Gm00498

Cell culture media and reagents were purchased from Gibco and Hyclone. Cell lines used in this study are A431 (ATCC CRL-1555), HEK293 (ATCC CRL-1573), HeLa (ATCC CCL-2), HepG2 (ATCC HB-8065), HT1080 (ATCC CCL-121), SH-SY5Y (ATCC CRL-2266), NIH-3T3 (ATCC CRL-1658), control fibroblasts GM00037 and GM00498 (Coriell). CLN5 patient fibroblasts #1 (homozygous c.694C > T, p.Gln232X) and #2 (c.671G > A, p.Trp224X and exon 4 deletion) were received from Massachusetts General Hospital CHGR NCL Disorders Clinical Database and Biorepository. All cells were grown and maintained in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 20 mM HEPES and gentamicin at 37 °C in a humidified incubator with 5% CO₂. For transfection, HEK293 cells were seeded in culture dishes for 24 h before transfection. The TransIT-LT1 transfection reagent (Mirus Bio) was used for overexpression of CLN5-Myc–His, and Lipofectamine RNA iMAX reagent (Life Technologies) was used for siRNA gene silencing. Transfections were done according to manufacturer's protocol. Opti-MEM reduced serum medium (Gibco) was used for reagent/nucleotides complexes formation.

Fibroblasts were generated from skin biopsies collected by K.A.R. and L.A.W. in patients previously described (28 (link)) and expanded by culturing in Dulbecco’s modified Eagle’s medium + 10% fetal bovine serum for 2 to 3 weeks. Two control lines, 162D and 165D, were from healthy siblings of NF1 patients. Line 13 was generated from fibroblast line GM00498 (Coriell). Fibroblast lines were reprogrammed with nonintegrative plasmids as previously described (37 (link)). Clonal colonies displaying iPSC morphology were manually selected and subsequently cultured on a Matrigel substrate (BD Biosciences) with mTeSR1 medium (Stem Cell Technologies). To determine pluripotency, teratomas were generated via three subcutaneous injections of 100,000 dissociated iPSCs in mTeSR1/30% Matrigel into nonobese diabetic mice/severe combined immunodeficient mice (Jackson Laboratory). After 2 to 3 months, tumors were surgically removed and sections were hematoxylin and eosin–stained by the Gladstone Histology Core at UCSF. All experiments were conducted with lines between passages 10 and 30.