Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors

BACKGROUND: Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. RESULTS: Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. CONCLUSION: AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination

Use of ordinal outcomes in vascular prevention trials: comparison with binary outcomes in published trials

Background and Purpose—Vascular prevention trials mostly count “yes/no” (binary) outcome events, eg, stroke/no stroke. Analysis of ordered categorical vascular events (eg, fatal stroke/nonfatal stroke/no stroke) is clinically relevant and could be more powerful statistically. Although this is not a novel idea in the statistical community, ordinal outcomes have not been applied to stroke prevention trials in the past. Methods—Summary data on stroke, myocardial infarction, combined vascular events, and bleeding were obtained by treatment group from published vascular prevention trials. Data were analyzed using 10 statistical approaches which allow comparison of 2 ordinal or binary treatment groups. The results for each statistical test for each trial were then compared using Friedman 2-way analysis of variance with multiple comparison procedures. Results—Across 85 trials (335 305 subjects) the test results differed substantially so that approaches which used the ordinal nature of stroke events (fatal/nonfatal/no stroke) were more efficient than those which combined the data to form 2 groups (P0.0001). The most efficient tests were bootstrapping the difference in mean rank, Mann–Whitney U test, and ordinal logistic regression; 4- and 5-level data were more efficient still. Similar findings were obtained for myocardial infarction, combined vascular outcomes, and bleeding. The findings were consistent across different types, designs and sizes of trial, and for the different types of intervention. Conclusions—When analyzing vascular events from prevention trials, statistical tests which use ordered categorical data are more efficient and are more likely to yield reliable results than binary tests. This approach gives additional information on treatment effects by severity of event and will allow trials to be smaller. (Stroke. 2008;39:000-000.)