Gene Transfer Techniques:
The introduction of functional (usually cloned) Genes into cells. A variety of techniques and naturally occurring processes are used for the gene transfer such as cell hybridization, Liposomes or microcell-mediated gene transfer, Electroporation, chromosome-mediated gene transfer, Transfection, and Genetic transduction. Gene transfer may result in genetically transformed cells and individual organisms.
JoVE General
Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation
1The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, 2Department of Developmental Biology, Washington University School of Medicine
This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.
JoVE General
RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus
Biology Department, California State University
In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.
JoVE General
Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences
Department of Cell and Developmental Biology, University of Pennsylvania
We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.
JoVE General
Nuclear Transfer into Mouse Oocytes
Department of Molecular and Cell Biology, Harvard
This movie and the protocol are intended to help learning nuclear transfer.
JoVE General
A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt
1Department of Biology, University of Dayton, 2Center for Tissue Regeneration and Engineering, University of Dayton
In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.
JoVE Neuroscience
Forebrain Electrophysiological Recording in Larval Zebrafish
A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.
JoVE Clinical and Translational Medicine
Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model
1Program in Molecular Medicine and Department of Cancer Biology, University of Massachusetts Medical School, 2Departments of Surgery and Medicine, Weill Cornell Medical College, 3Departments of Surgery and Medicine, New York Presbyterian Hospital
A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.
JoVE General
MISSION esiRNA for RNAi Screening in Mammalian Cells
Max Planck Institute of Molecular Cell Biology and Genetics
Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.
JoVE General
Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos
1Genetics Training Program, University of Wisconsin-Madison, 2Department of Anatomy, University of Wisconsin-Madison, 3Department of Zoology, University of Wisconsin-Madison, 4Cell and Molecular Biology Training Program, University of Wisconsin-Madison
This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.
JoVE General
Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells
Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.
JoVE General
Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster
1Department of Biology, University of Dayton, 2Department of Biology, Center for Tissue Regeneration and Engineering at Dayton, University of Dayton
Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.
JoVE Neuroscience
Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation
Department of Pathology and Immunology, Washington University School of Medicine
This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.
JoVE Neuroscience
Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells
1Department of Pediatric Oncology, University Children's Hospital Essen, 2Department of Developmental Pathology, Bonn Medical School, Institute of Pathology
To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.
JoVE General
Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes
Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University
In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.
JoVE Neuroscience
Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato
Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles
Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.
JoVE General
The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System
1Division of Developmental Neuroscience, United Centers for Advanced Research and Translational Medicine (ART), Tohoku University Graduate School of Medicine, 2The Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST)
Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.
JoVE General
Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit
1Genetically Engineered Models and Services, Charles River, 2Research Models and Services, Charles River
Here we demonstrate the newly developed I•Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I•Cryo kit and later successfully recovered by IVF.
JoVE General
Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran
1Department of Biological Sciences, Vanderbilt University, 2Department of Chemical and Systems Biology, Stanford University
This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.
JoVE General
A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis
Department of Surgery, Weill Cornell Medical College of Cornell University
Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.
JoVE General
Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging
This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.
JoVE General
RNAi Screening to Identify Postembryonic Phenotypes in C. elegans
Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center
We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.
JoVE Neuroscience
Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation
Oregon Hearing Research Center, Oregon Health & Science University
The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.
JoVE General
Imaging Cell Shape Change in Living Drosophila Embryos
1Program in Cell & Molecular Biology, Baylor College of Medicine (BCM), 2Verna & Marrs McLean Department of Biochemistry & Molecular Biology, Baylor College of Medicine (BCM)
Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.
JoVE General
Fertilization of Xenopus oocytes using the Host Transfer Method
Department of Biology, University of Iowa
Procedure for fertilizing Xenopus oocytes by the host transfer method.
JoVE Immunology and Infection
The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis
Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.
JoVE General
Purification of Transcripts and Metabolites from Drosophila Heads
1Department of Neurology, McKnight Brain Institute, University of Florida, 2Department of Entomology and Nematology, University of Florida, 3Genetics Institute, Department of Molecular Genetics and Microbiology, University of Florida, 4McKnight Brain Institute, Department of Neuroscience, Genetics Institute, Center for Translational Research on Neurodegenerative Diseases, and Center for Movement Disorders and Neurorestoration, University of Florida
We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.
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