Abstract
BackgroundInactivating 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.ResultsRecombinant 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.ConclusionAAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.
Highlights
Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system
Viral vectors deliver Cre to a cell line The primary objective of this part of the study was to determine whether the LV-Cre-EGFP and associated virus (AAV)-Cre viruses allow the delivery of Cre recombinase to mammalian cells. 293T cells were infected either with LV-Cre-EGFP or AAV-Cre viruses
Cultures infected with AAV-Cre showed many Cre-positive cells, with the enzyme predominantly localized in the nucleus (Fig. 1C)
Summary
Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Conventional null mutations of genes, which have important functions during development often show an embryonic lethal phenotype, making experiments on adult animals impossible. Two vectors in particular, recombinant adeno-associated virus (AAV) and lentivirus (LV) have been shown to have the potential to mediate the delivery and stable transduction of genes to both dividing and non-dividing cells in the nervous system, without inducing immune responses [410]. These characteristics have made such vectors useful for transferring a gene of interest into the CNS
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