Figure 2.

Generation of zebrafish nr1d1 mutants. (A) Diagram of the TALEN target fragment in zebrafish nr1d1. The left and right TALEN arms are underlined; the TALEN spacer containing a BslI restriction enzyme site (in red) is in small case. (B) Examine TALEN efficiencies by gel analysis. The nr1d1 target fragment was amplified by PCR from genomic DNAs of approximately 10 embryos microinjected with capped mRNAs of nr1d1 TALEN left and right arms at a concentration of 300 pg each, and digested with BslI. Mutagenesis efficiencies were estimated by the ratios of the uncleaved bands and the sum of the cleaved bands and the uncleaved bands quantified with software ImageJ. WT, wild type; M, marker. (C) Two mutated fish lines were identified with DNA sequencing. One has a 7-bp deletion, the other has a 124-bp insertion and a 2-bp deletion, and both lead to frame shift mutations resulting in truncated proteins. (D) The predicted truncated Nr1d1 proteins induced by TALEN. The 2 truncated Nr1d1 proteins each have only 57 amino acids.