2012
DOI: 10.1371/journal.pone.0043748
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Efficient Delivery of DNA and Morpholinos into Mouse Preimplantation Embryos by Electroporation

Abstract: Mouse preimplantation development is characterized by three major transitions and two lineage segregations. Each transition or lineage segregation entails pronounced changes in the pattern of gene expression. Thus, research into the function of genes with obvious changes in expression pattern will shed light on the molecular basis of preimplantation development. We have described a simplified and effective method–electroporation–of introducing plasmid DNA and morpholinos into mouse preimplantation embryos and … Show more

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Cited by 45 publications
(46 citation statements)
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“…Pretreatment of zygotes by acidic Tyrode's solution has been initially established to increase the permeability of the zona pellucida for large RNA molecules during electroporation and has been subsequently shown to be necessary for gene editing using Cas9 mRNA of about 4,500 nucleotides length [7,22]. Uptake of small molecules like morpholinos of only 25 nucleotides length, however, neither required nor greatly benefited from acidic Tyrode's pre-treatment of zygote prior to electroporation [12]. We accomplished highly efficient gene editing in have been successfully used for generation of mutant mice by CRISPR-PNI [23].…”
Section: Discussionmentioning
confidence: 99%
See 1 more Smart Citation
“…Pretreatment of zygotes by acidic Tyrode's solution has been initially established to increase the permeability of the zona pellucida for large RNA molecules during electroporation and has been subsequently shown to be necessary for gene editing using Cas9 mRNA of about 4,500 nucleotides length [7,22]. Uptake of small molecules like morpholinos of only 25 nucleotides length, however, neither required nor greatly benefited from acidic Tyrode's pre-treatment of zygote prior to electroporation [12]. We accomplished highly efficient gene editing in have been successfully used for generation of mutant mice by CRISPR-PNI [23].…”
Section: Discussionmentioning
confidence: 99%
“…However, several obstacles within the current protocols hinder the broader application of electroporation: Firstly, they either require the costly investment in a specialized electroporator device, or use corrosive pre-treatment of the embryo by acidic Tyrode's solution to allow uptake of the CRISPR/Cas9 components. Weakening of the zona pellucida, a vital part of the embryo, by acidic Tyrode's solution compromises embryo viability [10][11][12] while the necessary incubation time of as short as 10 s [7] is challenging to be reproducibly applied even for highly experienced users. Furthermore, the batch specific activity of acidic Tyrode's solution [8] challenges its safe and reproducible application.…”
Section: Introductionmentioning
confidence: 99%
“…Nevertheless, the introduction of plasmid DNA and mRNA into murine preimplantation embryos has been achieved by electroporation of zonaweakened [30] and zona-intact [31] embryos, suggesting that even macromolecules of high molecular weight can enter into the embryonic cytoplasm through ZP. Those findings lead us to investigate whether crotamine could deliver exogenous DNA through ZP of intact bovine preimplantation embryos.…”
Section: Discussionmentioning
confidence: 99%
“…Furthermore, MOs are highly target-specific because of their RNAse H-independent mechanism of action and inability to form small, transient RNA duplexes (Summerton, 2007). Previ-ously, MOs have been used in a variety of vertebrate and invertebrate organisms to study gene function, although the method of delivery has been largely restricted to microinjection (McMahon et al, 2010;Layden et al, 2013;Melvin et al, 2013) or electroporation (Peng et al, 2012). MOs have also shown high bioavailability and efficiency for target knockdown, however, when administered orally to adult rats (Arora et al, 2002).…”
Section: Introductionmentioning
confidence: 99%