A morpholino phenocopy of the mouse MOS mutation

Coonrod, Scott A.; Bolling, Laura; Wright, Paul; Visconti, Pablo E.; Herr, John C.

doi:10.1002/gene.1065

Cited by 36 publications

(30 citation statements)

References 10 publications

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“…Development 135, 1735Development 135, -1743Development 135, (2008 These problems in testing gene function delayed the field until early this century, when it was shown, with some fanfare [Editorial,Nature Genetics 26(2),[129][130], that MOs could be targeted to knockdown specific gene expression both in frog (Heasman et al, 2000) and in zebrafish (Ekker, 2000;Nasevicius and Ekker, 2000). The power of MOs to test gene function was quickly recognised and applied to other organisms, including another species of frog, X. tropicalis (Nutt et al, 2001), the chick Gallus gallus (Kos et al, 2001), an ascidian, Ciona savignyi (Satou et al, 2001), oocytes of the mouse Mus musculus (Coonrod et al, 2001), and the sea urchin,Strongylocentrotus purpuratus (Coffman et al, 2004). The importance of MO technology in advancing the field was heralded by an entire issue of the journal Genesis [30(3), July 2001], devoted to gene targeting studies employing MOs in a variety of organisms.…”

Section: Antisense Techniquesmentioning

confidence: 99%

Controlling morpholino experiments: don't stop making antisense

2008

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One of the most significant problems facing developmental biologists who do not work on an organism with welldeveloped genetics -and even for some who do -is how to inhibit the action of a gene of interest during development so as to learn about its normal biological function. A widely adopted approach is to use antisense technologies, and especially morpholino antisense oligonucleotides. In this article, we review the use of such reagents and present examples of how they have provided insights into developmental mechanisms. We also discuss how the use of morpholinos can lead to misleading results, including off-target effects, and we suggest controls that will allow researchers to interpret morpholino experiments correctly.

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Section: Antisense Techniquesmentioning

confidence: 99%

Controlling morpholino experiments: don't stop making antisense

2008

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“…The use of antisense MOs has proven to be a valuable tool for investigating gene function in zebrafish and xenopus embryos [13]. However, few studies have utilized this technology for the study of mammalian oocytes [14,15]. In this work, we investigated the feasibility of using antisense MOs to suppress mouse gene function and revealed the function of H1foo by microinjection of an antisense MO against H1foo into mouse GVstage oocytes.…”

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confidence: 99%

H1foo is Indispensable for Meiotic Maturation of the Mouse Oocyte

et al. 2007

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Abstract. Oocyte-specific linker histone H1foo is localized in the oocyte nucleus, either diffusely or bound to chromatin, during the processes of meiotic maturation and fertilization. This expression pattern suggests that H1foo plays a key role in the control of gene expression and chromatin modification during oogenesis and early embryogenesis. To reveal the function of H1foo, we microinjected antisense morpholino oligonucleotides (MO) against H1foo into mouse germinalvesicle stage oocytes. The rate of in vitro maturation of the antisense MO group was significantly lower than that of the control group. Eggs that failed to extrude a first polar body following injection of antisense MO arrested at metaphase I. Additionally, co-injection of in vitro synthesized H1foo mRNA along with antisense MO successfully rescued expression of H1foo and improved the in vitro maturation rate. There was no difference in the rate of parthenogenesis between the antisense MO and control groups. These results indicate that H1foo is essential for maturation of germinal vesiclestage oocytes.

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“…However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists 13 , a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated 7,12 . We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development [17][18][19] . Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation.…”

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confidence: 99%

“…Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development [17][18][19] . Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation.…”

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confidence: 99%

<em>In vivo</em> Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Hyde

Godwin

Thummel

2012

JoVE

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Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B) 6,7 . Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate ( Figure 1D The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is upregulated during specific stages of fin regeneration [9][10][11][12][13][14][15][16] . However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists 13 , a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated 7,12 . We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development [17][18][19] . Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation. Video LinkThe video component of this article can be found at https://www.jove.com/video/3632/ Protocol 1. Resuspend Morpholino 1. Dilute 300 nM of fluorescein-tagged morpholino into 100 μl of nuclease-free water to make an approximately 3 mM solution. The morpholino solution is aliquoted into multiple paraffin-sealed microcentrifuge tubes and stored at room temperature and away from light. 2. To determine the exact morpholino concentration, dilute 5 μl of morpholino solution (or water as a blank) in 95...

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A morpholino phenocopy of the mouse MOS mutation

Abstract: genesis 30:198–200, 2001. © 2001 Wiley‐Liss, Inc.

Cited by 36 publications

References 10 publications

Controlling morpholino experiments: don't stop making antisense

Controlling morpholino experiments: don't stop making antisense

H1foo is Indispensable for Meiotic Maturation of the Mouse Oocyte

<em>In vivo</em> Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

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