Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture

Calegari, Federico; Marzesco, Anne Marie; Kittler, Ralf; Buchholz, Frank; Huttner, Wieland B.

doi:10.1111/j.1432-0436.2004.07202002.x

Cited by 24 publications

(15 citation statements)

References 59 publications

(84 reference statements)

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“…For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse [1][2][3][4][5][6][7][8][9][10] . However, it is important to note that in all of these studies, investigators were not targeting soft tissues.…”

mentioning

confidence: 99%

Electroporation of Craniofacial Mesenchyme

Tabler

Liu

2011

JoVE

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Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse [1][2][3][4][5][6][7][8][9][10] . However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis [11][12][13] . In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14 . Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (˜100 million years closer) 15 . Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

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confidence: 99%

Electroporation of Craniofacial Mesenchyme

Tabler

Liu

2011

JoVE

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“…A variety of molecules can be efficiently introduced into cells by electroporation, including DNA, mRNA (Cerda et al, 2006;Chernet and Levin, 2012), dsRNA (Mellitzer et al, 2002;Soares et al, 2008), morpholinos (Falk et al, 2007;Mellitzer et al, 2002;Voiculescu et al, 2008), siRNA (Calegari et al, 2004;Paganin-Gioanni et al, 2011), proteins and drugs (Teissie et al, 2012). In this work we concentrated on DNA electroporation to provide a readout for transfection.…”

Section: Discussionmentioning

confidence: 99%

“…It has been widely used to introduce nucleic acids in model organisms (Calegari et al, 2004;Escoffre et al, 2009;Nakamura and Funahashi, 2012;Swartz et al, 2001). Unlike viral infection and lipofection, electroporation does not require specific sample preparation.…”

Section: Introductionmentioning

confidence: 99%

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

et al. 2014

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The ability to follow and modify cell behaviour with accurate spatiotemporal resolution is a prerequisite to study morphogenesis in developing organisms. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has been used with this aim in different model systems. However, current localised electroporation strategies suffer from insufficient reproducibility and mediocre survival when applied to small and delicate organisms such as early post-implantation mouse embryos. We introduce here a microdevice to achieve localised electroporation with high efficiency and reduced cell damage. In silico simulations using a simple electrical model of mouse embryos indicated that a dielectric guide-based design would improve on existing alternatives. Such a device was microfabricated and its capacities tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Transfection was efficiently and reproducibly restricted to fewer than four visceral endoderm cells without compromising cell behaviour and embryo survival. Combining targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos revealed that, like leading DVE cells, non-leading ones send long basal projections and intercalate during their migration. Finally, we show that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation stages to organ explants. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in the mouse embryo. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices.

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“…Moreover, while stereotaxic injection can be performed on mice of any age, optimal performance of in utero electroporation is limited from about E11 to E16. Stages prior to E11 would require either the use of an ultrasound backscatter microscope 18,19 or a whole embryo culture system 17,20 .…”

Section: Discussionmentioning

confidence: 99%

Expansion of Embryonic and Adult Neural Stem Cells by <em>In Utero</em> Electroporation or Viral Stereotaxic Injection

Artegiani¹,

Lange²,

Calegari³

2012

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Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1 . Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2 . However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4 . Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes [5][6][7][8] . By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9 . Both approaches, whose basic principles were already described by other video protocols [10][11][12][13][14] , were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15 . These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult ne...

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Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture

Cited by 24 publications

References 59 publications

Electroporation of Craniofacial Mesenchyme

Electroporation of Craniofacial Mesenchyme

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

Expansion of Embryonic and Adult Neural Stem Cells by <em>In Utero</em> Electroporation or Viral Stereotaxic Injection

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