Pmxs hc myc

iMEF cells were grown in DMEM supplemented with 10% FCS, 100 mM nonessential amino acids, 1 mM L-glutamine. Ezh2^flox/flox;ROSA26-CreERT2 or Ezh2^flox/Δ;ROSA26-CreERT2 MEF cells were isolated from 13.5-d-old embryos and subsequently infected with the following retroviral constructs: pMXs-hc-MYC (Addgene, 17220) to generate c-Myc iMEFs, pBABE-hygro p53 DD (Addgene, 9058) to generate p53-DN iMEFs, and Ndy1-MigR1 (kindly provided by Philip N. Tsichlis) to generate Ndy1 iMEFs. A clone was obtained by limiting dilution of a pool of c-Myc Ezh2^flox/Δ;ROSA26-CreERT2 iMEFs. For conditional deletion of Ezh2, cells were treated with 4-hydroxytamoxifen (Sigma) at a final concentration ranging from 1 nM to 1 µM. For Ezh1 and Ezh2 rescue experiments, cells were infected with MSCVhygro-Flag-Ezh1 or MSCVhygro-Flag-Ezh2 ecotropic retroviruses.
The Myc-CaP mouse prostate cancer cell line was generously provided by Charles L. Sawyers, and cells were grown in DMEM supplemented with 10% FCS, 100 mM nonessential amino acids, and 1 mM L-glutamine. To obtain optimal growth conditions, the growth medium was supplemented with a growth factor cocktail composed of 25 µg/mL bovine pituitary extract (Life Technologies), 5 µg/mL bovine insulin (Sigma), and 6 ng/mL recombinant human epidermal growth factor (Sigma).

Retrovirus was generated using constructs pMXs‐hOCT4, pMXs‐hSOX2, pMXs‐hKLF4, pMXs‐hc‐MYC (Addgene) as described previously (Hotta et al., 2009; Chang et al., 2013). To generate VSV‐G pseudotyped retrovirus, Plat‐GP cells were transfected with 15 mg of expression vector and 5 mg of pVSV‐G. Two days post‐transfection, retrovirus was collected and filtered through a 0.45‐mm filter. Transduction of 5 × 10⁵ patient fibroblasts was performed by the addition of polybrene to a final concentration of 4 mg mL⁻¹ to a retroviral cocktail either containing a combination of four factors or five factors. After transduction, fibroblasts were maintained in fibroblast medium for 6 days, then trypsinized and replated onto fresh mouse embryonic fibroblasts (MEFs). Fibroblast media was then replaced with hESC media and was changed daily. Approximately 20 days later, colonies resembling hESCs in morphology were mechanically picked and replated onto fresh MEFs. These iPSCs were mechanically dissociated for a few passages and then adapted to collagenase IV passaging.

iPS-NHDF-1 and iPS-NHDF-2 were derived from NHDFs (Lonza; CC-2511). Reprogramming plasmids were obtained from Addgene (17220: pMXs-hc-MYC, 17219: pMXs-hKLF4, 17218: pMXs-hSOX2, 17217: pMXs-hOCT3/4, 13354: pMXs-Nanog) [14] and packaged using the Plat-GP retroviral packaging cell line. Fibroblasts were infected on days 0 and 1with 5 µg/ml polybrene and spinoculation (1200×g for 45 minutes at 16°C). They were transferred onto mitomycin C-inactivated mouse embryonic feeder cells (MEFs; outbred Swiss mice [15] (link), [16] (link) established and maintained at the Department of Pathology, Oxford) on 0.1% gelatin coated plates on day 4. From day 5 onwards, cells were cultured in KnockOut™ serum replacement medium (Life Technologies) supplemented with 50 µg/ml ascorbic acid and 0.5 µM valproic acid. Medium was replaced (50%) on alternate days, and substituted with MEF-conditioned medium from day 10 onwards.
Colonies displaying iPSC morphology were picked on day 28 and transferred onto MEFs by manual dissection every 5–7 days. Prior to differentiation, iPSC lines were adapted to feeder-free conditions onto Matrigel-coated plates (BD Matrigel hESC-qualified Matrix) in mTeSR™1 (StemCell Technologies) supplemented with Rock inhibitor Y27632 (10 µM; Calbiochem) on the day of passage.