Targeted disruption of zebrafish miR-1-2.

(A) Frequency and spectrum of TALEN induced miR-1-2 mutations. The TALEN binding sites are shown in yellow background. DNA sequence encoding the mature miR-1-2 is underlined, with the seed sequence in bold. Deletions are indicated by dash lines and insertions are indicated by lowercase letters. The sizes of the insertions (+) or deletions (Δ) and the number of times each mutant allele appearing are shown on the right side of the mutant allele. (B) The hairpin structure of the wild-type pre-miR-1-2 and two pre-miR-1-2 mutants (MA and MB) in Panel A. (C–D) Functional suppression of target GFP expression by the wild-type or two mutated pre-miR-1-2. Messager RNA of GFP sensor (GFP-3XIPT-miR-1-2 or GFP-cnn2-32UTR) and RFP indicator expressing wild-type or mutant pre-miR-1-2 was co-injected into one-cell stage zebrafish embryos. Pictures were taken at 24 hours after injection. GFP-3XIPT-miR-1-2, GFP sensor containing three imperfect complementary sites to miR-1-2; GFP-cnn2–3′UTR, GFP sensor containing the 3′UTR sequence of cnn2 (a miR-1-2 target gene); dsRed-miR-1-2, RFP indicator expressing mature miR-1-2.