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The cnidarian sea anemone Nematostella vectensis has grown in popularity as a model system to complement the ongoing work in traditional bilaterian model species (e.g. Drosophila, C. elegans, vertebrate). The driving force behind developing cnidarian model systems is the potential of this group of animals to impact EvoDevo studies aimed at better determining the origin and evolution of bilaterian traits, such as centralized nervous systems. However, it is becoming apparent that cnidarians have the potential to impact our understanding of regenerative neurogenesis and systems neuroscience. Next-generation sequencing and the development of reverse genetic approaches led to functional genetics becoming routine in the Nematostella system. As a result, researchers are beginning to understand how cnidarian nerve nets are related to the bilaterian nervous systems. This chapter describes the methods for morpholino and mRNA injections to knockdown or overexpress genes of interest, respectively. Carrying out these techniques in Nematostella requires obtaining and preparing embryos for microinjection, designing and generating effective morpholino and mRNA molecules with controls for injection, and optimizing injection conditions.

Antisense morpholino oligonucleotides (MOs) have become a valuable method to knockdown protein levels, to block with mRNA splicing and to interfere with miRNA function. MOs are widely used to alter gene expression in development of Xenopus and Zebrafish, where they are typically injected into the fertilized egg or blastomeres. Here we present methods to use electroporation to target delivery of MOs to the central nervous system of Xenopus laevis or Xenopus tropicalis tadpoles. Briefly, MO electroporation is accomplished by injecting MO solution into the brain ventricle and driving the MOs into cells of the brain with current passing between 2 platinum plate electrodes, positioned on either side of the target brain area. The method is relatively straightforward and uses standard equipment found in many neuroscience labs. A major advantage of electroporation is that it allows spatial and temporal control of MO delivery and therefore knockdown. Co-electroporation of MOs with cell type-specific fluorescent protein expression plasmids allows morphological analysis of cellular phenotypes. Furthermore, co-electroporation of MOs with rescuing plasmids allows assessment of specificity of the knockdown and phenotypic outcome. By combining MO-mediated manipulations with sophisticated assays of neuronal function, such as electrophysiological recording, behavioral assays, or in vivo time-lapse imaging of neuronal development, the functions of specific proteins and miRNAs within the developing nervous system can be elucidated. These methods can be adapted to apply antisense morpholinos to study protein and RNA function in a variety of complex tissues.

Morpholino antisense oligonucleotides are used as routine tools in developmental biology to investigate gene function during early embryogenesis. These chemically modified oligos contain morpholine ring connected with phosphorodiamidate linkages as backbone but carry unmodified nucleobases. In this chapter, we describe the methods to further modify the nucleobases using palladium-catalyzed cross-coupling reactions. The key reactions used are halogenations of the nucleobases in suitable position and subsequent Pd-catalyzed Sonogashira and Suzuki reactions. The sequential synthetic steps are described in detail in this chapter, and the examples are shown in tables.

The induction of germ-line chimerism in fish is a strategy for the reproduction of endangered or genetically valuable fish species. Chimeras can be created by transplanting a single primordial germ cell or multiple blastomeres from a donor into a sterile host embryo. When the host reaches sexual maturity, it will produce donor-originating gametes throughout its reproductive life span. This technique provides unique experimental conditions for basic biology research in model fish species like zebrafish. The success of cell transplantation relies on the effective sterilization of host embryos, the correct identification of developing germ cells, and the synchronization of migratory cues between the host and the transplanted cells. Developments in non-transgenic methods of germ cell ablation and identification have made germ cell transplantation more applicable to use in conservation and aquaculture. In this chapter, we provide a protocol for germ cell labeling by injection of chimeric RNA or FITC-dextran, the sterilization of host embryos using an antisense morpholino oligonucleotide, and two methods for producing germ-line chimeras in zebrafish: single primordial germ cell transplant and blastomere transplant.

Disturbances in the general mRNA metabolism have been recognized as a major defect in a growing number of hereditary human diseases. One prominent example of this disease group is retinitis pigmentosa (RP), characterized by selective loss of photoreceptor cells. RP can be caused by dominant mutations in key factors of the pre-mRNA processing spliceosome. In these cases, the complex events leading to the RP phenotype can only insufficiently be analyzed in animal knockout models due to the essential functions of splice factors. Furthermore knockout animals frequently miss the specific phenotypes caused by knockdown of the respective genes. Here we introduce the zebrafish Danio rerio as a valuable vertebrate model system to study RP and related diseases in knockdown case scenarios.

