In vivo correction of a Menkes disease model using antisense

Erik C. Madsen*, Paul A. Morcos†, Bryce A. Mendelsohn*, and Jonathan D. Gitlin*‡

*Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110; and †GeneTools, LLC, Philomath, OR 97370

Edited by William S. Sly, Saint Louis University School of Medicine, St. Louis, MO, and approved January 22, 2008 (received for review November 15, 2007) Although the molecular basis of many inherited metabolic diseases tions that interfere with normal splicing of the pre-mRNA has been defined, the availability of effective therapies in such message (8). In such cases, the threshold of the WT transcript disorders remains problematic. Menkes disease is a fatal neurode- necessary to restore functional may require only a small generative disorder due to loss-of-function in the ATP7A shift in favor of the WT message to ameliorate the disease. encoding a copper-transporting P-type Atpase. To develop RNA targeting has recently emerged as a potential alternative therapeutic approaches in affected patients, we have identified a to more conventional approaches in gene therapy (9). Antisense zebrafish model of Menkes disease termed calamity that results oligonucleotides that bind to pre-mRNA and sterically alter from splicing defects in the zebrafish orthologue of the ATP7A processing have been used successfully in culture to dem- gene. Embryonic-recessive lethal mutants have impaired copper onstrate the utility of this approach (10–13). More recently, in homeostasis that results in absent melanin pigmentation, impaired murine models of Duchenne muscular dystrophy, systemic de- notochord formation, and hindbrain neurodegeneration. In this livery of oligonucleotides has been shown to effect current study, we have attempted to rescue these striking pheno- partial restoration of protein function on a cellular level in typic alterations by using a series of antisense morpholino oligo- mutant mice (14–16). A critical factor in such strategies for the directed against the splice-site junctions of two mutant treatment of human metabolic disorders is the availability of calamity alleles. Our findings reveal a robust and complete correc- robust disease models that permit rapid screening for effective tion of the copper-deficient defects of calamity in association with phenotypic rescue. In this current study, we have used such an the generation of the WT Menkes protein in all rescued mutants. approach to rescue the of calamity, demonstrating the Interestingly, a quantitative analysis of atp7a-specific transcripts applicability to complex organisms of several recent suggests that competitive translational regulation may account for studies using this technology in a number of human genetic the synthesis of WT protein in these . This in vivo correc- diseases (13, 16, 17). tion of Menkes disease through the rescue of aberrant splicing may provide therapeutic options in this fatal disease and illustrates the Results and Discussion potential for zebrafish models of human genetic disease in the Recently, our laboratory has described the mutant development of treatments based on the principles of interactions calamity (allele vu69) as a model of Menkes disease that of synthetic analogues with mRNA. recapitulates key aspects of the human phenotype (Fig. 1A)(7). The defect in calamityvu69 is caused by the creation of a new 3Ј ͉ ͉ ͉ copper metabolic disease morpholino zebrafish splice site, resulting in a 7-bp insertion into the mRNA (Fig. 1F). The subsequent frame shift creates an early stop codon and the enkes disease is an inherited metabolic disease due to loss of Ͼ95% of the protein product (Fig. 1 D and E). In the Mloss-of-function mutations in the ATP7A gene encoding a course of continued screening, we have identified a second allele P-type Atpase required for copper absorption and homeostasis of calamity, designated as gw246. This allele causes an identical (1, 2). Patients with Menkes disease have absent melanin phenotype to the first and is noncomplementary in a standard pigmentation, impaired extracellular matrix formation, and neu- genetic cross (Fig. 1 B and C). Cloning of the mRNA in this MEDICAL SCIENCES rodegeneration, resulting in intractable seizures, hypotonia, mutant revealed a 12-bp in-frame deletion at the 3Ј end of severe failure to thrive, and death in early infancy (3, 4). These 20, which removes 4 aa located in the critical and highly pleiotropic result from the loss of activity of essential conserved ATPase domain of Atp7a (Fig. 1F). This deletion at cuproenzymes within distinct subcellular compartments. Treat- the junction of two is due to the preferential use of a new ment with exogenous copper is largely ineffective (5). Absent the 5Ј splice site. Interestingly, genomic sequence analysis revealed development of directed pharmacotherapy, the restoration of the of neither the WT 5Ј splice site (splice donor) nor normal gene function remains the most viable treatment for the creation of a new GT splice donor pair. Rather, the mutation affected patients. Although partial correction of a murine model was 5 bp downstream from the new mutant splice donor. of Menkes disease was demonstrated by using a human Menkes Comparison of the mutant sequence to the previously estab- transgene (6), methods of safely introducing WT cDNAs into lished zebrafish splice donor consensus sequence indicated that humans have remained elusive. a new splice consensus site had been formed (18). Given that the We recently used chemical to obtain calamity,a mutant displays such a severe phenotype, we inferred that this zebrafish model of Menkes disease (7). Embryonic mutants reveal a striking phenotype with absent melanin pigment, im- paired notochord formation, and neurodegeneration. Because Author contributions: E.C.M., B.A.M., and J.D.G. designed research; E.C.M., B.A.M., and several calamity alleles arise from splicing defects in the ze- J.D.G. performed research; P.A.M. contributed new reagents/analytic tools; E.C.M., P.A.M., brafish orthologue of ATP7A, we reasoned that if normal splicing B.A.M., and J.D.G. analyzed data; and E.C.M., P.A.M., and J.D.G. wrote the paper. could be restored, sufficient WT message might be generated to The authors declare no conflict of interest. rescue these defects. Furthermore, because previous studies of This article is a PNAS Direct Submission. copper nutrition in the zebrafish revealed a hierarchy of Freely available online through the PNAS open access option. temporal and dosage-dependent phenotypes (7), therapeutic ‡To whom correspondence should be addressed. E-mail: [email protected]. windows might allow for long-term correction after transient This article contains supporting information online at www.pnas.org/cgi/content/full/ restoration of WT expression. Such an approach has broad 0710865105/DC1. applicability because many metabolic diseases arise from muta- © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710865105 PNAS ͉ March 11, 2008 ͉ vol. 105 ͉ no. 10 ͉ 3909–3914 Downloaded by guest on September 25, 2021 Fig. 1. Comparison of phenotype, genotype, and protein expression for two alleles of calamity. (A) calamityvu69, characterized in ref. 7, displays the copper-deficient phenotype of loss of pigmentation, notochord abnormalities (arrowheads), and an enlarged ventricle and is due to a loss of function of atp7a. (B) calamitygw246, a second allele of atp7a, has the same phenotype. (C) calgw246 fails to complement calvu69, confirming allelism. (D) Amino acid sequence changes caused by the mutations. calvu69 results in a frameshift and truncation of the C terminus of the protein. calgw246 contains an in-frame deletion in the highly conserved ATPase domain of Atp7a. (E) Immunoblot analysis using an antibody made to a C-terminal peptide from zebrafish Atp7a shows complete loss of WT protein in calvu69, calgw246, and in the compound heterozygote. (F) The mRNA and genomic sequences of calvu69 (Upper) and calgw246 (Lower), highlighting the mutation and subsequent changes in amino acid sequence due to missplicing of exons through the aberrant use of mutant splice sites (red boxes). Exon boundaries in the mRNA are illustrated by a change in color from magenta to blue. In the genomic sequence, are in lowercase and the mutations are the bases with the white background. The green boxes delineate the WT splice donor/acceptor pair and red boxes the mutant pair. For calgw246, a small percentage of transcripts splice at the WT splice junction retaining the nonsynonymous mutation, a Leu to Arg amino acid substitution (*).

