The miR-155* targeting TALEN pair causes mutations in the miR-155* sequence.
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293T cells were transfected with or without plasmids encoding the TALEN pair designed to target the miR-155* region. A GFP expressing plasmid was co-transfected. (A) 48 hours later, GFP+ cells were sorted by FACS and subjected to gDNA extraction. (B–F) The miR-155* targeted region was amplified by PCR and subjected to an HRMA analysis (B–D) or TOPO cloning and sequencing (E,F). (B) Schematic of the HRMA approach. A small region of the genome that includes different lengths of DNA deletions is amplified by PCR. Upon annealing, different types of homoduplex and heteroduplex dsDNA molecules are produced with different melting temperatures. (C) HRMA of the mir-155* PCR amplicons generated using gDNA from Wt (mock transfected 293t cells), Unsorted (293t cells with the TALEN pair transfection), Sorted GFP+ and sorted GFP-(293t cells with the TALEN pair and a GFP plasmid co-transfection followed by FACS sorting). (D) The results of the HRMA analysis are also shown as fluorescence difference plots using the normalized data. The Wt sample is used as the baseline. (E) PCR products from C were cloned into a TOPO vector and the length of the individual DNA fragment was assessed by gel electrophoresis. (F) Sequencing results of TOPO clones from E are shown. They are aligned with the wild-type miR-155* sequence. The left and right TALEN binding sites are highlighted in yellow and the miR-155* region is boxed in red.