Morpholinos: studying gene function in the chick (original) (raw)
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Chinese Science Bulletin, 2014
Gene transfection is an indispensable approach for studying gene function since it provides important information on gain-and/or loss-of-function. Chick embryos are also extensively employed for studying biological function since they are easily accessible and can be maintained alive after manipulation. The combination of both techniques presents a powerful approach to understanding how genes regulate embryo development. Furthermore, combining these approaches with tissue transplant techniques make even more attractive for elucidate gene function. Electroporation, employing parallelly fashioned electrodes, has been widely used in chick embryos. However, experimenters have been frustrated by unsuccessfully transfection in some embryonic tissue of interest because the electrodes were improperly positioned.
Spatially and temporally controlled electroporation of early chick embryos
2008
The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain-and loss-of-function experiments and studying gene regulatory elements in living embryos.
Methods for introducing morpholinos into the chicken embryo
Developmental Dynamics, 2003
The use of antisense morpholino oligos to inhibit the translation of a target transcript has been applied recently to studies of the chicken embryo. In contrast to other developmental systems such as in frog, sea urchin, and zebrafish that permit the direct microinjection of morpholinos into a blastomere, square pulse electroporation is used to introduce fluorescently tagged morpholinos into specific populations of chick embryo cells in ovo. This article reviews the methods that have proven successful, the types of controls that are necessary when performing knockdowns of gene expression in the chick embryo, and discusses the limitations of the current technique, as well as directions for further research. Developmental Dynamics 226: 470 -477, 2003.
Screening for gene function in chicken embryo using RNAi and electroporation
Nature Biotechnology, 2002
In the postgenomic era the elucidation of the physiological function of genes has become the rate-limiting step in the quest to understand the development and function of living organisms. Gene functions cannot be determined by high-throughput methods but require analysis in the context of the entire organism. This is particularly true in the developing vertebrate nervous system. Because of its easy accessibility in the egg, the chicken embryo has been the model of choice for developmental in vivo studies. However, its usefulness has been hampered by a lack of methods for genetic manipulation. Here we describe an approach that could compensate for this disadvantage. By combining gene silencing by dsRNA (through RNA interference, RNAi) with in ovo electroporation, we developed an efficient method to induce loss of gene function in vivo during the development of the chicken CNS. This method opens new possibilities for studying gene function not only by gain-of-function but also by loss-of-function approaches and therefore represents a new tool for functional genomics.
A detailed description of an economical setup for electroporation of chick embryos in ovo
Brazilian Journal of Medical and Biological Research, 2013
One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.
Sparking New Frontiers: Using in Vivo Electroporation for Genetic Manipulations
Developmental Biology, 2001
In vivo electroporation is a fascinating new approach by which gene expression, regulation, and function can be studied in developmental systems. This technique offers new opportunities for manipulations in animal models that lack genetic approaches, including avians. Furthermore, this approach is applicable to other embryo populations including mice, ascidians, zebrafish, Xenopus, and Drosophila. In this review, we discuss technical aspects of in vivo electroporation, review recent studies where this approach has been utilized successfully, and identify future directions.
Method for electroporation for the early chick embryo
Development, Growth & Differentiation, 2008
In vitro whole-embryo culture of chick embryos, originally invented by New, has been widely used for studies of early embryogenesis. Here, a method for electroporation using the New culture and its derivatives is described, to achieve misexpression of exogenous gene in a temporally and spatially controlled manner in gastrulating chick embryos. Detailed information for the devices and procedures, and some experimental examples are presented.
Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus
BMC Developmental Biology, 2007
Background: Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes.
New Tools for Gene Manipulation in Chicken Embryos
Oligonucleotides, 2003
Genomics has changed the pace by which genes are analyzed. Rather than looking at genes one by one, gene expression today is studied at the genome level. Unfortunately, the data we get from microarray analysis do not give us any clues about the function of these genes. Functional analyses are still refractory to large-scale, high-throughput studies, particularly in vertebrates.
Chapter 13 Electroporation into Cultured Mammalian Embryos
2014
Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms underlying complex developmental processes (Mak, 2007). However, our understanding of cell–cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, whi...