As new imaging methods are developed and our knowledge of neural development deepens, methods for growing neuronal cells in culture where they are readily manipulated and observed become more and more important. In the August issue of Cold Spring Harbor Protocols, Amy MacDermott and colleagues from Columbia University provide a series of protocols describing neuronal culture techniques that are particularly useful for studying synapse formation and interconnection.

Preparation of Coverslips for Neuronal Cultures describes setting up glass coverslips for three different types of cell culture: Mass Culture, which results in an extensive and widespread network of synapses, Microisland Culture, which allows easier identification of synaptically connected neurons, and Macroisland Culture, which strikes a balance between the better survival rates and better identification of synapses offered by the two methods above.

Survival of CNS neurons in culture is usually greatly improved if the neurons are plated on top of a confluent astrocyte layer, as is described in Dissection, Plating, and Maintenance of Cortical Astrocyte Cultures. Under these conditions, neurons attach more firmly and develop a more easily identifiable bi-dimensional neuritic tree than in culture conditions where primary cells are plated on top of collagen, laminin or other cell-free substrates.

Once established the astrocyte layer can be used to in the Dissection, Plating, and Maintenance of Dorsal Horn Neuron Cultures, which are suitable for electrophysiological, molecular and immunocytochemical studies. Dorsal horn neurons can be grown by themselves, or co-cultured with embryonic dorsal root ganglion neurons as described in Dissection, Plating, and Maintenance of Dorsal Root Ganglion Neurons for Monoculture and for Coculture with Dorsal Horn Neurons.

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.