Pmxs gw

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.

mTert-pBabe-puro (Addgene plasmid #36413) (Addgene, Watertown, MA, USA), pMXs-gw (Addegene plasmid #18656), and pBRPy-nuclear mCherry-IRES-Puro (Addegene plasmid #52409) were gifts from Dr. Marta Alvarez and Dr. Joseph Bidwell, Dr. Shinya Yamanaka, and Dr. Jacob Hanna, respectively. The original mTert-pBabe-puro harbors cDNA identical to BC127068.1 (NM001362388), which encodes a putative N-terminal–truncated mTert protein [10 (link)]. We constructed a modified mTert-pBabe-puro (mTert^FL-pBabe-puro) containing cDNA identical to NM009354. The resulting cDNA encodes a full-length mTert highly homologous to hTERT, which has been frequently used for cell immortalization. The cDNA fragment for replacement was PCR-amplified using E14.1 mouse embryonic stem cell cDNA and sequence-verified. The SV40tsA58 large T antigen (Ag) cDNA-harboring plasmid was previously described [11 (link)]. cDNA was subcloned into pMSCV-hygro (Clontech/TaKaRa Bio, Shiga, Japan). pMX-Bsd-mCherry-nls was constructed by removing an attR1-attR2 gateway cassette from pMXs-gw and inserting mCherry-nls cDNA, followed by the SV40-EM7-Bsd cassette (Invitrogen, Carlsbad, CA, USA). mCherry-nls cDNA was derived from pBRPy-nuclear mCherry-IRES-Puro.