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Figure 2. Rescue of SRF null ES cells with virally expressed SRFwt and SRF triple alanine substitution mutants reveal critical SRF contacts for NKX2-5 and Gata4. To quantitate and isolate reprogrammed SRF null ES cells, we used a FACS strategy in which lentiviral infection of SRF null ES with SRF mutants was detected by expression of lentiviral expressed IRES-GFP. (A) Schematic diagram of our lentiviral infection transduction protocol into freshly trypsin-dissociated SRF null ES cells mixed with lentivirus at a multiplicity of infection of 100 using 8 μg/mL polybrene (Sigma), containing doxycycline-induced control virus (rTTA), SRF, and other SRF(A3) scanning mutants. (B) Alanine scanning mutations (shown as the letter a) were generated by PCR mutagenesis from Amino Acids 141 to 155 in the MADS Box. (C) Schematic diagram of the full-length cDNA of SRF and SRF(A3) mutants were amplified by PCR and cloned into the doxycycline lentiviral vector rTA-responsive vector with Ptet-LTR1 promoter and the T7 plasmid vector. (D) Three days later, the upper 50% of EGFP+ cells were twice enriched and isolated by FACS (Beckman-Coulter Altra) cultured under growth conditions and FACS sorted again for further enrichment to approximately 90%. Enriched infected ES cells were suspended in hanging drops on Day 5 and then treated with Doxycycline for the next three days, which induced expression of SRF and SRF(A3) mutants, as evaluated by protein blots. On Day 7, cellular aggregates were collected and plated to Day 8 and collected for analysis. (E) Extracts of SRF null murine ES cells rescued by lentiviral expressed SRF(A3) mutants, spanning the N-terminal extension to the start of alpha-helix 1, were used in EMSA DNA binding assays with the [P³²]-labeled alpha-cardiac promoter in the presence of lentiviral co-expressed NKX2-5 and/or GATA4. (F) Luciferase reporter assay of viral transfected alpha cardiac actin promoter luciferase reporter with SRF mutants and virally co-expressed NKX2-5 and/or GATA4. SRF: Serum response factor; ES: embryonic stem.