RUSH and CRUSH: A rapid and conditional RNA interference method in mice
Summary RNA interference (RNAi) is a powerful approach to phenocopy mutations in many organisms. Gold standard conventional knock‐out mouse technology is labor‐ and time‐intensive; however, off‐target effects may confound transgenic RNAi approaches. Here, we describe a rapid method for conditional a...
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| Veröffentlicht in: | Genesis (New York, N.Y. : 2000) N.Y. : 2000), 2014-01, Vol.52 (1), p.39-48 |
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| container_title | Genesis (New York, N.Y. : 2000) |
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| creator | Brown, Juliana R. Zetsche, Bernd Jackson-Grusby, Laurie |
| description | Summary
RNA interference (RNAi) is a powerful approach to phenocopy mutations in many organisms. Gold standard conventional knock‐out mouse technology is labor‐ and time‐intensive; however, off‐target effects may confound transgenic RNAi approaches. Here, we describe a rapid method for conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells. RUSH and CRUSH RNAi vectors were designed for reversible or conditional knockdown, respectively, demonstrated using targeted replacement in an engineered ROSA26lacZ ES cell line and wildtype V6.5 ES cells. RUSH was validated by reversible knockdown of Dnmt1 in vitro. Conditional mouse model production using CRUSH was expedited by deriving ES cell lines from Cre transgenic mouse strains (nestin, cTnnT, and Isl1) and generating all‐ES G0 transgenic founders by tetraploid complementation. A control CRUSHGFP RNAi mouse strain showed quantitative knockdown of GFP fluorescence as observed in compound CRUSHGFP, Ds‐Red Cre‐reporter transgenic mice, and confirmed by Western blotting. The capability to turn RUSH and CRUSH alleles off or on using Cre recombinase enables this method to rapidly address questions of tissue‐specificity and cell autonomy of gene function in development. genesis 52:39–48, 2014. © 2013 Wiley Periodicals, Inc. |
| doi_str_mv | 10.1002/dvg.22718 |
| format | Article |
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RNA interference (RNAi) is a powerful approach to phenocopy mutations in many organisms. Gold standard conventional knock‐out mouse technology is labor‐ and time‐intensive; however, off‐target effects may confound transgenic RNAi approaches. Here, we describe a rapid method for conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells. RUSH and CRUSH RNAi vectors were designed for reversible or conditional knockdown, respectively, demonstrated using targeted replacement in an engineered ROSA26lacZ ES cell line and wildtype V6.5 ES cells. RUSH was validated by reversible knockdown of Dnmt1 in vitro. Conditional mouse model production using CRUSH was expedited by deriving ES cell lines from Cre transgenic mouse strains (nestin, cTnnT, and Isl1) and generating all‐ES G0 transgenic founders by tetraploid complementation. A control CRUSHGFP RNAi mouse strain showed quantitative knockdown of GFP fluorescence as observed in compound CRUSHGFP, Ds‐Red Cre‐reporter transgenic mice, and confirmed by Western blotting. The capability to turn RUSH and CRUSH alleles off or on using Cre recombinase enables this method to rapidly address questions of tissue‐specificity and cell autonomy of gene function in development. genesis 52:39–48, 2014. © 2013 Wiley Periodicals, Inc.</description><identifier>ISSN: 1526-954X</identifier><identifier>EISSN: 1526-968X</identifier><identifier>DOI: 10.1002/dvg.22718</identifier><identifier>PMID: 24166816</identifier><language>eng</language><publisher>United States: Blackwell Publishing Ltd</publisher><subject>Animals ; Cell Line ; Embryonic Stem Cells - metabolism ; ES cells ; Gene Knockdown Techniques ; Genetic Vectors ; HEK293 Cells ; Humans ; Mice ; Mice, Transgenic - genetics ; Models, Animal ; mouse models ; Reproducibility of Results ; RNA Interference ; RNAi ; transgenesis</subject><ispartof>Genesis (New York, N.Y. : 2000), 2014-01, Vol.52 (1), p.39-48</ispartof><rights>Copyright © 2013 Wiley Periodicals, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4818-59c251c59a2cfbae74bc343c8a7eab983974a21e395cc11ed27088c148ae131b3</citedby><cites>FETCH-LOGICAL-c4818-59c251c59a2cfbae74bc343c8a7eab983974a21e395cc11ed27088c148ae131b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fdvg.22718$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fdvg.22718$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>230,314,780,784,885,1416,27922,27923,45572,45573</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24166816$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Brown, Juliana R.</creatorcontrib><creatorcontrib>Zetsche, Bernd</creatorcontrib><creatorcontrib>Jackson-Grusby, Laurie</creatorcontrib><title>RUSH and CRUSH: A rapid and conditional RNA interference method in mice</title><title>Genesis (New York, N.Y. : 2000)</title><addtitle>genesis</addtitle><description>Summary
