Figure 1
TALEN-mediated gene disruption of FLT3 in the JM domain in leukemia cells.
(a) Schematic overview of the strategy to disrupt the JM domain of FLT3. The three exons that encode the JM domain, the TALEN pair used (TALEN L and TALEN R) and the primer pair (Primer F and Primer R) for PCR and DNA sequencing to screen the mutant clones were shown. The boxes indicate the TALEN binding sequences in the lower diagram in grey. (b) A T7 Endonuclease I assay presented as an electrophoretic trace (the left upper and lower panels) and a pseudo-gel image (the right panel). The blue-colored trace and the lane labeled with a blue bar at the top correspond to K562 cells transfected with the empty TALEN expression vector as a negative control. The red-colored trace and the lane labeled with a red bar at the top correspond to K562 cells transfected with the TALEN pair. The red arrows denote T7EI cleavage products of the appropriate size and the single black arrow denotes the original uncleaved PCR product in both the electrophoretic trace and the pseudo-gel image. The internal lane standards were labeled with a black bar. (c) Representative sequencing results of targeted K562 clones displaying either the wild-type or targeted mutant allele. The original base numbers in the genomic region of the wild-type FLT3 gene were shown. The dotted lines denote the 19bp deletion in the mutant allele. The colored bars below each chromatogram indicate the predicted proteins. The black bar with an asterisk represents the premature termination codon. (d) Summary of Sanger sequencing results of positive isogenic mutant clones derived from the K562 cell line transfected with TALENs targeting FLT3 exon14. The TALEN binding sequences are shown in bold letters in the wild-type clone. The deletions are indicated by dashes and insertion or base substitution is denoted by a black triangle or an asterisk, respectively. ECD, extracellular domain; TM, transmembrane domain; JM, juxtamembrane domain; TKD, tyrosine kinase domain; KI, kinase insert; TALEN, transcription activator-like effector nuclease; WT, wild-type; Mut, mutant; Bp, base pair; NHEJ, non-homologous end joining.
