प्रसवोत्तर माउस मस्तिष्क के विकास में जीन फंक्शन के वायरस आधारित Cre पुनर्संयोजन का उपयोग करके मोज़ेक विश्लेषण
Summary
एक<em> Vivo में</em> प्रसव के बाद दिमाग में जीन समारोह का परीक्षण विधि वर्णित है. पुनः संयोजक Cre और / या एक फ्लोरोसेंट प्रोटीन व्यक्त AAVs नवजात माउस मस्तिष्क में इंजेक्ट कर रहे हैं. मोज़ेक जीन निष्क्रियता और विरल neuronal लेबलिंग हासिल कर रहे हैं, तंत्रिका सर्किट विकास के लिए महत्वपूर्ण प्रक्रियाओं में जीन समारोह का तेजी से विश्लेषण की अनुमति है.
Abstract
Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development, including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice and rats.
Protocol
Discussion
नवजात वायरल इंजेक्शन विधि यहाँ प्रस्तुत करने के लिए एक सरल और तेजी से प्रसव के बाद मस्तिष्क के विकास के अध्ययन के लिए vivo मोज़ाइक में उत्पन्न तरीका प्रदान करता है. विधि floxed alleles कि वर्तमान में उपलब्ध …
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
इस काम एनआईएच (NINDS) से एक RO1 अनुदान द्वारा समर्थित है.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| rAAV8-Cre, -GFP, -DsRed, | Vector Biolabs | #7060, #7061, custom order | http://www.vectorbiolabs.com |
| Harvard Pump 11 Plus | Harvard Apparatus | #702208 | (No foot pedal port) |
| Retinal Pigment Epithelium Injection Kit | World Precision Instruments | RPE-KIT | Contains connective tubing, injection needles (36G), and needle holder |
| NanoFil Syringe, 100μl | World Precision Instruments | NANOFIL-100 | Includes reusable loading needle |
| D-PBS | Invitrogen | 14040-117 | |
| GFP antibody | Aves | GFP-1020 | |
| DsRed antibody | Clontech | 632496 | |
| Heating Block | VWR | 97042-610 | |
| ROSA26R mouse | Jackson Laboratory | 003309 |
Referencias
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