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that the role of calpains in cell migration and other processes has been extensively studied in vitro, the same does not apply to cell migration and morphogenetic events during embryogenesis, in vivo. Herein, we describe the use of three different methods to selectively block calpain activity in vivo in order to investigate the impact on Xenopus gastrulation and neurulation, namely, a calpain inhibitor, a dominant negative, and a morpholino antisense oligonucleotide (MO). We also provide methods to determine the effectiveness of the calpain inhibition and effect on cell fate specification and morphogenetic movements, during embryogenesis in vivo.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by a mutation in SMN1 that stops production of SMN (survival of motor neuron) protein. Insufficient levels of SMN results in the loss of motor neurons, which causes muscle weakness, respiratory distress, and paralysis. A nearly identical gene (SMN2) contains a C-to-T transition which excludes exon 7 from 90% of the mature mRNA transcripts, leading to unstable proteins which are targeted for degradation. Although SMN2 cannot fully compensate for a loss of SMN1 due to only 10% functional mRNA produced, the discovery of the intronic splicing silencer (ISS-N1) opened a doorway for therapy. By blocking its function with antisense oligonucleotides manipulated for high specificity and efficiency, exon 7 can be included to produce full-length mRNA, which then compensates for the loss of SMN1. Nusinersen (Spinraza), the first FDA-approved antisense oligonucleotide drug targeting SMA, was designed based on this concept and clinical studies have demonstrated a dramatic improvement in patients. Novel chemistries including phosphorodiamidate morpholino oligomers (PMOs) and locked nucleic acids (LNAs), as well as peptide conjugates such as Pip that facilitate accurate targeting to the central nervous system, are explored to increase the efficiency of exon 7 inclusion in the appropriate tissues to ameliorate the SMA phenotype. Due to the rapid advancement of treatments for SMA following the discovery of ISS-N1, the future of SMA treatment is highly promising.

Efficient intracellular delivery is critical to the successful application of synthetic antisense oligonucleotides (ASOs) to modulate gene expression. The conjugation of cell-penetrating peptides (CPPs) to ASOs has been shown to significantly improve their intracellular delivery. It is important, however, that formation of the covalent linkage between the peptide and oligonucleotide is efficient and orthogonal, to ensure high yields and a homogeneous product. Described herein are efficient and facile methodologies for the conjugation of peptides to ASOs, and their subsequent labeling with various moieties such as fluorescent dyes for intracellular tracking studies.

Duplications of one or more dystrophin exons that disrupt the reading frame account for about 15% of all Duchenne cases, and like the more common genomic deletions, most pathogenic duplications of single or multiple dystrophin exons are also amenable to targeted exon skipping. However, additional considerations must be taken into account: (1) skipping of all duplicated exons, and, flanking exons as necessary, will frequently be required to restore the reading frame and generate an in-frame Becker muscular dystrophy-like mRNA, (2) the phosphorodiamidate morpholino oligomer chemistry is more effective than the 2'-O-methyl modified oligonucleotides at inducing multiple exon skipping, and (2) the apparent efficiency of exon skipping can be confounded by the choice of RT-PCR system. Standard RT-PCR systems can preferentially amplify the shorter amplicons, implying more efficient exon skipping than may actually be induced. Unless high fidelity RT-PCR systems are used, strand slippage during annealing/elongation steps will generate normal length transcripts that are artifacts of the amplification.

Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is among the more promising approaches available for the treatment of several neuromuscular disorders, including Duchenne muscular dystrophy. The main weakness of this treatment arises from the low efficiency and sporadic nature of delivery of the neutrally charged PMO into muscle fibers, the mechanism of which is unknown.Recently, using wild-type and dystrophic mdx52 mice, we showed that muscle fibers took up PMO more efficiently during myotube formation. Interestingly, through in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. Next, we tested the therapeutic potential of PMO in laminin-alpha2 (laminin-α2) chain-null dy 3K/dy 3K mice, a model of merosin-deficient congenital muscular dystrophy 1A (MDC1A) with active muscle regeneration. We confirmed the recovery of the laminin-α2 chain following skipping of the mutated exon 4 in dy 3K/dy 3K mice, which prolonged the life span of the animals slightly. These findings support the theory that PMO entry into fibers is dependent on the developmental stage in myogenesis rather than on dystrophinless muscle membranes, and provide a platform for the future development of PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration. Herein, we describe the methods for PMO transfection/injection and evaluation of the efficacy of exon skipping in the laminin-α2-deficient dy 3K/dy 3K mouse model both in vitro and in vivo.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal-dominant disorder characterized by progressive heterotopic ossification. More than 95% of cases are caused by a recurrent mutation (617G>A; R206H) of ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. Recent studies revealed that ACVR1R206H induces heterotopic ossification by aberrant activation in response to activin A. Because ACVR1R206H is a hyperactive receptor, a promising therapeutic strategy is to decrease the activity of ACVR1 in patients. Here, we describe a method to reduce ACVR1 expression in FOP patient cells by exon skipping in ACVR1 mRNAs using phosphorodiamidate morpholino oligomers (PMOs). This strategy can be applied to the screen to select antisense oligomers to knockdown not only ACVR1 but also genes which cause other autosomal-dominant genetic diseases.

Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (ASOs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Phosphorodiamidate morpholino oligomer (PMO) is one of the safest among therapeutic ASOs for patients and has recently been approved under the accelerated approval program by the US Food and Drug Administration (FDA) as the first ASO-based drug for Duchenne muscular dystrophy (DMD). Multi-exon skipping utilizing ASOs can theoretically treat 80-90% of patients with DMD. Here, we describe the systemic delivery of a cocktail of ASOs to skip exon 51 and exons 45-55 in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting, and the evaluation of their efficacies in vivo.

Electroporation allows targeting of genetic materials (e.g., DNA, RNA, antisense morpholinos) to the tissue of interest with high spatial and temporal specificity. Here, we describe a highly efficient and reproducible electroporation strategy for targeting the central nervous system in Xenopus. This versatile approach can be combined with live imaging or other existing experimental procedures to aid the investigation of different research questions.

Morpholino oligomers have great therapeutic potential for treatment of a broad range of human diseases, including viral, bacterial, age-related, and genetic diseases, but they suffer from poor systemic delivery into cells. Although various approaches have been undertaken to address the delivery problem, it remains as the major barrier of morpholinos to be used as effective therapeutics. This slow development is in part due to the cost of materials and the animal models used for screening the efficacy and safety of those delivery approaches. The need to have an inexpensive vertebrate model for assessing in vivo delivery of morpholinos is evident. Therefore, we have produced a novel transgenic zebrafish model containing a dual reporter cassette for determination of in vivo delivery, bio-distribution, and safety of a morpholino. The levels of morpholino delivered to the cells in various tissues can be determined by changes in reporter gene expressions caused by morpholino-induced exon skipping. This chapter provides a description of the reagents, equipment, and procedure for successful retro-orbital injection of a peptide-conjugated morpholino into the blood stream of the adult zebrafish to cause targeted exon skipping in the heart of the zebrafish.

The application of antisense oligonucleotides (AONs) to modify pre-messenger RNA splicing has great potential for treating genetic diseases. The strategies used to redirect splicing for therapeutic purpose involve the use of AONs complementary to splice motifs, enhancer or silencer sequences. AONs to block intronic splicing silencer motifs can efficiently augment exon 7 inclusion in survival motor neuron 2 (SMN2) gene and have demonstrated robust therapeutic effects in both preclinical studies and clinical trials in spinal muscular atrophy (SMA), which has led to a recently approved drug. AONs with phosphorodiamidate morpholino oligomer (PMO) backbone have shown target engagement with restoration of the defective protein in Duchenne muscular dystrophy (DMD) and their safety profile lead to a recent conditional approval for one DMD PMO drug. PMO AONs are also effective in correcting SMN2 exon 7 splicing and rescuing SMA transgenic mice. Here we provide the details of methods that our lab has used to evaluate PMO-mediated SMN2 exon 7 inclusion in the in vivo studies conducted in SMA transgenic mice. The methods comprise mouse experiment procedures, assessment of PMOs on exon 7 inclusion at RNA levels by reverse transcription (RT-) PCR and quantitative real-time PCR. In addition, we present methodology for protein quantification using western blot in mouse tissues, on neuropathology assessment of skeletal muscle (muscle pathology and neuromuscular junction staining) as well as behaviour test in the SMA mice (righting reflex).

