Figure 2.
The eFlut vectors enable efficient tagging of CDKN1A with combinations of colors and markers. (A) Schema of endogenous and engineered locus (exons are indicated by E# and a thick a line). (B) PCR amplification of genomic DNA from un-engineered MCF7 cells or cell lines engineered with CDKN1A tagged with YFP (1A plasmid) or T2A-GFP (1B plasmid). All three primers diagramed in 2A (F1′, R1′, R2′) were used in each reaction (see Table S2 for primer sequences). (C) MCF7 cells with CDKN1A tagged were cloned from single cells treated with Nutlin3A for 8 hours to induce p21 expression and subsequently lysed and probed for CDKN1A and GFP expression. The WT MCF7 cells show CDKN1A expression 8hr after Nutlin3A treatment, but no GFP signal. The CDKN1A-YFP cell line show a CDKN1A-YFP fusion protein which runs at the expected ~48kD. The CDKN1A-T2A-GFP-NLS clone (expressing the transcriptional reporter 1B) shows endogenous CDKN1A running at its wild type molecular weight, and an additional GFP-NLS species induced by Nutlin3A. * represents a non-specific band for the GFP antibody. (D) CDKN1A was tagged in MCF7 cells with the listed vectors and measured before and 5hrs after induction of DNA breaks by Neocarzinostatin (NCS) treatment. (E) CDKN1A was tagged with T2A cleaved GFP-NLS sequences (plasmid 1B) and imaged before and 5hrs after NCS treatment. (F) MCF7 cells with an N-terminally tagged BBC3 (PUMA – transcript isoform NM_014417, plasmid 1C) before and 8hrs after induction of the BBC3 transcriptional activator p53 by Nutlin3A. (G) MCF7 cells with CDKN1A tagged with both YFP and RFP (1A plasmids) show a tight correlation 8hrs after DNA damage induced by NCS.