Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors
| Autor: | Sablitzky Fred, Niclou Simone P, Levelt Christiaan, Zhang Jing, Hermens Wim TJMC, Eggers Ruben, Chakravarthy Sridhara, Ahmed Bushra Y, Anderson Patrick N, Lieberman AR, Verhaagen Joost |
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| Přispěvatelé: | Netherlands Institute for Neuroscience (NIN) |
| Rok vydání: | 2003 |
| Předmět: |
Genetic Vectors
Green Fluorescent Proteins Gene Expression Mice Transgenic Neocortex Hippocampus lcsh:RC321-571 Cell Line Mice Viral Proteins Genes Reporter Journal Article Animals Transgenes lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry Neurons Recombination Genetic Integrases Research Support Non-U.S. Gov't lcsh:QP351-495 Lentivirus Gene Transfer Techniques Dependovirus Recombinant Proteins Luminescent Proteins lcsh:Neurophysiology and neuropsychology Research Article |
| Zdroj: | BMC Neuroscience BMC Neuroscience, 5. BioMed Central BMC Neuroscience, Vol 5, Iss 1, p 4 (2004) |
| ISSN: | 1471-2202 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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