new consensus sequence creates a dominant splice donor to the (Fig. 1E). This lack of protein product in the gw246 allele near exclusion of the WT donor. High-resolution separation of suggests that there may be an early processing defect that causes a small PCR product from mutant cDNA demonstrates that this rapid and early degradation of the protein. is indeed the case (Fig. 2B, lane 1). In this mutant, any WT The presence of the WT splice sites in both alleles of calamity splicing that does occur retains a single base change that causes immediately caused us to consider the use of a nonconservative amino acid change from Leu to Arg at directed to specific sequences near the mutant splice sites in a bid position 1316 (Fig. 1F). Immunoblots with an antibody raised to force WT splicing events. There were no data available against a C-terminal peptide from zebrafish Atp7a revealed indicating the precise location that would be best suited for near-complete loss of protein product in both vu69 and gw246 accomplishing this goal. At the same time, the nature of both alleles provided an ideal system for investigating this very question. In the case of gw246, we had two competing splice donors sufficiently far apart that we might interfere with one or the other, but close enough that we also might block both. Design of a series of morpholinos that would walk along the exon– boundary sequence was based on a comparison with the known consensus sequences (Fig. 2A). The basic 5Ј splice site consensus sequence is small and well defined, making targeting straightforward for the gw246 mutant (18). Wherever a mor- pholino sequence overlapped a mutation, the mutant sequence was used at that . The first series of morpholinos was injected at a dose of 1.44 pmol per embryo (12 ng per embryo) into intercrosses of gw246 heterozygous carriers. At 48 h after fertilization, embryos were examined for phenotypic effects, and mutants were collected for mRNA extraction and RT-PCR. The Fig. 2. A series of morpholinos causes alterations of splicing in calgw246.(A) effects on splicing are shown in Fig. 2B. Across the series of Schematic and sequence information of the morpholino design for exploring morpholinos, several splice forms were seen. We confirmed that alterations in splicing caused by variable morpholino placement with respect each band is a splice form of atp7a via direct sequencing of the to WT and mutant splice donors. Exon sequence is uppercase. The mutation is RT-PCR product. Several patterns emerged from these exper- red. Lines indicate the binding site of each listed morpholino. (B) Injection of iments. First, and most interesting, the blocking of the mutant the series of morpholinos in A into calgw246. Splice forms were amplified by PCR of cDNA using the primers illustrated in A. The WT and three alternate splice splice donor did indeed restore a significant amount of WT splice forms were seen on polyacrylamide gel stained with ethidium bromide. Each product (lanes e2 and e3). However, this finding was not was sequenced to identify exact locations of splicing that are illustrated in A. accompanied by a rescue of the phenotype. We surmised that All cryptic splice forms were located within exon 20. this result was due to the retention of a single base change caused

