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”. Read the rest of this entry »

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 »