Rtta2s m2

The coding regions of mouse Aid (NM_009645.2, NCBI) and mouse Apobec1 (NM_001134391, NCBI) were cloned from mouse ES cells by RT-PCR. The PCR products were sequenced and subcloned into pENTR-D-TOPO (Invitrogen) and recombined with pMXs-gw [1] (link) using LR recombinase according to manufacturer’s instructions (Invitrogen). Mouse dominant-negative Apobec1 (H61K/C93S/C96S) [20] (link) was generated by PCR-based site-directed mutagenesis. To generate lentivirus vectors encoding doxycycline-inducible reprogramming factors, TRE, the Gateway cassette (Invitrogen) and rtTA2s-M2 (Clontech) were introduced into a pLKO.1 backbone (#10878, Addgene). Then coding sequences of Oct3/4, Sox2, Klf4 and c-Myc were inserted by the LR reaction to make pLV-TRE-rtTA2s-M2-Oct3/4, -Sox2, -Klf4 and -c-Myc. psPAX2 (#12260) and pMDG.2 (#12259) were obtained from Addgene. The primers used for the construction of plasmids are listed in Table S7.

HEK293-Tet-On cells stably expressing the reverse tetracycline-controlled transactivator (rtTA2^S-M2) were purchased from Clontech (Cat-No. 630931; Heidelberg, Germany) and cultivated in Dulbecco’s Modified Eagle Medium-High Glucose with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO₂.