Figure 1
CRISPR-Cas9-directed generation of the Y537S mutation in the ESR1 gene in MCF7 breast cancer cells. (a) Schematic representation of the ESR1 gene, exons 5-8, annotated for the positions of PCR primers used for RT-PCR analysis. (b) RT-PCR of MCF7 (WT) and MCF7-Y537S cell lines using primers in a. Expression of wild-type and mutant ER alleles was confirmed by RT-PCR, using primers in ESR1 Exon 5 (Primer 1, 5′-CCAGGGAAGCTACTGTTTGC-3′) and Exon 8 (Primer 3, 5′-GATGCATGCCGGAGTGTATG-3′), which generate a 700 bp product. The ESR1 transcript arising from the Y537S mutant allele was amplified as a 466 bp product using the exon 5 primer and the knock-in-specific primer. (Primer 2, 5′-TAGTGGGCGCGTGAAGTCTA-3′). (c) Sequencing chromatogram of the RT-PCR products for the exon 8 coding region for MCF7-Y537 S cells, showing expression of both mutant and wild-type alleles. (d) Frequency of RNA-seq reads for the Y537 and 537S codons in MCF7 and MCF7-Y537S cells. (e) Normalized intensity of ER tryptic peptide containing amino acid 537 from liquid chromatography mass spectrometry. Results are shown for three independent immunoprecipitated ER samples for MCF7 and MCF7-Y537S lines. No signal was obtained for the mutant peptide in MCF7 cells. For the MCF7-Y537S samples, the normalized intensity for each replicate sample is shown by circles of the same colour, demonstrating similar amounts of the two peptides in MCF7-Y537S cells.