RNA interference (RNAi) is a powerful approach to phenocopy mutations in many organisms. Gold standard conventional knock‐out mouse technology is labor‐ and time‐intensive; however, off‐target effects may confound transgenic RNAi approaches. Here, we describe a rapid method for conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells. RUSH and CRUSH RNAi vectors were designed for reversible or conditional knockdown, respectively, demonstrated using targeted replacement in an engineered ROSA26lacZ ES cell line and wildtype V6.5 ES cells. RUSH was validated by reversible knockdown of Dnmt1 in vitro. Conditional mouse model production using CRUSH was expedited by deriving ES cell lines from Cre transgenic mouse strains (nestin, cTnnT, and Isl1) and generating all‐ES G0 transgenic founders by tetraploid complementation. A control CRUSHGFP RNAi mouse strain showed quantitative knockdown of GFP fluorescence as observed in compound CRUSHGFP, Ds‐Red Cre‐reporter transgenic mice, and confirmed by Western blotting. The capability to turn RUSH and CRUSH alleles off or on using Cre recombinase enables this method to rapidly address questions of tissue‐specificity and cell autonomy of gene function in development. genesis 52:39–48, 2014. © 2013 Wiley Periodicals, Inc.</description><subject>Animals</subject><subject>Cell Line</subject><subject>Embryonic Stem Cells - metabolism</subject><subject>ES cells</subject><subject>Gene Knockdown Techniques</subject><subject>Genetic Vectors</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Mice</subject><subject>Mice, Transgenic - genetics</subject><subject>Models, Animal</subject><subject>mouse models</subject><subject>Reproducibility of Results</subject><subject>RNA Interference</subject><subject>RNAi</subject><subject>transgenesis</subject><issn>1526-954X</issn><issn>1526-968X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kU1v1DAQhiNERT_gwB9AkbiUQ1qPv-JwQFotZVu0KlJLl4qL5TizrUsSb-1sof8et9tdARInj8bPPBrNm2WvgRwAIfSwubs6oLQE9SzbAUFlUUl1-XxdC365ne3GeEMIEYrSF9k25SClArmTTc4uzo9z0zf5-KF6n4_yYBaueWxZ3zducL43bX52OspdP2CYY8DeYt7hcO2b1Ms7Z_FltjU3bcRXT-9edvHp6Ov4uJh-mZyMR9PCcgWqEJWlAqyoDLXz2mDJa8s4s8qUaOpKsarkhgKySlgLgA0tiVIWuDIIDGq2l31YeRfLusPGYj8E0-pFcJ0J99obp__-6d21vvJ3mlVKcEaSYP9JEPztEuOgOxcttq3p0S-jBl5RWVKQLKFv_0Fv_DKkYyRKpkuCIpQm6t2KssHHGHC-WQaIfohHp3j0YzyJffPn9htynUcCDlfAT9fi_f9N-uNsslYWqwkXB_y1mTDhh5YlK4X-djrR4vwzo7PpTH9nvwHyV6cH</recordid><startdate>201401</startdate><enddate>201401</enddate><creator>Brown, Juliana R.</creator><creator>Zetsche, Bernd</creator><creator>Jackson-Grusby, Laurie</creator><general>Blackwell Publishing Ltd</general><general>Wiley Subscription Services, Inc</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QP</scope><scope>7T7</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>201401</creationdate><title>RUSH and CRUSH: A rapid and conditional RNA interference method in mice</title><author>Brown, Juliana R. ; Zetsche, Bernd ; Jackson-Grusby, Laurie</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4818-59c251c59a2cfbae74bc343c8a7eab983974a21e395cc11ed27088c148ae131b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Animals</topic><topic>Cell Line</topic><topic>Embryonic Stem Cells - metabolism</topic><topic>ES cells</topic><topic>Gene Knockdown Techniques</topic><topic>Genetic Vectors</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>Mice</topic><topic>Mice, Transgenic - genetics</topic><topic>Models, Animal</topic><topic>mouse models</topic><topic>Reproducibility of Results</topic><topic>RNA Interference</topic><topic>RNAi</topic><topic>transgenesis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Brown, Juliana R.</creatorcontrib><creatorcontrib>Zetsche, Bernd</creatorcontrib><creatorcontrib>Jackson-Grusby, Laurie</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Genesis (New York, N.Y. : 2000)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Brown, Juliana R.</au><au>Zetsche, Bernd</au><au>Jackson-Grusby, Laurie</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>RUSH and CRUSH: A rapid and conditional RNA interference method in mice</atitle><jtitle>Genesis (New York, N.Y. : 2000)</jtitle><addtitle>genesis</addtitle><date>2014-01</date><risdate>2014</risdate><volume>52</volume><issue>1</issue><spage>39</spage><epage>48</epage><pages>39-48</pages><issn>1526-954X</issn><eissn>1526-968X</eissn><abstract>Summary
RNA interference (RNAi) is a powerful approach to phenocopy mutations in many organisms. Gold standard conventional knock‐out mouse technology is labor‐ and time‐intensive; however, off‐target effects may confound transgenic RNAi approaches. Here, we describe a rapid method for conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells. RUSH and CRUSH RNAi vectors were designed for reversible or conditional knockdown, respectively, demonstrated using targeted replacement in an engineered ROSA26lacZ ES cell line and wildtype V6.5 ES cells. RUSH was validated by reversible knockdown of Dnmt1 in vitro. Conditional mouse model production using CRUSH was expedited by deriving ES cell lines from Cre transgenic mouse strains (nestin, cTnnT, and Isl1) and generating all‐ES G0 transgenic founders by tetraploid complementation. A control CRUSHGFP RNAi mouse strain showed quantitative knockdown of GFP fluorescence as observed in compound CRUSHGFP, Ds‐Red Cre‐reporter transgenic mice, and confirmed by Western blotting. The capability to turn RUSH and CRUSH alleles off or on using Cre recombinase enables this method to rapidly address questions of tissue‐specificity and cell autonomy of gene function in development. genesis 52:39–48, 2014. © 2013 Wiley Periodicals, Inc.</abstract><cop>United States</cop><pub>Blackwell Publishing Ltd</pub><pmid>24166816</pmid><doi>10.1002/dvg.22718</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete |
| subjects | Animals Cell Line Embryonic Stem Cells - metabolism ES cells Gene Knockdown Techniques Genetic Vectors HEK293 Cells Humans Mice Mice, Transgenic - genetics Models, Animal mouse models Reproducibility of Results RNA Interference RNAi transgenesis |
| title | RUSH and CRUSH: A rapid and conditional RNA interference method in mice |
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