DNA Delivery/Gene Transfer

Zinc finger nucleases (ZFNs) are artificial restriction enzymes made by fusing an engineered zinc finger DNA-binding domain to the DNA cleavage domain of a restriction enzyme. ZFNs can be used to generate targeted genomic deletions of large segments of DNA in a wide variety of cell types and organisms. In the August issue of Cold Spring Harbor Protocols, Jin-Soo Kim and colleagues present Analysis of Targeted Chromosomal Deletions Induced by Zinc Finger Nucleases, a detailed protocol for the detection and analysis of large genomic deletions in cultured cells introduced by the expression of ZFNs. The method described allows researchers to detect and estimate the frequency of ZFN-induced genomic deletions by simple PCR-based methods. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.

While it is possible to analyze the global lipid composition of a cell, a deeper understanding of what lipids are doing within that cell is more difficult to come by. Though the lipid components may be known, finding their exact position, how dynamically they change location, and how rapidly they are metabolized presents an experimental challenge. The obvious approach would be the addition a fluorescent tag, which would allow for imaging of lipids in cells. Unfortunately, most commonly used fluorescent tags are as large as the lipid itself and are likely to have a strong effect on lipid location and metabolism.

In the July issue of Cold Spring Harbor Protocols, Joachim Goedhart and colleagues present a suite of protocols to get around these problems and allow for live imaging of lipids in cells. Their introduction to the topic explains the approach:

To circumvent this problem, two solutions have been developed–namely, the use of fluorescently labeled proteins that specifically recognize lipids and a chemical method to introduce the fluorescent tag inside the cell.

Protocols are provided for Transfection of Cells with DNA Encoding a Visible Fluorescent Protein-Tagged Lipid-Binding Domain, Labeling Lipids for Imaging in Fixed Cells, and Labeling Lipids for Imaging in Live Cells.

The zebrafish (Danio rerio) has rapidly become a favored model organism for studying developmental biology. One of the most commonly used methods for genetic manipulation in the zebrafish is the delivery of plasmids or oligonucleotides to cells within the living embryo via electroporation. When cells are exposed to brief electrical fields, transient membrane destabilization occurs and nucleic acids can cross the plasma membrane. When the electrical field is removed, the membrane seals and the nucleic acids are trapped inside the cell. In vivo electroporation has proven particularly effective for delivering fluorescent protein expression vectors for imaging and loss-of-function reagents such as morpholinos or RNA interference (RNAi) constructs for the knockdown of gene function. In the July issue of Cold Spring Harbor Protocols, Jack Horne and colleagues present Targeting the Zebrafish Optic Tectum Using In Vivo Electroporation, a modification of the technique that can be used to specifically target the developing optic tectum, the midbrain’s visual processing center. Instructions are given for the construction of electroporation electrodes, preparation and injection of DNA, and electroporation of the DNA into the embryonic brain.

The generation of transgenic plants can be a lengthy and difficult process. Transient expression assays have been developed as faster and more convenient alternatives for investigating gene function. These assays often take advantage of the ability of Agrobacterium to transfer foreign DNA into plant cells with intact cell walls. Agrobacterium-mediated transformation is, however, inefficient and shows great variability. In the May issue of Cold Spring Harbor Protocols, Andreas Nebenführ and colleagues from the University of Tennessee present FAST Technique for Agrobacterium-Mediated Transient Gene Expression in Seedlings of Arabidopsis and Other Plant Species, a quick, efficient and economical assay for gene function in intact plants. The technique involves cocultivation of young plant seedlings and Agrobacterium in the presence of Silwet-77. The Silwet-77 facilitates transformation, thus replacing a wounding or device-dependent vacuum step. As one of May’s featured articles, it is freely available to subscribers and non-subscribers alike.

Neurons are organized into anatomical and functional groups called “circuits”. The activity of these circuits is traditionally monitored using conventional electrophysiological techniques. But some cells, such as the submandibular ganglia, are difficult to impale for intracellular recordings. Instead, viral vectors can be used to deliver fluorescent calcium sensors for detecting activity in a living animal. Calcium Imaging of Neuronal Circuits In Vivo Using a Circuit-Tracing Pseudorabies Virus, from Lynn Enquist and colleagues at Princeton University, provides detailed instructions for the use of the pseudorabies virus (PRV) as a vector for imaging connectivity and activity of neuronal circuits. PRV has a broad host range but does not infect higher-order primates, and it travels along chains of synaptically connected neurons. The PRV strain used in this procedure encodes G-CaMP2, a sensitive fluorescent calcium sensor protein. Available in the April issue of Cold Spring Harbor Protocols, the method allows for reliable detection of endogenous circuit activity at single-cell resolution. As one of April’s featured articles, it is freely available to subscribers and nonsubscribers alike.

