Plat e

PlatE (Cell Biolabs), HEK293T (ATCC), and SNL cells (a gift from Allan Bradley, Sanger Institute, Cambridge, UK) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), 2 mM L-glutamine and antibiotics, 50 U/ml penicillin and 50 μg/ml streptomycin. Human adult dermal fibroblasts (hADFs, Cascade Biologicals) were grown in alpha­-modified minimum essential medium (α-MEM) with 20% FBS, 2 mM L-glutamine and the antibiotics. Reprogramming was carried out with the hiPSC medium containing DMEM-F12 with 20% knockout serum replacement (KOSR), 2 mM L-GlutaMAX, 0.1 mM minimal essential medium–nonessential amino acids (MEM-NEAA) solution, 0.11 mM β-mercaptoethanol, 10 ng/ml basic fibroblast growth factor (bFGF) and antibiotics. For the culture of hiPSC colonies 10 ng/ml bFGF was used. The embryoid body (EB) suspensions were maintained in hiPSC medium without bFGF, and the attached EBs were maintained in the same medium with 10% ES cell grade FBS instead of KOSR. All the cell culture reagents were purchased from Life Technologies.

The SST-REX method was performed as described3 (link)4 (link). Total RNA was purified from LNCaP-CR and LNCaP cells using Trizol (Invitrogen) and mRNA was isolated using Fast-Track 2.0 kits (Invitrogen). cDNA was synthesized using random hexamers with SuperScript Choice System (Invitrogen) according to the manufacturer’s instructions and inserted into BstXI sites of pMX-SST vector3 (link)4 (link) using BstXI adaptors (Invitrogen). The ligated vectors were electroporated into E. coli using an E-coli Pulser (Bio-Rad) at 1.8 kV. Then, the cDNA library was prepared by culturing the transfected E. coli. High-titer retroviruses from the above cDNA library were produced using the packaging cell line Plat-E (Cell Biolabs). Ba/F3 cells were infected with the retroviruses using Polybrene (Chemicon). Ba/F3 clones that grew in the absence of IL-3 were selected. Genomic DNA extracted from the IL-3-independent Ba/F3 clones was applied to PCR to recover the integrated cDNAs using the PCR primers, 5′-TAATACGACTCACTATAGGGCGCGCAGCTGTAAACGGTAG-3′ and 5′-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3′. The PCR products were sequenced using BigDye Terminator v3.1 Cycle Sequencing kits with 5′-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3′ as a primer.