Transgenic Technology


The “Brainbow” strategy was originally developed in mice, as a system for labeling neurons in a variety of different colors, allowing one to follow multiple cells regardless of their proximity. Brainbow uses a construct that carries sequences for red, blue and green fluorescent proteins in tandem array, with two pairs of lox sites flanking the first two fluorescent proteins. Recombination occurs in the presence of the Cre recombinase, and one gets a variety of outcomes, resulting in the production of a red, blue or green label. When more than one copy of the Brainbow cassette exists within a cell, the primary tones can be mixed, providing more possible color combinations. This provides a powerful platform for studying neuronal morphology and cell movements. In the January issue of Cold Spring Harbor Protocols, Alex Schier and colleagues offer Multicolor Brainbow Imaging in Zebrafish. This protocol translates the system for use in zebrafish, which offer the advantages of easy visualization of transparent embryos and efficient generation of labeled subjects.

Visualizing mammalian development presents an obvious problem: embryos must develop in utero. That makes them a lot more difficult to see under a microscope than a zebrafish or a frog that develops as a free-standing egg. Extensive work has been done to develop embryonic culture techniques for external development of mouse embryos, allowing imaging approaches to be applied. Early efforts by members of Scott Fraser’s lab (including myself) provided a protocol for growing d 6.5-9.5 mouse embryos on the microscope stage. The December issue of Cold Spring Harbor Protocols features Imaging Cell Movements in Egg-Cylinder Stage Mouse Embryos from Oxford University’s Shankar Srinivas. The article describes a method for isolating and culturing much earlier mouse embryos, as well as an approach for time-lapse imaging as those embryos develop. While cell movements can be followed using light microscopy alone, the increasing variety of transgenic fluorescent reporter mice makes studies of cell movement easier and more informative. As one of December’s featured articles, the protocol is freely available to subscribers and non-subscribers alike.

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.

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.

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.

The May issue of Cold Spring Harbor Protocols is out and it contains a set of articles detailing the use of adenovirus vectors for gene transfer. Genetically modified adenoviruses serve as one of the most versatile and efficient gene delivery systems in use today. Laboratories throughout the world use adenoviruses for the delivery of DNA to cells for basic science and for gene therapy applications. Unlike most other vectors, adenoviruses can infect post-mitotic cells, which makes them particularly useful as vectors for gene delivery into cells like neurons.

In one of May’s featured articles, Robin Parks and colleagues from the Ottawa Health Research Institute provide Construction and Characterization of Adenovirus Vectors, a set of detailed instructions for the generation, propagation, purification, and characterization of adenovirus vectors. Like all of our featured articles, the protocol is freely accessible to subscribers and non-subscribers alike.

In addition, the May issue also contains a set of methods for Cell and Tissue Targeting from David Curiel and colleagues. Transfecting specific cells in a mixed population can be a difficult process. Adenovirus vectors are well-characterized, so they are excellent candidates for modification for targeting to specific cell types. The protocols here describe the creation of adenovirus vectors that enable targeting at the level of binding and entry in targeted cells through primary and/or secondary receptors (transduction), and protein expression of the transgene in the targeted cells (transcription/translation). The articles are:
Construction of Adenovirus Vectors with RGD-Modified Fiber for Transductional Targeting
Construction of Fusion Proteins for Transductional Targeting
and
Construction of Adenovirus Vectors for Transcriptional Targeting

The chicken has long been a superb model system for developmental biology. The patterns of gene expression and overall development of avians and mammals are close enough to make comparisons meaningful. And windowing an egg to view an embryo, then sealing it with scotch tape is a lot easier than performing survival surgery on a pregnant mouse. The big drawback to chicken as a model system has been the lack of genetics, the inability to generate transgenic and knockout lines of birds. Though some success has been reported with chicken ES cells, the large size of the animals, the space requirements and the long generational times makes them unfeasible as laboratory animals for this purpose.

The Japanese Quail, however (Coturnix coturnix japonica), has all of the advantages of the chicken, but with a smaller sized adult, short time to sexual maturity, and prodigious egg production. In the January issue of CSH Protocols, Caltech’s Rusty Lansford and colleagues have contributed a set of papers detailing methods for generating transgenic quail via lentiviral vectors. The resultant transgenic birds can be housed and raised in a standard animal facility, with no more space requirements than mouse.

An overview is available here, and protocols for Generation of High-Titer Lentivirus, Injection of Lentivirus and Screening for Transgenic Offspring are available.

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