Abnormal splicing of the chloride channel 1 (CLCN1) gene causes myotonic dystrophy type 1 (DM1). Therefore, controlling the alternative splicing process of this gene by antisense oligonucleotides can be a promising treatment for DM1. In this study, we describe an efficient phosphorodiamidate morpholino oligomer (PMO) delivery method by ultrasound-mediated bubble liposomes, which is a known gene delivery tool with ultrasound exposure, to treat skeletal muscles in a DM1 mouse model, HSALR. Effective delivery of PMO using this technique can help control the alternative splicing of the Clcn1 gene via exon skipping and enhance the expression of Clcn1 protein in skeletal muscles and the amelioration of myotonia. Thus, exon skipping by PMO delivery with ultrasound-mediated BLs may be feasible in myotonic dystrophy model mice.

The zebrafish (Danio rerio), a small tropical fish, has become a powerful model for the study of early vertebrate development, human diseases, and drug screening. Zebrafish provides large numbers of optically clear embryos, and its development is very rapid. Overexpression or under-expression of proteins can be effectively achieved by microinjection of mRNA or morpholino antisense oligonucleotides (MOs), respectively, into developing embryos at the 1-2 cell stage. The function of a particular protein can be revealed by correlating gene expression patterns with the phenotypes observed from over- or under-expression. We defined the expression pattern of zebrafish progranulin A (zfPGRN-A), an orthologue to the single human PGRN by whole-mount in situ hybridization (ISH) and immunofluorescence (IF). The MO-mediated knockdown of zfPGRN-A expression generated embryos that display abnormal motor neuron development resulting in touch-evoked swimming deficits.

In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.

Exon skipping therapy using synthetic DNA-like molecules called antisense oligonucleotides (ASOs) is a promising therapeutic candidate for overcoming the dystrophin mutation that causes Duchenne muscular dystrophy (DMD). This treatment involves splicing out the frame-disrupting segment of the dystrophin mRNA, which restores the reading frame and produces a truncated yet functional dystrophin protein. Phosphorodiamidate morpholino oligomer (PMO) is the safest ASO for patients among ASOs and has recently been approved under the accelerated approval pathway by the U.S. Food and Drug Administration (FDA) as the first drug for DMD. Here, we describe the methodology and protocol of PMO transfection and evaluation of the exon skipping efficacy in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting. The mdx52 mouse model offers advantages over the mdx mouse, a spontaneous DMD model with a nonsense mutation in exon 23, in terms of the deletion in a hotspot of deletion mutations in DMD patients, the analysis of caveolae and also Dp140 and Dp260, shorter dystrophin isoforms.

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration, caused by nonsense or frameshift mutations in the dystrophin (DMD) gene. Antisense oligonucleotides can be used to induce specific exon skipping; recently, a phosphorodiamidate morpholino oligomer (PMO) has been approved for clinical use in DMD. However, an efficient PMO delivery strategy is required to improve the therapeutic efficacy in DMD patients. We previously developed polyethylene glycol (PEG)-modified liposomes containing ultrasound contrast gas, "Bubble liposomes" (BLs), and found that the combination of BLs with ultrasound exposure is a useful gene delivery tool. Here, we describe an efficient PMO delivery strategy using the combination of BLs and ultrasound exposure to treat muscles in a DMD mouse model (mdx). This ultrasound-mediated BL technique can increase the PMO-mediated exon-skipping efficiency, leading to significantly increased dystrophin expression. Thus, the combination of BLs and ultrasound exposure may be a feasible PMO delivery method to improve therapeutic efficacy and reduce the PMO dosage for DMD treatment.

Giardia lamblia, a major parasite, is an emerging model organism due to its compact genomic arrangement and composition. The most popular reverse genetic technique, RNAi, is ineffective in Giardia. In contrast, protein depletion by translation blocking morpholinos is suitable for most gene targets and provides up to 80% depletion of the target protein. The method is fast, reliable, and specific. After antisense morpholino oligomer delivery into Giardia trophozoites by electroporation, the cells can be used for many subsequent analyses 8-48 h after treatment. In this chapter, suitable gene tags, plasmids, and techniques necessary for proper morpholino targeting are described.

The control processes that underlie the progression of development can be summarized in maps of gene regulatory networks (GRNs). A critical step in their assembly is the systematic perturbation of network candidates. In sea urchins the most important method for interfering with expression in a gene-specific way is application of morpholino antisense oligonucleotides (MOs). MOs act by binding to their sequence complement in transcripts resulting in a block in translation or a change in splicing and thus result in a loss of function. Despite the tremendous success of this technology, recent comparisons to mutants generated by genome editing have led to renewed criticism and challenged its reliability. As with all methods based on sequence recognition, MOs are prone to off-target binding that may result in phenotypes that are erroneously ascribed to the loss of the intended target. However, the slow progression of development in sea urchins has enabled extremely detailed studies of gene activity in the embryo. This wealth of knowledge paired with the simplicity of the sea urchin embryo enables careful analysis of MO phenotypes through a variety of methods that do not rely on terminal phenotypes. This article summarizes the use of MOs in probing GRNs and the steps that should be taken to assure their specificity.

