DNA Delivery/Gene Transfer

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
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.

April’s issue of CSH Protocols features a set of articles on the production and use of retroviral vectors for gene transfer from Kenneth Cornetta, Karen Pollok and Dusty Miller. Retroviral Vectors for Gene Transfer provides an overview of the subject, drawing on the more than twenty years of experience researchers have with the use of these vectors. The advantages of retroviral vectors are detailed (efficiency, integration and ease of production) along with the disadvantages (inactivation, a requirement for cell division and possible oncogenic activation). The authors discuss important aspects of vector design and choice of packaging cell lines.

Four protocols are provided, two for production of viral vectors, and two for their use in transducing cells. Detailed methods are offered for Retroviral Vector Production by Transient Transfection, and for the Generation of Stable Vector-Producing Cells. Once vectors are generated, they can easily be used to Transduce Cell Lines which are actively proliferating. However, using retroviral vectors with primitive progenitor or stem cells, which are not continuously dividing, is much less efficient. In Transduction of Primary Hematopoietic Cells by Retroviral Vectors, the authors describe two interventions to improve efficiency of transfer, the use of cytokines and other growth factors to stimulate cell cycling, and the use of matrix proteins to mediate colocalization of target cells and vector.

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.

Fate-mapping, the tagging of specific cells or tissues in an embryo, and following their movements and development over time, has a long history as a valuable method. The earliest fate-maps date back to the 1880’s. The first “modern” fate-maps were created in 1929 by Walter Vogt, who applied vital dyes to regions of the amphibian embryo. This allowed him to track which embryonic regions developed into which adult tissues. Two methods, featured in the December issue of CSH Protocols and freely available to non-subscribers, present new fate-mapping techniques, which overcome some serious experimental barriers.
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I know I’m a week or so late, but congratulations are in order for Martin Evans, Oliver Smithies and Mario Capecchi for winning the 2007 Nobel Prize in Physiology or Medicine “for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells”. (more…)

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