Topo cloning technology

A 5.1-kb polycistronic cassette containing Oct4-P2A-Sox2-T2A-Klf4-E2A-c-Myc^{56 (link)} was cloned using KAPA HiFi HotStart ReadyMix (KAPA Biosystems). This cassette was inserted into pCR8-GW-TOPO using TOPO cloning technology (Invitrogen), transferred into the Rosa26 tetO-attR1-ccdB-attR2-ires-mCherry targeting vector using the Gateway technology (Thermo Fisher Scientific), and used as a targeting vector.

Freshly collected pancreatic tissues were frozen in liquid nitrogen and grounded into powder using mortar. Genome DNA of the pancreas was extracted using Purelink Kit (Invitrogen) according to the manufacturer’s protocol. Bisulfite treatment was performed using EZ DNA Methylation-Gold Kit (Zymo Research). The target sequence was cloned by Ex Taq HS (Takara Bio) and ligated into pCR4-Topo vector using Topo cloning technology (Invitrogen). These plasmids were transformed into DH5α at 37 °C, overnight. Colony PCR was performed using Go taq Master Mix (Promega) and ascertained ligation of the cassettes. After cleanup of the PCR products by exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (Takara Bio), the sequence reaction was performed using M13 Rv primer (5′-CAGGAAACAGCTATGAC-3′) and was sequenced with ABI 3500 xL (Applied Biosystems). Methylation rates were measured using Quantification tool for Methylation Analysis (QUMA) software (Riken).