Photoactivatable morpholinos (MO) allow specific temporal and spatial inhibition of gene function, which is not possible with conventional morpholino or genetic global gene knock-out approaches. Here, we describe an application of commercially available photoactivatable MO technology for specific gene inhibition in a zebrafish embryonic model and discuss the required controls related to the specificity and efficacy of this method. A similar approach should be also applicable to other model organisms.

In the zebrafish regenerating fin, specific gene-targeting morpholinos have been widely utilized to assess gene function. Unlike in embryos, injection of standard morpholinos in the adult regenerating fin is not sufficient for cellular uptake. Rather, morpholinos are first injected extracellularly into the blastemal compartment, followed by electroporation for cellular uptake. Knockdown phenotypes are evaluated 1-4 days post electroporation. This chapter provides a description of the reagents, equipment, and procedure for successful injection and electroporation of morpholinos into the regenerating fin.

A good Morpholino experiment starts with oligos that have been carefully designed to minimize off-target RNA binding. Performing a successful, reproducible, and well-controlled Morpholino experiment requires oligos that are single stranded and in solution at a known concentration. The outcome of treatment with the oligo needs to be checked for specificity, that is, that the observed outcome is due to interaction with the intended RNA and not an interaction with an unexpected RNA. In this chapter, I will discuss Morpholino use mostly in the context of embryonic microinjection experiments, though many techniques and warnings will be applicable to cell culture or adult animal experiments as well. Controls are critical to a good experiment, but good techniques in designing, preparing, storing, and using the oligos can improve the strength and specificity of the knockdown. Finally, it is important to know the solution concentration of the oligo to ensure that the results are reproducible.

Differing from the conventional direct-targeting strategy in which a probe or payload is directly loaded onto a targeting molecule that binds to the native target, pretargeting is an improved targeting strategy. It converts the native target to an artificial target specific for a secondary targeting molecule loaded with the probe or payload (effector). The effector is small and does not accumulate in normal tissues, which accelerates the targeting process and generates high target to nontarget ratios. DNA/cDNA analogs can serve as the recognition pair, i.e., the artificial target and the secondary targeting effector. Morpholino oligomers are so far the most investigated and the most successful DNA/cDNA analog recognition pairs for pretargeting. Herein, we describe the pretargeting principles, the pretargeting strategy using Morpholino oligomers, and the preclinical success so far achieved.

Influenza viruses replicate primarily in the lung tissue of different host species. For efficient replication the virus utilizes host factors that are expressed in target cells. Cell-penetrating peptide-conjugated Morpholino oligomers (PPMOs) designed to target viral proteins have shown promising results as potential antiviral drugs in tissue culture and animal models. However, since viruses tend to have high rates of mutations, targeting viral proteins may result in viral escape mutants. An alternative approach to inhibit virus replication with PPMOs is to target host factors that are required for virus replication. Delivery of PPMO through the intranasal route has been shown to be effective in knockdown of host factors or microbial genes leading to protection against respiratory pathogens and reduced microbial burden. In addition, protective host innate antiviral immune responses in the lung can be studied by knockdown of immune signaling factors using PPMOs. Here we describe a successful approach using PPMOs to knockdown either proviral or antiviral host factors leading to changes in influenza virus replication in the lungs of mice, providing a tool to investigate immune responses and host-virus interactions in vivo.

Regulation of alternative splicing can be harnessed by antisense-based compounds to control gene expression. Antisense-mediated splicing interference has become a valuable molecular tool to modulate endogenous alternative splicing patterns, to correct cryptic or aberrant splicing, to reduce gene expression by triggering nonsense-mediated mRNA decay, and to activate intronic polyadenylation, both in vitro and in vivo. Here, we describe methods to induce and analyze the modulation of RNA processing, using modified splice-switching antisense oligonucleotides, such as phosphorodiamidate morpholino (PMO).

Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (AONs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Multiple exon skipping utilizing a cocktail of AONs can theoretically treat 80-90% of patients with Duchenne muscular dystrophy (DMD). The success of multiple exon skipping by the systemic delivery of a cocktail of AONs called phosphorodiamidate morpholino oligomers (PMOs) in a DMD dog model has made a significant impact on the development of therapeutics for DMD, leading to clinical trials of PMO-based drugs. Here, we describe the systemic delivery of a cocktail of PMOs to skip multiple exons in dystrophic dogs and the evaluation of the efficacies and toxicity in vivo.

