Downregulation of Gene Expression with Negatively Charged Peptide Nucleic Acids (PNAs) in Zebrafish Embryos

Wickstrom, Eric; Urtishak, Karen A.; Choob, M. V.; Tian, Xiaofeng; Sternheim, Nitzan; Cross, Laura M.; Rubinstein, Amy L.; Farber, Steven

doi:10.1016/s0091-679x(04)77008-9

Cited by 21 publications

(15 citation statements)

References 45 publications

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“…The main antisense chemistry used in zebrafish are morpholino phosphorodiamidate oligonucleotides (morpholinos, MOs) 11. MOs are composed of a phosphorodiamidate backbone with a morpholine ring and the same bases as DNA ( A ) 26,50. Standard use is through steric hindrance of the normal endogenous splicing ( B ) or translation ( E ) mechanisms.…”

Section: Figuresmentioning

confidence: 99%

A Primer for Morpholino Use in Zebrafish

et al. 2009

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Morpholino oligonucleotides are the most common anti-sense “knockdown” technique used in zebrafish (Danio rerio). This review discusses common practices for the design, preparation, and deployment of morpholinos in this vertebrate model system. Off-targeting effects of morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting morpholino. Finally, new uses of morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.

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Section: Figuresmentioning

confidence: 99%

A Primer for Morpholino Use in Zebrafish

et al. 2009

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“…Its transparent body makes it easier to observe the morphology of organs and cells, and of reporter gene expression. Forward genetic screens using chemical mutagens have been successful in discovering many developmental mutations (15), but strategies for reverse genetics have mostly been limited to the use of antisense oligonucleotides that are effective for a limited period of time only (16,17). Recently, methods have been developed for effectively selecting random mutations in a gene of interest by resequencing or enzyme digestion assays (TILLING) after chemical mutagenesis (18,19).…”

Section: Introductionmentioning

confidence: 99%

Characterization of zebrafish Rad52 and replication protein A for oligonucleotide-mediated mutagenesis

Takahashi¹

2005

Nucleic Acids Research

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Zebrafish has become a favorite model organism not only in genetics and developmental biology, but also for the study of cancer, neuroscience and metabolism. However, strategies for reverse genetics in zebrafish are mostly limited to the use of antisense oligonucleotides, and therefore the development of other targeting methods is highly desirable. Here, we report an approach to gene targeting in this system in which single-stranded oligonucleotides and zebrafish Rad52 protein are employed. It has been proposed that a single-stranded oligonucleotide containing a mutation can be incorporated into the genome by annealing to the single-stranded region of the lagging strand of the replication fork. Rad52 is expected to accelerate the annealing step. In vitro experiments using purified truncated Rad52 proteins and replication protein A (RPA) showed that annealing of oligonucleotides is accelerated by Rad52 in the presence of RPA. We developed a simple and sensitive PCR-based method to detect point mutations in the genome. In exploratory experiments, we found that microinjection of single-stranded oligonucleotide targeted to a specific gene together with truncated Rad52 into zebrafish embryos resulted in a low level of recombinant copies in 3 of the 80 embryos tested under these conditions.

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“…Binding of the morpholino to the mRNA prevents its translation into protein and, thus, produces a knockdown or morphant for that particular protein. Furthermore, antisense chimeric oligos, composed of negatively charged peptide and nucleic acid molecules, can produce knockdown zebrafish [Wickstrom et al, 2004]. These forward and reverse genetic approaches in zebrafish not only enable rapid progression from gene discovery to gene function but also secure successful genetic models for the study of human disorders [Keller and Murtha, 2004;Rubenstein, 2003].…”

Section: Techniques Used For Studying Gene Function In Zebrafish and mentioning

confidence: 99%

Fish Pharming: Identifying Gene Function and Therapeutic Targets Using Pharmacologic Treatment of Zebrafish Embryos

Kamkar

Steggles

Martin

2006

CPG

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One of the remaining challenges for the post-genomic era researcher is the systematic assignment of gene function to a sequenced genome. The zebrafish is an effective model organism for conducting comparative analyses of the human genome. The ability to obtain large numbers of zebrafish embryos, grow them in a 96-well dish, and then expose them to molecules dissolved in their water, underline the valuable characteristics of this organism that is currently being explored by academic and private researchers. This high throughput strategy is being applied to evaluate the specificity and the cellular toxicity of environmental toxins and new drug therapies. Similarly, small molecules, metabolic inhibitors, enzyme antagonist and agonist or receptor/ligand substitutes have been used to enhance our understanding of developmental and physiological pathways and gene cascades. These studies also introduce valuable systems that can be employed to investigate the pathologies that can occur when these pathways are disrupted. Pharmacologics act primarily at the post-translational level, the proteome. These results are further supported by comparative genomic studies using the numerous zebrafish mutants that are available or by targeted gene knockdowns using antisense morpholino oligonucleotides. The conservation of genetic mechanisms across a wide range of phyla ensures that the results obtained from fish can be directly transferred to humans.

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Downregulation of Gene Expression with Negatively Charged Peptide Nucleic Acids (PNAs) in Zebrafish Embryos

Cited by 21 publications

References 45 publications

A Primer for Morpholino Use in Zebrafish

A Primer for Morpholino Use in Zebrafish

Characterization of zebrafish Rad52 and replication protein A for oligonucleotide-mediated mutagenesis

Fish Pharming: Identifying Gene Function and Therapeutic Targets Using Pharmacologic Treatment of Zebrafish Embryos

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