3) The intensity and spread of YFP expression increased over the

3). The intensity and spread of YFP expression increased over the following week, reaching levels by P7 that were almost identical to the adult brain (Fig. 3). Fluorescent labeling allowed us to observe postnatal neuronal migration and structural maturation throughout the brain. Particularly striking were C59 wnt in vivo the formation of the hippocampal dentate gyrus (middle column of Fig. 3) and dendritic outgrowth of cerebellar Purkinje cells (right column of Fig. 3). The unexpected speed of functional transgene expression following intraventricular AAV injection offers a powerful new tool for studying early postnatal brain development. The AAV serotype

influences tissue tropism, cellular specificity, and transduction efficiency (Passini et al., 2003; Broekman et al., 2006; Wu et al., 2006; Cearley et al., 2008). We set out to determine whether innate serotype properties could be used to bias which neurons or cell types are manipulated by AAV transgenesis, comparing the transduction patterns of AAV8 with AAV1 and AAV6. Preliminary experiments were performed to determine what titer of

each virus yielded similar expression intensity, ending with ICR pups receiving 1.3 × 1010 particles/ventricle of AAV1, 1.2 × 1010 particles/ventricle of AAV6, or 1.3–4.0 × 109 particles/ventricle of AAV8. All vectors were controlled by the CBA promoter and encoded either three copies of YFP connected by a 2A self-cleavage sequence (triple YFP) (AAV1 and AAV8) or tdTomato (AAV6 and JAK activation AAV8) as a readout for expression. Transduction patterns were analysed at P2, P4, P7, P14 and P21 (n = 4–7 per time point for each serotype). As expected, each serotype produced different expression patterns with varying levels of intensity across

different brain regions. AAV1 and AAV6 were both most strongly expressed in the ventricular ependymal cell layer, suggesting that they do not penetrate the parenchyma as well as AAV8 (Fig. 4). Within the neocortex, AAV1 expressed most strongly in superficial layers, which contrasted sharply with Fossariinae the even distribution of transduced neurons observed with AAV8. AAV1 produced dense transduction within the olfactory bulb and caudal neocortex, but was notably excluded from the rostral neocortex. AAV6 transduction was more sparse than either AAV1 or AAV8, but more evenly distributed throughout the forebrain than AAV1. AAV6 stood out for its relative ability to infect ventral lobules of the cerebellum VIII, IX, and X, where fluorescence within Purkinje cells matched that of pyramidal neurons in the neocortex. Like AAV8, expression of both AAV6 and AAV1 was apparent at the earliest time point examined (P2) although, compared with AAV8, both AAV1 and AAV6 reached maximal expression levels later than AAV8 and produced less intense fluorescence overall.

Leave a Reply

Your email address will not be published. Required fields are marked *


You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>