The use of antisense oligonucleotides to target specific mRNA sequences represents a promising therapeutic strategy for neurological disorders. Recent advances in antisense technology enclose the development of phosphorodiamidate morpholino oligomers (MO), which is one of the best candidates for molecular therapies due to MO's excellent pharmacological profile.Nevertheless, the route of administration of antisense compounds represents a critical issue in the neurological field. Particularly, as regards motor neuron diseases, intracerebroventricular (ICV) injection is undoubtedly the most efficient procedure to directly deliver therapeutic molecules in the central nervous system (CNS). Indeed, we recently demonstrated the outstanding efficacy of the MO antisense approach by its direct administration to CNS of the transgenic mouse models of Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS).Here, we describe methods to perform the ICV delivery of MO in neonatal SMA mice and in adult ALS mice.

Alternative polyadenylation is increasingly being recognized as an important layer of gene regulation. Antisense-mediated modulation of alternative polyadenylation represents an attractive strategy for the regulation of gene expression as well as potential therapeutic applications. In this chapter, we describe methods to upregulate the functional Kv11.1 isoform expression by blocking intronic polyadenylation signal sequences with antisense morpholinos.

In vivo delivery of morpholino antisense oligonucleotides (MAO) directly into the uterine lumen of a peri-implantation period pregnant sheep is an effective technique for evaluation of gene products for conceptus development. The highly phagocytic conceptus is undergoing rapid morphological change, thereby the available MAO are readily consumed and delivered to developing cells. Here, we describe the method for preparation and surgical delivery of MAO-Endo-Porter complex to developing ovine conceptus on day 8 postmating. Also outlined are methods for posttreatment sample recovery on day 16 postmating.

Modifications at either end, both ends, or in-between the ends of a Morpholino oligo provide functional groups for further conjugation. Amino groups are the most useful and efficient reactive entities for chemical bonding with other molecules. The combination of modifications at both ends, especially with double functionalization at the 3'-end, yields myriad opportunities for diverse applications. An orthogonally protected diamine for advanced 3'-end double modification on the solid phase synthesis support allows the convenient assembly of a vast variety of custom-designed molecules. A particular application is the assembly of a class of Vis-Vivo-Morpholino where at the 3'-end an optically visible fluorophore is installed at one side for fluorescent detection and an in vivo delivery moiety is attached at the other side for intracellular activity studies.

Antisense morpholino oligonucleotides have been commonly used in zebrafish to inhibit mRNA function, either by inhibiting pre-mRNA splicing or by blocking translation initiation. Even with the advent of genome editing by CRISP/Cas9 technology, morpholinos provide a useful and rapid tool to knockdown gene expression. This is especially true when dealing with multiple alleles and large gene families where genetic redundancy can complicate knockout of all family members. miRNAs are small noncoding RNAs that are often encoded in gene families and can display extensive genetic redundancy. This redundancy, plus their small size which can limit targeting by CRISPR/Cas9, makes morpholino-based strategies particularly attractive for inhibition of miRNA function. We provide the rationale, background, and methods to inhibit miRNA function with antisense morpholinos during early development and in the adult retina in zebrafish.

Diagnostic applications of morpholinos take advantage of their unique properties including backbone charge neutrality, a weak impact of ionic strength on their hybridization behavior, and their resistance to enzymatic degradation. This chapter overviews how these properties have advanced transduction and other capabilities useful for the analysis of nucleic acids. In many cases, the benefits stem from electrostatic mechanisms; for example, use of low ionic strengths improves sensitivity of detection while decreasing background signals because only the nucleic acid analyte is charged. While most literature reports focus on in vitro assays in buffer, morpholinos have been also used for biodistribution measurements of species such as fungal rRNA and miRNA. After reviewing the diagnostic applications of morpholinos, the chapter describes preparation of morpholino monolayers on metal supports for electrochemical diagnostics and the procedure for performing label-free detection of DNA from changes in surface capacitance.

Electroporation enables delivering bionanomolecules, such as DNAs, RNAs, siRNAs, and morpholinos, into chick embryos in a spatially and temporally restricted fashion. Recent advances in electroporation techniques allowed us to deliver transgenes into the restricted area of the limb bud and to analyze the function of the enhancers in the limb field. Here, we describe the introduction of transgenes by electroporation in the limb field and its application on enhancer analysis.