3910 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710865105 Madsen et al. Downloaded by guest on September 25, 2021 by the mutation in the final, properly spliced transcript that (19). Conversely, this would mean that just under two-thirds of results in the nonconservative L1316R substitution. Such a large random amino acid changes will result in functional protein change in polarity and charge in a highly conserved region of this sequence. Together with alternate approaches, such as forced ATPase might be expected to disrupt function. Consistent with , the probability of rescuing any given splice this hypothesis, when we introduced this mutation into the WT mutation with morpholinos remains fairly high. cDNA, it resulted in an ATPase that fails to deliver copper to Unlike the gw246 allele, the rescue of vu69 would not cause tyrosinase in transiently transfected ATP7A-deficient fibroblasts, inclusion of the mutated base, but would fully restore the WT indicating that this mutation results in an inactive transporter sequence without alteration. Also unlike gw246, the mutation [supporting information (SI) Fig. 5]. Alternatively, the mutation alters the splice acceptor. The length and placement of consen- may render the protein unstable, leading to early degradation sus splicing sequences around the splice acceptor are much less and subsequent loss of function. well defined and more degenerate, making targeting predictions Second, the injection of the series of morpholinos revealed much more cumbersome (18). Given the proximity of the vu69 several other weak cryptic splice sites all contained within exon splice acceptor to the polypyrimidine tract, we reasoned that 20, immediately 5Ј to the mutation (Fig. 2). This is most clearly directly overlapping the mutant splice acceptor with a morpho- seen when the morpholino overlies both the WT and mutant sites lino would interfere with WT splicing. Similarly, we assumed (lanes e4 and e5). There was no evidence of exon skipping that both mutant and WT splice acceptors would use the same induced by any of the morpholinos (data not shown). Bands polypyrimidine tract, making it difficult to preferentially target corresponding to the inclusion of the entire intron in the final one splice acceptor by direct targeting of the polypyrimidine product were seen, but we could not totally rule out the tract. However, the inherent degeneracy of the U2AF recogni- possibility of genomic contamination of the cDNA as the source tion sequence might permit a morpholino placed nearby to of these bands (data not shown). sterically interfere and alter the exact location of binding of the Third, the morpholino that accomplished the most robust U2AF to the polypyrimidine tract (20). No structural data rescue of mRNA splicing (lane e3) overlay the mutant splice existed to aid in the design of such a morpholino. Therefore, we site on either side and remained at least 6 bp 5Ј to the WT GT took a similar approach using a series of morpholinos that ‘‘walk’’ pair. Moving only four bases in either direction resulted in across the splice acceptor region, but we expanded our target either an increased use of the mutant site or an increase in the area to include sequences up to 73 bp 5Ј to the mutant acceptor upstream cryptic sites due to blockage of the WT and mutant (Fig. 3A). Briefly, morpholinos i1 and i2 both caused a more sites (lanes e4 and e5). severe phenotype in calamity mutants and heterozygotes, im- Last, when the morpholino target sequence was located 3Ј to plying an interference with WT splice events (data not shown). the mutant splice donor, the mutant again became the dominant Morpholinos i5 to i7 had no effect on phenotype. Morpholinos splice form (lane e7). WT embryos injected with this same i3, i3.5, and i4 caused robust rescue of the phenotype of mutant morpholino fully displayed the calamity phenotype (SI Fig. 6B). calamity embryos (Fig. 3 B–G). Melanin pigmentation was Combined with the RT-PCR data, this indicates that when the restored, and the notochord did not contain any discernible WT 5Ј splice site is blocked by a morpholino, the next most defects, compared with calamity-mutant embryos. The rescue robust cryptic site is the one used in the mutant. Injection of WT was seen in 100% of the mutant embryos, with all injected embryos also resulted in the same pattern of splicing, illustrating mutants containing at least a few melanocytes and 95% having why the mutation is so easily capable of coopting splicing to the Ͼ20. No injected mutant embryo had a discernable notochord cryptic site in the mutant (SI Fig. 6A). defect when scored at 48 h after fertilization (Fig. 3H). The These specific observations on a trial-and-error approach rescue effect was still clearly visible at 6 days after fertilization, allow us to propose a set of generalities regarding morpholino when Ϸ75% of the uninjected calamity embryos were either targeting to an exon–intron junction. First, morpholinos targeted dead or dysmorphic (Fig. 3I), compared with nearly normal- within 30 bp of a 5Ј splice site will have an effect on splicing even looking rescued embryos (Fig. 3J). Lowering the dose of the if it is subtle. Second, in some cases, it appears that the morpholino resulted in similar pigmentation, with intermediate

