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Viruses are effective vectors for delivery of these valuable research tools to mammalian brains. Recombinant viruses are optimized to mediate regulatable, long\u2010term, and cell\u2010specific gene expression. Here, we describe production methods for three of the most commonly used types of recombinant viruses in neurobiology research: adeno\u2010associated virus (AAV), retrovirus\/lentivirus, and glycoprotein\u2010deleted rabies virus. These viral constructs are frequently used for calcium imaging or to deliver neural tracers and optogenetic tools. Popular constructs are readily obtained commercially; however, customized virus production through commercial sources is time consuming and costly. This article aims to provide readers with detailed technical information for rapid production and validation of high\u2010quality viral particles in a laboratory setting while highlighting advantages and limitations of each viral type. \u00a9 2019 by John Wiley &amp; Sons, Inc.<\/jats:p>","DOI":"10.1002\/cpns.66","type":"journal-article","created":{"date-parts":[[2019,3,18]],"date-time":"2019-03-18T15:30:08Z","timestamp":1552923008000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics"],"prefix":"10.1002","volume":"87","author":[{"given":"Shih\u2010Heng","family":"Chen","sequence":"first","affiliation":[{"name":"Viral Vector Core, National Institute of Environmental Health Sciences, NIH\/DHHS, Research Triangle Park  North Carolina"},{"name":"Neurobiology Laboratory, National Institute of Environmental Health Sciences, NIH\/DHHS, Research Triangle Park  North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juhee","family":"Haam","sequence":"additional","affiliation":[{"name":"Neurobiology Laboratory, National Institute of Environmental Health Sciences, NIH\/DHHS, Research Triangle Park  North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mitzie","family":"Walker","sequence":"additional","affiliation":[{"name":"Viral Vector Core, National Institute of Environmental Health Sciences, NIH\/DHHS, Research Triangle Park  North Carolina"},{"name":"Neurobiology Laboratory, National Institute of Environmental Health Sciences, NIH\/DHHS, Research Triangle Park  North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erica","family":"Scappini","sequence":"additional","affiliation":[{"name":"Signal Transduction Laboratory, Fluorescence Microscopy and Imaging Center, National Institute of Environmental Health Sciences, NIH\/DHHS, Research Triangle Park  North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Naughton","sequence":"additional","affiliation":[{"name":"Gene Transfer, Targeting and Therapeutics (GT3) Core, Salk Institute for Biological Studies  La Jolla California"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Negin P.","family":"Martin","sequence":"additional","affiliation":[{"name":"Viral Vector Core, National Institute of Environmental Health Sciences, NIH\/DHHS, Research Triangle Park  North Carolina"},{"name":"Neurobiology Laboratory, National Institute of Environmental Health Sciences, NIH\/DHHS, Research Triangle Park  North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2019,3,18]]},"reference":[{"key":"e_1_2_9_2_1_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0800027105"},{"key":"e_1_2_9_2_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ydbio.2011.03.004"},{"key":"e_1_2_9_2_3_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mb1508s73"},{"key":"e_1_2_9_2_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nn1525"},{"key":"e_1_2_9_2_5_1","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.0409-15.2015"},{"key":"e_1_2_9_2_6_1","doi-asserted-by":"publisher","DOI":"10.1089\/hum.2013.189"},{"key":"e_1_2_9_2_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nn.4593"},{"key":"e_1_2_9_2_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpns.67"},{"key":"e_1_2_9_2_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41593-018-0091-7"},{"key":"e_1_2_9_2_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2017.06.014"},{"key":"e_1_2_9_2_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/mtm.2016.2"},{"key":"e_1_2_9_2_12_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471143030.cb2611s51"},{"key":"e_1_2_9_2_13_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.72.11.8463-8471.1998"},{"key":"e_1_2_9_2_14_