Neonatal in vivo electroporations and retinal explant electroporations have been widely employed in understanding the effects of loss or gain of function of protein-coding genes in retinal development. Here, we describe a rapid and efficient delivery of morpholinos to add another tool to perturb gene expression during mouse retinal development.

Noncoding RNAs have essential biochemical functions in different areas of cellular metabolism, including protein synthesis, RNA splicing, protein secretion, and DNA replication. We have successfully used Morpholino antisense oligonucleotides for the functional inactivation of small noncoding RNAs required for DNA replication (Y RNAs in vertebrates and stem-bulge RNAs in nematodes). Here we discuss specific issues of targeting functional noncoding RNAs for inactivation by Morpholino antisense oligonucleotides. We present protocols for the design, preparation, and efficacy controls of Morpholino antisense oligonucleotides, as well as brief descriptions for their delivery into vertebrate and nematode embryos.

Beginning with my concept in 1969 to treat disease at the nucleic acid level using antisense nucleic acids, antisense has evolved to the current Morpholino oligos. Morpholinos have been the dominant gene knockdown system in developmental biology. Lack of delivery technologies has limited their use in adult animals (including humans), though alteration in muscles in Duchenne muscular dystrophy (DMD) allows delivery into adult muscle. Morpholinos are currently in Phase 3 clinical trials for DMD and a Morpholino oligo for skipping dystrophin exon 51 has been approved by the US FDA. With improved delivery techniques, such as those in development at Gene Tools, therapeutic Morpholinos for many difficult-to-treat diseases will be possible. Initial applications are expected to be custom cocktails of delivery-enabled Morpholinos for treating cancers.

Morpholino oligomers are effective antisense molecules to regulate gene expression and the US FDA has approved a Morpholino drug for the treatment of Duchenne muscular dystrophy. However, it has been observed that the antisense activities of aqueous solutions of some Morpholinos decrease over time. We hypothesize that the decreased activity is caused by the formation of soluble aggregates of the Morpholinos. Here, we analyzed three Morpholino sequences by size exclusion chromatography and found two of them have over time formed soluble aggregates in water. The degree of aggregation is sequence-, temperature-, and time-dependent. We describe a simple procedure for detecting and breaking down the aggregates to return the Morpholinos to their monomeric forms.

The chick embryo is a well-established representative of amniote embryos, which has been used to make many discoveries, including many major concepts, which have moved our knowledge of developmental biology in hugely important ways. The chick has a relatively compact genome and is easily amenable to embryological manipulations and in vivo imaging. Morpholino gene manipulations have been used in a variety of contexts, and constitute a quick and versatile molecular tool. Here we describe methods to deliver morpholinos to chick embryos, allowing targeting of specific cell populations at defined developmental stages, using two stages as examples: the epiblast of the embryo in the first day of incubation, when the primary germ layers of the embryo are specified, and in ovo electroporation of the neural tube as an example of a later stage. With slight modifications, these general methods can be used to target other embryonic tissues.

The cohesin protein complex regulates multiple cellular events including sister chromatid cohesion and gene expression. Several distinct human diseases called cohesinopathies have been associated with genetic mutations in cohesin subunit genes or genes encoding regulators of cohesin function. Studies in different model systems, from yeast to mouse have provided insights into the molecular mechanisms of action of cohesin/cohesin regulators and their implications in the pathogenesis of cohesinopathies. The zebrafish has unique advantages for embryonic analyses and quantitative gene knockdown with morpholinos during the first few days of development, in contrast to knockouts of cohesin regulators in flies or mammals, which are either lethal as homozygotes or dramatically compensated for in heterozygotes. This has been particularly informative for Rad21, where a role in gene expression was first shown in zebrafish, and Nipbl, where the fish work revealed tissue-specific functions in heart, gut, and limbs, and long-range enhancer-promoter interactions that control Hox gene expression in vivo. Here we discuss the utility of the zebrafish in studying the developmental and pathogenic roles of cohesin.