magnitude of the effect of the morpholino depends not only on notochord defects consistent with previous observations on the MEDICAL SCIENCES its distance from the splice site, but also on the general strength developmental hierarchy of copper metabolism that revealed a of the splice consensus (cf. lanes e1 and e4) and surrounding critical threshold of lysyl oxidase activity necessary for normal cryptic sites. Third, optimal blocking occurs in two forms: (i) the notochord development (data not shown and refs. 7 and 21). morpholino is entirely exonic except for a 2-bp overlap with the Sequencing of genomic DNA confirmed that the rescued GT pair (lanes e2 and e5), or (ii) the morpholino extends at least embryos were indeed mutant (data not shown). 6 bp on either side of the exon–intron junction. Fourth, mor- Because the mutant embryos had clear phenotypic rescue, we pholinos can reveal unappreciated enhancers and suppressors of wanted to confirm that this was the result of restored protein specific splicing events located outside of the consensus se- expression. Immunoblot analysis of injected and rescued mu- quence (lane e10) and can be used to change the hierarchy of tants with a zebrafish Atp7a-specific antibody shows the resto- cryptic splicing events. Validation of these rules will require ration of full-length WT protein in the rescued embryos, com- separate experiments in a different mutant with a similar defect pared with no visible protein in uninjected mutants (Fig. 4A). To and distinct phenotype; however, they do establish a framework our surprise, however, this did not correspond with an increase that can guide further testing. in WT splice products as measured via quantitative (q)RT-PCR. Taken together, these results demonstrate the utility of using Only three consistent patterns emerged from these experiments. morpholinos to correct aberrant splicing in an embryonic ver- The first is the presence of a small amount of amplifiable WT tebrate in cases where WT splice donors are not affected. transcript in all of the mutants, consistent with the presence of Morpholino e3 strongly rescues the mRNA. Unfortunately, we a WT band when endpoint RT-PCR products are run on a did not see a corresponding rescue of the phenotype. That this polyacrylamide gel (Fig. 4C and SI Fig. 7 B and C). The second mutant protein was incapable of rescue indicates one of the is that there is decreased total atp7a transcript in the mutants potential caveats of this system: that the retention of a mutation probably due to nonsense-mediated decay of mutant transcripts in the rescued mRNA can just as strongly affect the phenotype (SI Fig. 7D). The third is that there is a significant reduction of of the organism as the misspliced sequence. Promisingly, how- mutant transcript in the injected embryos. The most likely ever, it has been estimated that the probability that a random explanation for this reduction is the shifting of this transcript to amino acid change would cause protein inactivation is Ϸ34% a third splice form, which is corroborated by the RT-PCR gel