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1214441110"},{"key":"e_1_2_9_2_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrdp.2017.91"},{"key":"e_1_2_9_2_16_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mb1002as75"},{"key":"e_1_2_9_2_17_1","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.2129-12.2012"},{"key":"e_1_2_9_2_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/nn.2148"},{"key":"e_1_2_9_2_19_1","doi-asserted-by":"publisher","DOI":"10.1089\/hum.2009.179"},{"key":"e_1_2_9_2_20_1","doi-asserted-by":"publisher","DOI":"10.1089\/hum.2011.111"},{"key":"e_1_2_9_2_21_1","doi-asserted-by":"publisher","DOI":"10.1089\/hum.2010.055"},{"key":"e_1_2_9_2_22_1","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/10.19.2109"},{"key":"e_1_2_9_2_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2013.094"},{"key":"e_1_2_9_2_24_1","doi-asserted-by":"publisher","DOI":"10.3389\/fnmol.2015.00014"},{"key":"e_1_2_9_2_25_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.nsa03bs38"},{"key":"e_1_2_9_2_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2016.01.004"},{"key":"e_1_2_9_2_27_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142301.ns0421s37"},{"key":"e_1_2_9_2_28_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142905.hg1210s54"},{"key":"e_1_2_9_2_29_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mb1006s85"},{"key":"e_1_2_9_2_30_1","doi-asserted-by":"publisher","DOI":"10.1016\/0165-0270(91)90128-M"},{"key":"e_1_2_9_2_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2016.09.021"},{"key":"e_1_2_9_2_32_1","doi-asserted-by":"publisher","DOI":"10.1038\/mt.2009.255"},{"key":"e_1_2_9_2_33_1","doi-asserted-by":"publisher","DOI":"10.1089\/hum.2010.921"},{"key":"e_1_2_9_2_34_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2016.11.045"},{"key":"e_1_2_9_2_35_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.gt.3300938"},{"key":"e_1_2_9_3_2_1","unstructured":"https:\/\/www.addgene.org\/"},{"key":"e_1_2_9_3_3_1","unstructured":"Addgene 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The \u201cPrincipal Investigator\u201d section of Addgene contains recombinant rabies plasmids deposited by the laboratories of Edward Callaway Thomas Jessell and Ian Wickersham."},{"key":"e_1_2_9_3_4_1","unstructured":"https:\/\/www.janelia.org\/"},{"key":"e_1_2_9_3_5_1","unstructured":"The Janelia research campus website provides researchers with the latest news about the developed molecular and imaging tools for neurobiological research."},{"key":"e_1_2_9_3_6_1","unstructured":"http:\/\/www.gensat.org\/cre.jsp"},{"key":"e_1_2_9_3_7_1","unstructured":"GENSAT is a gene\u2010expression nervous system atlas that contains a list of several BAC\u2010Cre recombinase driver lines. Transfer vectors for AAVs retroviruses and lentiviruses can be designed to include recombinase\u2010sensitive sequences and to target specific neuronal populations in these strains."},{"key":"e_1_2_9_3_8_1","unstructured":"https:\/\/www.salk.edu\/science\/core\u2010facilities\/viral\u2010vector\u2010core\/"},{"key":"e_1_2_9_3_9_1","unstructured":"The Salk Institute Viral Vector Core SADB19 rabies dG packaging cell lines and plasmids."},{"key":"e_1_2_9_3_10_1","unstructured":"https:\/\/www2.mrc\u2010lmb.cam.ac.uk\/group\u2010leaders\/t\u2010to\u2010z\/marco\u2010tripodi\/"},{"key":"e_1_2_9_3_11_1","unstructured":"Dr. Tripodi's laboratory website: SiR\u2010SADB19 rabies dG packaging cell lines and plasmids."}],"container-title":["Current Protocols in Neuroscience"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpns.66","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpns.66","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpns.66","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,9,10]],"date-time":"2023-09-10T21:51:48Z","timestamp":1694382708000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpns.66"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,3,18]]},"references-count":45,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2019,4]]}},"alternative-id":["10.1002\/cpns.66"],"URL":"https:\/\/doi.org\/10.1002\/cpns.66","archive":["Portico"],"relation":{},"ISSN":["1934-8584","1934-8576"],"issn-type":[{"value":"1934-8584","type":"print"},{"value":"1934-8576","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,3,18]]},"assertion":[{"value":"2019-03-18","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e66"}}