{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2023,9,13]],"date-time":"2023-09-13T19:13:07Z","timestamp":1694632387489},"reference-count":2,"publisher":"Cold Spring Harbor Laboratory","issue":"8","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cold Spring Harb Protoc"],"published-print":{"date-parts":[[2008,8]]},"abstract":"<jats:title>INTRODUCTION<\/jats:title><jats:p>This protocol describes the use of lentiviral vectors to deliver small interfering RNA (siRNA)-mediated silencing cassettes. The combination of these two technologies allows for the development of a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo. It combines the specificity of RNA interference with the versatility of lentiviral vectors to stably transduce a wide range of cell types. In this method, a small hairpin (shRNA) is cloned initially into an entry vector (pENTR\/U6) immediately downstream from an hU6 promoter. The silencing cassette is flanked by recombination sites from bacteriophage \u03bb (<jats:italic>attL1<\/jats:italic> and <jats:italic>attL2<\/jats:italic>). Once an effective shRNA is obtained, it can be transferred to the destination vector. The destination vector is a lentiviral vector carrying a marker (green fluorescent protein [GFP] or a selection marker) with a destination cassette cloned upstream of the marker (<jats:italic>attR1<\/jats:italic> and <jats:italic>attR2<\/jats:italic> flanking a <jats:italic>ccdB<\/jats:italic> toxic gene). Thus, the silencing cassette can be transferred from the entry vector to the destination vector in a simple Gateway LR cloning reaction. The positioning of the silencing cassette upstream of the marker expression cassette avoids down-regulation of the marker.<\/jats:p>","DOI":"10.1101\/pdb.prot5010","type":"journal-article","created":{"date-parts":[[2008,8,1]],"date-time":"2008-08-01T15:14:52Z","timestamp":1217603692000},"page":"pdb.prot5010","source":"Crossref","is-referenced-by-count":2,"title":["Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version B: Figure 1."],"prefix":"10.1101","volume":"2008","author":[{"given":"Gustavo","family":"Tiscornia","sequence":"first","affiliation":[]},{"given":"Oded","family":"Singer","sequence":"additional","affiliation":[]},{"given":"Inder M.","family":"Verma","sequence":"additional","affiliation":[]}],"member":"246","published-online":{"date-parts":[[2010,12,3]]},"reference":[{"key":"2021111008024830000_2008.8.pdb.prot5010.1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0437912100"},{"key":"2021111008024830000_2008.8.pdb.prot5010.2","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.prot5009"}],"container-title":["Cold Spring Harbor Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/pdb.prot5010","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,10]],"date-time":"2021-11-10T16:59:38Z","timestamp":1636563578000},"score":1,"resource":{"primary":{"URL":"http:\/\/www.cshprotocols.org\/lookup\/doi\/10.1101\/pdb.prot5010"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2008,8]]},"references-count":2,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2010,12,3]]},"published-print":{"date-parts":[[2008,8]]}},"alternative-id":["10.1101\/pdb.prot5010"],"URL":"https:\/\/doi.org\/10.1101\/pdb.prot5010","relation":{},"ISSN":["1940-3402","1559-6095","1559-6095"],"issn-type":[{"value":"1940-3402","type":"print"},{"value":"1559-6095","type":"electronic"},{"value":"1559-6095","type":"print"}],"subject":[],"published":{"date-parts":[[2008,8]]}}}