ornithine (Sigma) coated 100 mm dishes. Primary cultures from individual cerebella were cultured overnight at 37°C, 5% CO2, in granule neuron growth media (Dulbecco's Modified Eagle Medium [DMEM, Gibco BRL #11995-065], 10% fetal bovine serum [Sigma #F-2442], supplemented with 24 mM KCl). Infection was performed the following day with defective retrovirus transducing the temperature-sensitive tsA58/U19 large T antigen and a selection neomycin-resistance cassette [27], as previously described [28]. Following infection, cultures were shifted to the tsA58/U19 permissive growth temperature of 33°C and selection was in the same growth media as above, with 200 μg/ml G418. Conditionally immortalized colonies were isolated 3–9 weeks post-infection and expanded for frozen stocks and further sub-clone isolation. Multiple clonal lines were obtained for each genotype and all phenotypes were confirmed in at least 2 independent CbCln3 cell lines. CbCln3 cell lines were maintained on 0.01% poly-ornithine coated dishes at 30–90% confluency, in 33°C and 5% CO2 atmosphere. Passage number was recorded (up to ~20 passages), but had no apparent impact on phenotype. Neuronal differentiation was as previously described [29] with the following cocktail: 10 ng/ml α-FGF, 250 μM IBMX, 200 nM TPA, 50 μM forskolin, 5 μM dopamine (Sigma).