Supplementary Figure 11: Cloning of the BKSM polycistronic cassette.
From: Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage
(A) A schematic representation of the cloning strategy to generate the BKSM lentiviral vector cassette. A DNA fragment consisting of the Oct4 ORF, followed by an F2A peptide and part of the Klf4 ORF, was excised from the STEMCCA (OKSM) vector using NotI and BstZ17I and replaced by a fragment corresponding to Brn4-F2A-Klf4, generated by overlapping PCR. (B) Representative immunofluorescence images for Oct4 expression in MEFs 3 days after transduction with the tetOP-OKSM (top) and tetOP-BKSM (bottom) cassettes. Note that Oct4+ nuclear staining is detected only in cells that were infected with the tetOP-OKSM cassette. (C) Representative immunofluorescence images for Brn4 in MEFs 3 days following transduction with the tetOP-BKSM (top) or tetOP-OKSM (bottom) cassettes. Note that Brn4+ nuclear staining is detected only in cells that were infected with the tetOP-BKSM cassette.