Madsen et al. PNAS ͉ March 11, 2008 ͉ vol. 105 ͉ no. 10 ͉ 3911 Downloaded by guest on September 25, 2021 Fig. 3. Morpholinos rescue the phenotype of calvu69 embryos. (A) Schematic and sequence information for the design of morpholinos directed toward the rescue of the mutant splicing in calvu69. The WT exon is uppercase. The mutation is highlighted in red, and the WT splice acceptor is in green. Each morpholino-binding site is indicated by a line located 5Ј to the polypyrimidine tract. Morpholinos that cause phenotypic rescue are highlighted in pink. Morpholino i1 served as a control to demonstrate that blocking of proper splicing at this exon–exon boundary would result in the calamity phenotype (data not shown). (B and C) Side-by-side comparisons of zebrafish 48 h after fertilization. (Top) WT sibling from calvu69 intercross clutch. (Bottom) calvu69/vu69-mutant embryo. (Middle) calvu69/vu69 embryo rescued by injection of morpholino i3. (D and E) Close-up view of calvu69/vu69-uninjected embryo showing lack of melanin pigmentation over head region and notochord abnormalities (arrowhead). (F and G) Close-up view of morpholino i3-rescued calvu69/vu69 embryo showing restoration of melanin pigmentation and a notochord absent of defects. (H) Quantification of the extent of rescue. A single group of embryos derived from several clutches, half injected with rescuing morpholino (red bars; uninjected, blue bars), was scored for the presence of any or a significant number of melanocytes (Ͼ20) and the presence of notochord defects. The scale of the graph reflects the expected 25% ratio of homozygous mutants derived from the heterozygous intercross. (I and J) Six days after fertilization, embryos were either uninjected (I) or injected (J) with rescuing morpholino i3.5. Rescued embryos maintain near-normal body morphology, whereas unrescued embryos swell, possibly because of decreased extracellular matrix integrity due to the loss of lysyl oxidase activity.

