Fig. 4. Germ-line transmission of ZFN-induced noggin mutations. (A) Cel-1 digests of noggin amplicons from sibling heterozygous mutant and homozygous wild-type F1 tadpoles produced from three mutant line founders. Bands migrating at 450 bp are full-length noggin amplicons. Bands migrating at 300 and 150 bp are Cel-1 digest products. (B) Sequence alignments of the targeted noggin locus from Cel-1�positive F1 mutants. Genomic and translated sequences are shown for each mutant line. Asterisk indicates a stop codon. Red nucleotides and amino acids indicate insertions and dashes represent deletions. (C) Schematic of synthetic RNA injections into ventral vegetal blastomeres of four-cell-stage embryos to test functionality of the induced mutant noggin alleles. (D) Quantification of secondary axis induction following wild-type or mutant noggin RNA injection. Bars represent results of two (5 pg) or three (10 pg) independent experiments (�SD). Black bars show 5-pg RNA injections; white bars show 10-pg RNA injections. Two asterisks indicate significantly different (P < 0.01) from uninjected controls. (E, F, G, and G′) Uninjected control embryos. (H, I, J, and J′) Embryos injected with 10 pg of 4-bp insertion mutant noggin and 200 pg LacZ RNA. (K�M) Embryos injected with 10 pg of 12-bp deletion mutant noggin and 200 pg LacZ RNA. (N�P) Embryos injected with 10 pg of 3-bp insertion mutant noggin and 200 pg LacZ RNA. (Q�S) Embryos injected with 10 pg wild-type noggin and 200 pg LacZ RNA. (E, H, K, N, and Q) Dorsal view of stage 19. (F, I, L, O, and R) Dorsal view of stage 28. (G, J, M, P, and S) Embryos stained with 12/101 antibody. (G, J, and M) Lateral view. (G′, J′, P, and S) Dorsal view. Arrows show weak ectopic dorsal axis induction.
Image published in: Young JJ et al. (2011)
Copyright © 2011. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
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