Generation of hoxb1a and hoxb1b germ line mutants using ZFN and TALEN technologies. A. Diagram outlining experimental strategy. Active ZFNs and TALENs were identified by their ability to disrupt the sequence of a diagnostic restriction site in genomic DNA. Embryos injected with active ZFNs and TALENs were then raised and screened for founders that transmit frameshift mutations via their germ line. B. Identification of active nucleases. Genomic DNA was prepared from pools of injected embryos and digested with a diagnostic restriction enzyme. The Zb1b-3, Zb1b-4 and Tb1a-2 injected pools contain undigested material (arrows), indicating that the diagnostic restriction site has been disrupted. C. Characterization of germ line transmitted hoxb1a and hoxb1b mutations. Six mutant hoxb1a (top) and three mutant hoxb1b (bottom) alleles were identified to cause frameshift mutations. Nucleotide and peptide sequences are indicated for each mutant allele. Gaps in the nucleotide sequence indicate deletions, while red nucleotides indicate insertions. Numbers to the right of each nucleotide sequence indicate the net size of insertions/deletions. For the peptide sequence, gray boxes indicate residues read out of frame prior to encountering a premature stop codon. Amino acid numbers below each peptide sequence indicate the residue affected by the frameshift mutation, while numbers to the right indicate the length of the mutant peptide. HD = homeodomain. Black wedge indicates site of single intron in each sequence.