Manipulating gene expression in projection-specific neuronal populations using combinatorial viral approaches

After identifying the brain regions to be studied, coordinates for stereotaxic injections must be optimized as follows: All surgical procedures should be performed only in accordance with a protocol approved by your Institutional Animal Care Use Committee, which might require different methods for anesthesia, surgical procedures, pain management, and post-surgical care and follow-up. Make sure all surgical equipment is sterilized prior to use. Adeno-associated virus (AAV) has become widespread in neurobiological studies due to its ease of use, stable expression, and low tropism (Scammell et al., 2003). Production of AAV requires a shuttle vector containing your construct of interest and a packaging vector encoding additional components necessary for virus production (Gregorevic et al., 2004). Many shuttle vectors already contain the sequences required for Cre-dependent gene expression (Schnutgen et al., 2003). The primary limitation of AAV is its small capacity. The typical size limitation for a cDNA to be cloned into a conditional AAV shuttle vector is approximately 2.3 Kb (assuming a promoter of approximately 1 Kb, such as the CAG promoter (Judge and Chamberlain, 2005). To effectively transduce DK-ZEO cells requires 50–100 viral particles per cell. Each 15 cm dish will contain ~7.7 × 107 cells, requiring a total of ~3.85–7.7 × 109 viral particles. Sectioning of brain tissue for post-hoc histology is critical not only for the initial optimization of injection coordinates, but also to ensure consistency among all animals used for behavioral or other experiments. Many methods for cutting and mounting of brain sections are available, we present one simple method here. We are experimenting with display styles that make it easier to read articles in PMC. Our first effort uses eBook readers, which have several 'ease of reading' features already built in. These PMC articles are best viewed in the iBooks reader. You may notice problems with the display of certain parts of an article in other eReaders. Source.


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