Ubiquitination is a dynamic and reversible posttranslational modification. Much effort has been devoted to characterize the function of ubiquitin pathway genes in the cell context, but much less is known on their functional role in the development and maintenance of organs and tissues in the organism. In fact, several ubiquitin ligases and deubiquitinating enzymes (DUBs) are implicated in human pathological disorders, from cancer to neurodegeneration. The aim of our work is to explore the relevance of DUBs in retinal function in health and disease, particularly since some genes related to the ubiquitin or SUMO pathways cause retinal dystrophies, a group of rare diseases that affect 1:3000 individuals worldwide. We propose zebrafish as an extremely useful and informative genetic model to characterize the function of any particular gene in the retina, and thus complement the expression data from mouse. A preliminary characterization of gene expression in mouse retinas (RT-PCR and in situ hybridization) was performed to select particularly interesting genes, and we later replicated the experiments in zebrafish. As a proof of concept, we selected ups45 to be knocked down by morpholino injection in zebrafish embryos. Morphant phenotypic analysis showed moderate to severe eye morphological defects, with a defective formation of the retinal structures, therefore supporting the relevance of DUBs in the formation and differentiation of the vertebrate retina, and suggesting that genes encoding ubiquitin pathway enzymes are good candidates for causing hereditary retinal dystrophies.

Western Blotting has been used extensively for the identification of the protein factors that regulate mammalian oocyte meiosis. However, the limitations in collecting sufficient numbers of oocytes can hinder the efficiency of the technique. Here we provide a detailed protocol for the accurate preparation of mouse oocyte samples for Western Blotting analysis.

High-throughput screening with a loss-of-function strategy is a logical and efficient way to identify novel genes involved in biological processes of interest. In zebrafish, morpholinos have been developed as a convenient tool to knock down gene expression. Here, we describe procedures for systematic screening using morpholinos in zebrafish to identify novel deubiquitylases involved in convergent extension during gastrulation. In this example, we examine candidates based on embryonic morphology and molecular signals of whole mount in situ hybridization assay.

Axolotls are well known for their remarkable ability to regenerate complex body parts and structures throughout life, including the entire limb and tail. Particularly fascinating is their ability to regenerate a fully functional spinal cord after losing the tail. Electroporation of DNA plasmids or morpholinos is a valuable tool to gain mechanistic insight into the cellular and molecular basis of regeneration. It provides among other advantages a simple and fast method to test gene function in a temporally and spatially controlled manner. Some classic drawbacks of the method, such as low transfection efficiency and damage to the tissue, had hindered our understanding of the contribution of different signaling pathways to regeneration. Here, we describe a comprehensive protocol for electroporation of the axolotl spinal cord that overcomes this limitations using a combination of high-voltage and short-length pulses followed by lower-voltage and longer-length pulses. Our approach yields highly efficient transfection of spinal cord cells with minimal tissue damage, which now allows the molecular dissection of spinal cord regeneration.

Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δβ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

The many advantages of the zebrafish model provide a unique opportunity to integrate the tools of developmental embryology, transgenesis, and functional assays to elucidate the molecular pathways underlying hematopoiesis and for modeling human blood diseases. These methodologies have recently been applied to the zebrafish mast cell lineage and have resulted in a better understanding of vertebrate mast cell biology. By employing whole-mount in situ hybridization alone and in combination with co-localization approaches, fluorescence-activated cell sorting (FACS), and morpholino gene knockdown studies, new insights into early mast cell transcriptional regulation and ontogeny have been exposed in vivo. Transgenic strategies have permitted the modeling of human mast cell diseases, like systemic mastocytosis in zebrafish, which can subsequently be exploited for high-throughput chemical screens to identify potential therapies in these conditions. Mast cell functional assays have been adapted to zebrafish providing the opportunity to utilize this model for interrogating the cellular players in innate and adaptive immunity and as a live animal readout for drug responses in allergic and inflammatory reactions. These techniques are detailed in the following chapter.

A mutation in the dehydrodolichol diphosphate synthase (DHDDS) was recently identified as the cause of a subtype of recessive retinitis pigmentosa (RP). Molecular modeling indicates that this mutation could result in low enzymatic efficiency of DHDDS. To investigate the possible link between insufficient DHDDS activity and photoreceptor degeneration, the expression of DHDDS was knocked down by morpholino oligonucleotides (MO) injected into zebrafish one cell embryos. The general appearance and behavior of 4-day-old MO-injected fish were normal, but they failed to respond to light-off, suggesting loss of visual function. Morphological analysis showed that photoreceptor outer segments in retinas of MO-injected fish are very short and in many cases completely missing. Peanut agglutinin (PNA) staining confirmed the absence of cone outer segments. These results demonstrate that suppression of DHDDS expression in zebrafish leads to the loss of photoreceptor outer segments and visual function. These results support the hypothesis that insufficient DHDDS function leads to retinal degeneration.