analysis showing a third splice form containing an 800-bp consistent with observations on translational competition in intronic insertion due to the use of a cryptic 3Ј splice site within Drosophila (22) and is supported by our finding that knockdown the intron (Fig. 4C). Sequence analysis of the alternate splice of the mutant transcript best correlates with rescue of the form indicates a frame shift and early stop codon, which is calamity phenotype through restoration of protein levels (Fig. 4). analogous to the original mutation. It is important to note that, Although this proposed mechanism for the increase in functional because the antibody used for immunoblot recognizes the C protein is not directly testable under these conditions, if plau- terminus of the WT zebrafish protein, the identification of the sible, this significantly broadens the applicability of the mor- full-length protein will only occur when WT transcripts are pholino approach because it demonstrates that loss of one present. particular mutant form may be sufficient to cause rescue of Both the reduction in mutant transcript and the lack of any protein function and phenotype, with only minimal increases in increase in WT transcript levels as measured by qRT-PCR were the amount of WT mRNA. consistent across three embryo ages 12, 24, and 48 h after Taken together, the data reported here have several implica- fertilization (data not shown), indicating that there is not an early tions of direct relevance to the development of effective thera- correction of splicing that decreases as the morpholino is diluted peutics in Menkes disease. Experimental (7, 21) and clinical during development. The lack of a measurable substantial observations in affected patients (23) reveal that the mainte- increase in WT transcript in these studies could simply reflect nance of CNS function in this disease is prioritized under the limits of quantitation for the threshold necessary to result in circumstances of limited copper availability or decreased rescue. If so, then it suggests that only small changes in WT ATP7A function. This hierarchy of copper delivery suggests that mRNA can result in a significant phenotypic improvement on an the prevention of the neurodegenerative features in Menkes organismal level, at least in the case of Menkes disease. Alter- disease may be possible with therapeutic interventions that result natively, the data suggest that the increase in protein levels could in a modest increase in ATP7A within a specific developmental result from the removal of inhibition on of the WT window. The in vivo rescue observed in these studies directly transcript. Such inhibition could arise from the competition for supports these pathophysiological concepts and raises the po- translation from the mutant transcript, a concept that would be tential for in utero interventions in this disease. The data in this

3912 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710865105 Madsen et al. Downloaded by guest on September 25, 2021 both AB and WIK strains to rule out the possibility of strain-dependent polymorphisms.

Morpholino Synthesis and Injection. Morpholinos were synthesized by Gene- Tools, LLC. Morpholinos were dissolved to 2 mM in water and stored at Ϫ80°C. Injection of morpholinos was performed on a microinjection apparatus (Har- vard Instruments). A constant volume (1.44 nl) of morpholinos was injected at different dilutions of the stock (1:2 to 1:10) to optimize the dose that gave the strongest phenotypic effect while minimizing off-target effects.

Molecular Cloning and PCR. Total RNA was extracted from embryos by using the TRIzol reagent (Invitrogen). Random hexamer-primed cDNA was synthe- sized by using SuperScriptIII according to the manufacturer’s protocol (Invitro- gen). PCR was performed by using Phusion DNA polymerase (Finnzymes). High-resolution separation of products was performed on 10% TBE- polyacrylamide gels. All sequences were verified by direct sequencing. The L1316R clone of zebrafish atp7a was generated from the WT sequence by using the QuickChangeII site-directed mutagenesis kit (Stratagene). The al- tered clone was sequenced, and other PCR errors were excluded.

