Table 1.
Target sequences and in vitro activity of CompoZr ZFNs.
Table 2.
Target sequences for CoDA ZFNs: 2 pairs were selected for each gene.
Figure 1.
Flowchart of step-by-step experimental procedures for generating mutant zebrafish lines with ZFNs.
Steps involving ZFN design, mRNA injections, and toxicity assessment are shown in blue color, efficiency testing using somatic lesion analysis in green color and founder screening steps in orange color.
Table 3.
Efficiency of the tested CompoZr and CoDA ZFNs.
Figure 2.
Fluorescent PCR strategy and examples of mutant peaks from founder screening and F1 genotyping.
A) Schematic of the fluorescent PCR strategy. Target region is shown as two black lines. Forward primers are denoted as FAM (blue star) -labeled M13F primer (M13F-FAM) and gene-specific primer with M13F tail (M13F-tailed forward primer). Reverse primer is denoted as the gene-specific part and the PIG-tail sequence (PIG-tailed reverse primer). B and C) Founder screening for ak2 using 4 pooled embryos per well (B) and genotyping of heterozygous adults from F1 progeny of the corresponding transmitting founders (C). Fragment size scales are shown on the top and each fragment's size is marked underneath the peak. Vertical scale marks the intensity of the peaks. Black arrows mark the 257 bp peak corresponding to the Wild-type allele observed in all samples. The top panel in B is a Wild-type control DNA sample, the middle panel is a founder transmitting a 1 bp insertion mutation (258 bp, marked by a red arrow) and the bottom panel shows a founder transmitting two mutations, a 13 bp deletion (244 bp, marked by a green arrow) and a 4 bp insertion (261 bp, marked by a blue arrow). In C each panel shows a heterozygous adult zebrafish and color-matched arrows mark the mutant peaks.
Figure 3.
List of all mutations identified by founder screening and genotyping of F1 adults.
For each target gene, the total number of mutations, total number of germline-transmitting founders, and number of mutations transmitted by each founder are given. For example, for cbfb 4×1, 1×2, 1×3 indicates 4 founders each transmitted a single mutation, one founder transmitted two mutations and another founder transmitted three mutations, for a total of 9 mutations. In all cases, the Wild-type sequences with spacer marked in red are shown at the top followed by the sequences of the mutant alleles. Deletions are marked by red dashes highlighted in yellow and insertions by lower case letters highlighted in blue color. The nature of each mutation is indicated on the right side of the sequence, Δ indicates deletion, + indicates insertion. In some cases, identical mutations were transmitted by multiple founders and these are indicated by x # Fo (meaning times # of founders). Finally, additional mutations whose sequences were not determined are listed at the bottom for each gene.
Table 4.
Rate of germline transmission of ZFN-induced mutations.