The baculovirus expression vector system has been widely used to produce proteins originating from both prokaryotic and eukaryotic sources. It offers easy cloning techniques and abundant viral propagation, and since it is based on an insect cell environment, it provides eukaryotic posttranslational modification machinery. Surface modifications of the viral capsid enable specific targeting. Such modifications can be used to enhance viral binding and entry to a wide variety of both dividing and nondividing mammalian cells, as well as to produce antibodies against the displayed antigen. In addition, the technology should enable modifications of intracellular behavior, i.e., trafficking of recombinant “nanoparticles,” a highly relevant feature for studies of targeted gene or protein delivery. In the March issue of Cold Spring Harbor Protocols, Christian Oker-Blom and colleagues provide a suite of articles detailing the use of baculovirus-based display and gene delivery systems. Their protocol for Creation of Baculovirus Display Libraries is a featured article for March, and is freely available, along with nearly 90 other featured articles.

Cold Spring Harbor Protocols is hosting the movie figures that accompany the new lab manual, Live Cell Imaging, Second Edition, edited by Robert Goldman, Jason Swedlow and David Spector, . These movies are freely accessible to all, and worth a look if you’re interested in seeing the state of the art in time lapse imaging.

Phage-based E. coli homologous recombination systems have been extensively developed in recent years, and these recombination-mediated genetic engineering (“recombineering”) methods are now the preferred technique for carrying out genetic modifications in chromosomes and plasmids. Recombineering is efficient and precise and circumvents many of the problems of traditional genetic engineering methods, primarily the need to locate specific restriction enzyme sites. Construction of Gene-Targeting Vectors by Recombineering, from Pentao Liu and colleagues at the Wellcome Trust Sanger Institute gives detailed instructions for using recombineering to construct targeting vectors for the generation of conditional knockout mice. As one of September’s Featured Articles in Cold Spring Harbor Protocols, the method is freely available to subscribers and non-subscribers alike.

As noted earlier in the week, our featured article focus in August’s Cold Spring Harbor Protocols is on gene transfer into stem cells. The first featured protocol presented a method for using lentiviral vectors as the means for getting your gene of interest expressed. Alhough viral vectors are highly efficient, their use can raise concerns about recombination, immune responses and other safety issues. In contrast, DNA transposons offer an effective, alternative method for nonviral gene transfer that avoids the safety concerns associated with viral vectors. Use of the Sleeping Beauty Transposon System for Stable Gene Expression in Mouse Embryonic Stem Cells from Catherine Krull and colleagues at the University of Michigan provides a method for stable integration and reliable long-term expression of a transgene. Sleeping Beauty transposon-based transfection is a two-component system consisting of a transposase and a transposon containing inverted repeat/direct repeat sequences that result in precise integration into a TA dinucleotide. Like all of our featured articles, this protocol is freely accessible to subscribers and non-subscribers alike.

Our featured articles in the August issue of Cold Spring Harbor Protocols focus on methods for gene transfer in stem cells. Vectors derived from retroviruses are useful tools for long term gene transfer, because they allow stable integration of transgenes and propagation into daughter cells. Lentiviral vectors are preferred because they can transduce non-proliferating cellular targets. These vectors can be engineered to target specific tissues, and an overview of approaches to modify lentivirus vectors for use in gene transfer can be found in Engineering the Surface Glycoproteins of Lentiviral Vectors for Targeted Gene Transfer. Along with this overview, François-Loïc Cosset and colleagues from Ecole Normale Superieure de Lyon present a method for targeting hematopoietic stem cells using engineered viral vectors. The article, Hematopoietic Stem Cell Targeting with Surface-Engineered Lentiviral Vectors is one of our featured articles for August, and like all our featured articles, is freely available to subscribers and non-subscribers alike.

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