See also figure 5figure supplement 1, figure 5video 1, and figure 5source data 1 and 2. Highperformance and sitedirected in utero electroporation. Apr 26, 20 in utero electroporation has been extensively used to study a variety of developmental questions in the developing brain. Electroporationbased methods for in vivo, whole mount. We support the frontiers for the edge of the science. In utero electroporation required material square wave pulse electroporator cuy21sc electrode cuy650p3 or cuy650p5 depends on the stage of an embryo microinjection system electroporation procedure 1. We present a new protocol for zebrafish brain electroporation. A schematic drawing illustrating the orientation of the electrode paddles for conventional in utero electroporation. Jul 24, 20 here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. Explore btx systems designed for your specific application. In utero electroporation followed by primary neuronal. A transportable, inexpensive electroporator for in utero electroporation.
Jul 17, 2012 in utero electroporation iue is a simple and quick procedure to efficiently perform in vivo genetic manipulation of specific cell types at different brain locations 1,2. In utero intraventricular injection and electroporation of e15 mouse. Tweezertrodes may be used for many in vivo applications, including in utero and exvivo gene transfection, eletroporation therapy, and transdermal drug delivery. Many genetic risk factors for major mental disorders have key roles in brain development. This video is part of the steam 360 video beta program. After installing the steam 360 video tool in the steam client, you can watch this video in a supported virtual reality headset or on your desktop monitor. Gallery in utero electroporation in mouse embryos with rnai on the left and control on the contralateral side.
Tweezertrodes electrodes are reusable noninvasive, tweezerstyle electrodes for drug or gene delivery in animal tissues. Targeted in vivo genetic manipulation of the mouse or rat. The value cr is known as the time constant of the voltage decay. New and improved tools for in utero electroporation studies. Briefly, multiparous rats or mice wistar or cd1, charles river, wilmington, ma, usa, at 15 and 14 days gestation, were anesthetized with ketaminexylazine 10010 mixture, 0. Whether it is specific regions in a mouse embryonic brain such. A piece of sterile gauze with a hole cut in the center is placed over the incision, and one uterine horn is drawn out through the hole in the gauze.
New and improved tools for in utero electroporation. Early in 1992, cobain told rolling stone that he was sure that in utero would showcase both of the extremes of its sound, saying itll. The first demonstration that electroporation could be used for transfection and expression of a foreign gene was made in 1982, and the term electroporation was coined. In utero electroporation as a tool for genetic manipulation in vivo. Video 12 topographic organization of corpus callosum imaged using twophoton microscopy 25x, x19 tiles. We will provide an overview of the our protocol for in utero electroporation of rat embryos, focusing on the most important details, followed.
Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. Directional in utero electroporation specifically targets forebrain interneurons. Search over 1,000 protocols by system, cell type or transfectant. In utero electroporation has been extensively used to study a variety of developmental questions in the developing brain. After the disappointment that was the 20th anniversary of nevermind poor loudness war mastering being a big complaint of many it is a relief and a delight to report that the 20th anniversary edition of in utero is great. In utero electroporation is a rapid and powerful technique to study the development of many brain regions. With in utero retinal electroporation, we can misexpress or downregulate specific genes in rgcs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. One drawback is the inability to efficiently and reliably manipulate gene. In utero electroporation parameters technical drawing of cuy650p1, p3 electrodes technical drawing of cuy6505, p5, 7, p7 electrodes photo of mouthcontrolled micropipette system. Movie s9 continuous imaging of a whole chimeric embryo, injected with egfplabeled mescs in blastocyst stage, for 12 h, from e11.
Dec 09, 2019 all brain samples for each group of gk were collected from offspring of at least three independent in utero electroporation surgeries. Yoshida and mayumi kubota and masaharu ogawa and tomomi shimogori, title journal of visualized experiments. A cinematic rumination on life in the womb and its lasting impact on human development, human behavior, and the state of the world. However, we unexpectedly faced a technical problem. In utero electroporation surgeries begin by exposing the embryonic pups of a pregnant animal, usually a rodent. In utero electroporation is a powerful tool to transfect and manipulate neural precursor cells of the rodent parietal cortex and their progeny in. Recently, a threeelectrode system was designed for efficient electroporation in utero of neural cells.
Citeseerx document details isaac councill, lee giles, pradeep teregowda. To simultaneously visualize microglia and neurallineage cells that interact with each other, we applied the in utero electroporation iue technique, which has been widely used for genetransfer in neurodevelopmental studies, to cx3cr1gfp mice males and females. Here you can find all pieces from nirana album in utero. In utero electroporation iue is an effective transfection method for delivering plasmid dna into neural progenitor cells and neurons of mammalian neocortex in vivo.
In utero in vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. Highperformance and reliable sitedirected in vivo genetic manipulation of mouse and rat brain by in utero electroporation with a tripleelectrode probe. In utero electroporation of mouse cerebellar purkinje cells. Both egfp fluorescence f and mrfp1 fluorescence g can be observed in a single cell. Dna is injected into the pups ventricles, and electrode paddles direct the uptake of the injected dna constructs. Electroporation methods have also been reported for xenopus, zebrafish, and mouse.
Metadata of the chapter that will be visualized online. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene. Targeted gene delivery to telencephalic inhibitory neurons. In vivoin uteroin ovoex vivo electroporation electrodes.
