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.

Cold Spring Harbor Laboratory’s courses have long been tremendous community resources, training generation after generation of scientists in the latest cutting-edge techniques. The highly competitive nature of the courses means that not everyone who wants to attend can do so, and one of our missions at CSH Protocols is to help disseminate course material to the scientific community at large. The course instructors have been generously providing CSH Protocols with articles based on their lectures and laboratories, some of which you can see collected here.

November’s issue of CSH Protocols features several methods from the renowned Molecular Embryology of the Mouse course. This long-running course (25-plus years) has long been the absolute standard for training mouse biologists and has resulted in three editions of the well-known manual, Manipulating the Mouse Embryo. What’s interesting about the course as of late, is that the focus has shifted away from just the generation of transgenic and knock-out animals, and more towards the analysis of phenotypes in those animals. November’s featured articles present methods for analyzing specific tissues in the mouse.
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The January Issue of CSH Protocols features several articles detailing the use of nanoparticles for gene delivery. Drug delivery methods using nanoparticles have revolutionized the field. The traditional methods for drug delivery, via oral and intravenous routes, are inefficient, non-specific and expensive. Nanoparticles allow for much greater control over delivery, targeting to specific tissues, higher stability (which allows lower doses to be used) and they can be manufactured cheaply in large quantities. Nanoparticles made from natural polymers are preferred over synthetic ones because of their greater biocompatibility and biodegradibility.

These advances in therapeutic drug delivery techniques also bring benefits to researchers at the laboratory bench. Just as nanoparticles can be used for drug delivery, they can also be used for DNA delivery. Once inside the cell, the key to efficient transfection is getting the DNA through the nuclear membrane. Mansoor Amiji’s group at Northeastern University contribute a series of articles on the use of gelatin nanoparticles for gene delivery, including a general overview, preparation and loading of gelatin nanoparticles, studying intracellular trafficking using TEM and gold-encapsulated nanoparticles, and analysis of transfection using fluorescence microscopy and FACS. In the same issue, you’ll find a protocol for preparation and transfection using biodegradable nanoparticles made from biocompatible polymers such as poly(D,L-lactide-co-glycolide) (PLGA) or polylactide (PLA) from Vinod Labhasetwar’s group at the University of Nebraska.

You can also find several related articles in previous issues of CSH Protocols, including Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery, Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery and An Overview of Condensing and Noncondensing Polymeric Systems for Gene Delivery.

January’s issue of CSH Protocols is now available online, and it contains a set of protocols from Cathy Krull’s lab at the University of Michigan. The articles provide methods for electroporating your gene of interest into somites, neural crest cells and motor neurons. The accessibility of the chick embryo has long made it a standard model organism for developmental biology, and methods like these greatly enhance our abilities to tag and track cells, as well as to genetically manipulate the embryo. They’re even valuable for labs not working with avian systems, particularly mouse labs, because they offer the opportunity to get a quick and easy look at expression and potential effects of experimental constructs. Unlike making a transgenic mouse, an expensive and time-consuming process, working with chick eggs is inexpensive, and relatively rapid. Testing your mouse constructs in the chick embryo is a great way to fine tune the constructs themselves to ensure proper expression. It can also give insight into potential effects of construct expression, which can save valuable time once your transgenic mice are available, as you may already know where to start analyzing.

With the rapid growth of siRNA techniques in so many experimental systems, it’s important to know your options for getting those RNAs into your cells or organism of choice. This month CSH Protocols presents four different methods for delivering siRNAs and shRNAs into various organisms. Read the rest of this entry »

Our April issue is now online, and one of our featured protocols this month is a classic that, in the age of GFP and live imaging, has held up remarkably well and is still used with surprising frequency. Read the rest of this entry »