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.

Mutational analysis has long been a valuable tool for deciphering gene function. However, systematic repeated targeting of a single locus is difficult and is not a routine approach in multicellular organisms. Yikang Rong and colleagues at the National Cancer Institute have developed the Site-specific Integrase mediated Repeated Targeting (SIRT) method to facilitate targeted mutagenesis in Drosophila melanogaster. SIRT targets a landing site for the phage phiC31 integrase and allows the generation of several genetic variants at a locus of interest without having to perform multiple experiments. SIRT requires the construction of a series of plasmid vectors with varying arrangements of DNA elements. By taking advantage of bacterial recombineering approaches, SIRT bypasses the shortcomings of traditional cloning techniques that rely on the availability of convenient restriction enzyme cut sites. SIRT Combines Homologous Recombination, Site-Specific Integration, and Bacterial Recombineering for Targeted Mutagenesis in Drosophila, is one of June’s featured articles in Cold Spring Harbor Protocols. Like all of our featured articles, the protocol 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

Way back in 2003, we published RNAi: A Guide To Gene Silencing, which was one of, if not the first major treatises on the subject. One of the problems with being the first to publish on a fast-moving field is that a book can date quickly. While there’s still much valuable information in RNAi, I’ve been asking authors to update their protocols, which have evolved over the last 5 years or so.

Last month, Esther Stoeckli and colleagues provided an update to her method for Gene Silencing by Injection and Electroporation of dsRNA in Avian Embryos.

This month’s issue brings a tour de force updating and expansion of Petr Svoboda and Paula Stein’s chapter on RNAi in mouse oocytes and early embryos. They’ve written up a general topic introduction on the subject, explanations of how to choose the sequence of dsRNA for RNAi and how to clone and sequence an inverted repeat, and protocols for Cloning a Transgene for Transgenic RNAi in Mouse Oocytes, Preparation of dsRNA for Microinjection, Microinjection of dsRNA into Fully-Grown Mouse Oocytes, Microinjection of dsRNA into Mouse One-Cell Embryos, and Microinjection of Plasmids into Meiotically Incompetent Mouse Oocytes.

Next month will bring an update of Savithramma Dinesh-Kumar’s protocol for using viral vectors for RNAi in plants. More on that in February.

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.

August’s issue of CSH Protocols is now available, and one of the featured protocols this month comes from Inder Verma’s lab, and covers the Design and Cloning of an shRNA into a Lentiviral Vector. Combining the specificity of small interfering RNA (siRNA) silencing with the versatility of lentiviral vectors gives researchers a powerful tool for the investigation of gene function both in vivo and in vitro. There’s also an alternative method available. In the featured method, one undesirable consequence of this procedure is that the siRNA target sequence is also present in the mRNA expressing the marker gene, resulting in somewhat lower expression of the marker. In the alternative method, the position of the silencing cassette is upstream of the marker expression cassette, thus avoiding down-regulation of the marker. But, because the silencing cassette is not in the 3′ LTR, only one copy of the silencing cassette is delivered per viral particle (as opposed to two copies in the featured method).

All of our monthly featured articles are freely available to subscribers and non-subscribers alike.