Transgenic Technology

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. (more…)

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. (more…)

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