The retina and its sole output neuron, the retinal ganglion cell rgc, comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation1. In utero electroporation was performed as previously published 12,14. Embryonic neocortical microglia express tolllike receptor. In utero electroporation iue has become a method of choice for rapid gain and loss of function studies in embryonic cerebral cortex. This video will introduce the key principles behind in utero electroporation in addition to. Nepagene has developed tools and techniques for transfecting embryonic tissues, or even whole embryos, in mice, rats and chickens. Free access to all jove education videos until june 15. Inutero invivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the. In addition to this, in utero electroporation targets areas of the brain where no specific cre line exists, and will limit embryonic lethality 3,4. In utero invivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. More information can be found in the steam 360 video faq. Chapter title in utero electroporation methods in the study of cerebral cortical.
Article information, pdf download for in utero electroporation as a tool. Jul 07, 2015 electroporation, or electropermeabilization, is a molecular biology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing. Thus, exploring the roles for these genetic factors for brain development at the molecular, cellular, and n. Figures and data in sequential phosphorylation of ndel1 by. In addition to this, in utero electroporation targets areas of the brain where no specific cre line exists. In utero is the third and final studio album by american rock band nirvana, released on september 21, 1993, by dgc records. In neuroscience research, electroporation has been particularly useful for in utero preparations figure 11. Five to 10 minutes after injection, toe pinch and corneal re. Anterior and posterior regions of the cerebral cortex layer iiiii neurons were labeled with tdtomato magenta and egfp green, respectively, using in utero electroporation. Cortex, hippocampus, thalamus, hypothalamus, lateral. Electroporationbased methods for in vivo, whole mount and. All brain samples for each group of gk were collected from offspring of at least three independent in utero electroporation surgeries. In utero intraventricular injection and electroporation of. Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or dna to be introduced into the cell also called electrotransfer.
In utero electroporation resource file kindly also note the following in utero brain applications. Bex also has a collection of the services for the bioscience researchers like oligo dna synthesis, peptide synthesis, protein expression, and cell str analysis. This technique is now widely used to study the processes involved in corticogenesis by overexpressing or knocking down genes and observing the effects. Electroporation, or electropermeabilization, is a molecular biology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing. A hippocampal neuron 7 days after electroporation of 1.
C electroporation by holding the embryo through the uterus with forcepstype electrodes. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Neuroscience, issue 120, brain, development, cortex, electrophysiology, corpus callosum, electroporation. Adult hippocampus stained with neun green, gfap red, and torpo blue. Inutero invivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle.
Fetal origins experts, research scientists, psychologists, doctors and midwives, as well as examples from popular culture and mythology, collectively demonstrate how our experiences in utero shape our future. In chick embryos it has been a particularly powerful technique for the spatial and temporal control of gene expression in developmental studies. Prepare fast green colored endotoxinfree advanced transfectiongrade plasmid solution containing 1,5 pcaggs as previously described 12. In utero electroporation as a tool for genetic manipulation. Highperformance and sitedirected in utero electroporation by a. Put the mouse on a working plate with the abdomen upside. Therefore, the method is only described briefly in the following figure 1. This technique was originally described in the osumi lab 3 and our protocol is based upon one developed in the loturco lab 4. In utero invivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle.
Heterotopia formation in rat but not mouse neocortex after. Copy citation download citations reprints and permissions. Piggybac transposonmediated cellular transgenesis in mammalian forebrain by in utero electroporation fuyi chen 1, brady j. Tweezertrodes electrodes are reusable noninvasive, tweezerstyle electrodes for drug or. In utero electroporation is an important technique for studying the molecular mechanisms that guide.
Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. Keywords in utero electroporation, brain development, genetic risk. Procedure in utero electroporation 1 a 2 cm midline incision is then made in the abdominal wall along the linea alba using a set of forceps and scissors. Protocol micro in utero electroporation for efficient gene targeting in mouse embryos tomomi shimogori cold spring harb protoc. Piggybac transposonmediated cellular transgenesis in. Pdf in utero electroporation methods in the study of cerebral. Bex is a manufacturer of the electroporator cuy21edit, and cell fusion nuclear transfer unit lf101.
Gallery jan 05, 2015 nepagene has developed tools and techniques for transfecting embryonic tissues, or even whole embryos, in mice, rats and chickens. Nirvana intended for the record to diverge significantly from the polished, refined production of its previous album, nevermind 1991. See also figure 5figure supplement 1, figure 5 video 1, and figure 5source data 1 and 2. Parallel fixed 2 needle straight tip electrode 10mmtip, 0. The basic protocol for in utero electroporation has been described in detail in another jove article from the kriegstein lab 1, 2. When a capacitor, charged to a voltage v 0, is discharged into cells, the voltage applied to the cells decreases over time in an exponential curve such that the voltage v at any given time t is given by v v 0 etrc. Tranformation of bacteria, yeast,and plant protoplasts tissues in vivo, for in utero applications as well as in ovo transfection.
This protocol aims to provide the basic knowledge for a beginner to get familiar with the technique. In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by. Efficient gene transfer into the embryonic mouse brain. Embryonic neocortical microglia express tolllike receptor 9.
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