Fig. 4. Rescue morpholinos cause restoration of full-length protein without qRT-PCR. qRT-PCR was performed by using primers specific to either the detectable changes in mRNA. (A) Phenotypic rescue by morpholinos i3 and i3.5 mutant or WT form of the Menkes transcript. This was accomplished by is accompanied by the restoration of the WT protein. Forty-eight hours after designing primers that were complementary to the exon–exon junction af- fertilization, lysates from embryos of the indicated genotype were run on a fected in the mutant (SI Fig. 7A). The following primer sequences were used: SDS/6% PAGE gel and blotted for zebrafish Atp7a by using an antibody common forward primer, ATGATGAGCTCCGGACAGAC; calamity-specific re- generated to the zebrafish protein. ␤-catenin was used as a loading control. verse, GGAATGATCTTTTCCACCTGAG; WT-specific reverse, GGAATGATCTTT- Morpholino injection restored protein to 30–45% of WT levels in this exper- TCCACTGTCG; total atp7a primers, TGGAGCTTGTGGTCAGAGG and iment. (B) qRT-PCR using primers specific for WT and mutant atp7a transcript AGGGCAACTGAAGCGTAGAG; and ornithine decarboxylase primers, ATCTG- were used to measure the amounts of each in single embryos. Normalization GATCTCCGTTTTGCT and CCGTTTTACGCAGTGAAGTG. Primer efficiencies ϩ/ϩ Ϫ/Ϫ of WT transcript was to cal embryos and mutant transcript to cal were calculated in triplicate for each primer set by using either WT or mutant embryos. Error bars indicate one standard deviation around the mean. (C) cDNA, respectively. Random hexamer-primed cDNA was synthesized by using Polyacrylamide gel of RT-PCR products reveals a band containing 800 bp of SuperScript III (Invitrogen) according to the manufacturer’s protocol. qRT-PCR intronic sequence caused by morpholino injection. This band was sequenced was carried out on an iCycler (BioRad) with iQ Sybr green SuperMix (BioRad). Ј and represents use of a cryptic 3 splice site located within the intron. Also seen Calculations were performed by using the Pfaffl method, and each bar in the are mutant and WT transcripts in uninjected and rescued embryos. A nonspe- figures is the average of three to five individually prepared embryo RNA cific band was present in both uninjected and injected mutant embryos (*). samples. Relative to WT, all cal embryos had a 50% decrease in total atp7a transcript (data not shown).

article demonstrate the utility of zebrafish for rapid screens to Immunoblots. Forty-eight hours after fertilization, 20–30 zebrafish embryos identify specific antisense sequences effective in phenotypic were gently homogenized in sucrose homogenization buffer [250 mM sucrose rescue. Although morpholino delivery remains problematic, and 5 mM Tris (pH 7.4)] plus protease inhibitors (Roche) and then spun at given the robustness of the assay reported here and the increas- 350,000 ϫ g in a table-top Beckman ultracentrifuge for 30 min. The pellet was ing number of zebrafish models of human genetic disease (24), then resuspended in 50 ␮l of RIPA buffer. Alternatively, 48 h after fertilization, these findings support further study of this technology in the embryos were manually deyolked and homogenized directly in RIPA buffer. development of effective therapeutics for inherited metabolic Equal amounts of protein were loaded on 6% polyacrylamide gels. After disease. transfer to nitrocellulose, immunoblotting was performed by using a rabbit polyclonal antibody raised to a C-terminal peptide of zebrafish Atp7a. Anti-

Methods rabbit secondary antibody and ECL reagents were purchased from Pierce and MEDICAL SCIENCES used according to the manufacturer’s recommendations. Animal Maintenance. Fish stock maintenance was performed according to institutionally approved procedures. Embryos for experiments were obtained through in vitro fertilization and incubated at 28.5°C. The WT fish were of the ACKNOWLEDGMENTS. We thank Amy Koerber for expert zebrafish hus- bandry and Stephen Johnson, Lou Muglia, and Kerry Kornfeld for careful AB strain. The mutant calamity has been described (7). Mutagenesis of WT fish review of the manuscript. E.C.M. is grateful to Dr. Moses Lee for considerable was performed by using the chemical mutagen, ethyl nitrosourea. Mutants and meaningful early support and mentoring. This work was supported by were screened for the calamity phenotype comprising lack of pigmentation National Institutes of Health Grants DK44464 and DK61763 (to J.D.G.), Medical and notochord defects. Putative mutants were crossed to calamity to verify Scientist Training Program Grant T32 GM07200 (to E.C.M. and B.A.M.), and the allelism. These were then out-crossed to AB fish and grown to adulthood. Chancellor’s Hartwell Prize for Innovative Research from Washington Univer- Regions of genomic DNA containing putative mutations were sequenced in sity (to J.D.G.).

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