Minimal cross-recombination between wild-type and loxP511 sites in vivo facilitates truncating both ends of large DNA inserts in pBACe3.6 and related vectors
Contrary to several earlier reports, we find that cross-recombination between wild-type and the mutant loxP511 sites is
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| Veröffentlicht in: | Nucleic acids research 2005-01, Vol.33 (13), p.e118-e118 |
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| container_title | Nucleic acids research |
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| creator | Shakes, Leighcraft A. Garland, Douglas M. Srivastava, Deepak K. Harewood, Ken R. Chatterjee, Pradeep K. |
| description | Contrary to several earlier reports, we find that cross-recombination between wild-type and the mutant loxP511 sites is |
| doi_str_mv | 10.1093/nar/gni119 |
| format | Article |
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The finding enabled us to develop a procedure to truncate DNA progressively from both ends of large genomic inserts flanked by these two loxP sites in pBACe3.6 and related vectors with transposons carrying either a wild-type or a loxP511 sequence. Newly constructed loxP511 transposons contained either a kanamycin resistance gene or no marker. Insert DNA ends in deletions were sequenced with primers unique to each transposon-end remaining after the respective recombination. End-sequencing 223 deletions confirmed that the low level of cross-recombination, observed between those sites during the P1 transductions, does not complicate the procedure: truncations from the unintended end of genomic inserts did not occur. Multiple BACs pooled together could also be processed in a single tube to make end-deletions. This deletion technology, utilizing the very minimal cross-recombination between the mutant and wild-type loxP sites of most BAC clones in the public domain and a heterologous one inserted as a transposon, should facilitate functionally mapping long-range gene regulatory sequences and help to isolate genes with defined functional boundaries in numerous projects including those of therapeutic interest.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/gni119</identifier><identifier>PMID: 16061933</identifier><identifier>CODEN: NARHAD</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Bacteriophage P1 - genetics ; Base Sequence ; Chromosomes, Artificial, Bacterial ; Deoxyribonucleases, Type II Site-Specific - metabolism ; DNA - chemistry ; DNA Primers ; DNA Transposable Elements ; Integrases - metabolism ; Methods Online ; Mutagenesis, Insertional ; Plasmids ; Recombination, Genetic ; Regulatory Sequences, Nucleic Acid ; Sequence Deletion ; Transduction, Genetic ; Viral Proteins - metabolism</subject><ispartof>Nucleic acids research, 2005-01, Vol.33 (13), p.e118-e118</ispartof><rights>Copyright Oxford University Press(England) 2005</rights><rights>The Author 2005. Published by Oxford University Press. All rights reserved 2005</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c441t-1c5dec15609d7b1c35f42edfcb0e86e4e7dea429c3d26e647e957ed737e5ddd13</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1182172/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1182172/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16061933$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shakes, Leighcraft A.</creatorcontrib><creatorcontrib>Garland, Douglas M.</creatorcontrib><creatorcontrib>Srivastava, Deepak K.</creatorcontrib><creatorcontrib>Harewood, Ken R.</creatorcontrib><creatorcontrib>Chatterjee, Pradeep K.</creatorcontrib><title>Minimal cross-recombination between wild-type and loxP511 sites in vivo facilitates truncating both ends of large DNA inserts in pBACe3.6 and related vectors</title><title>Nucleic acids research</title><addtitle>Nucl. Acids Res</addtitle><description>Contrary to several earlier reports, we find that cross-recombination between wild-type and the mutant loxP511 sites is <0.5% of that between two wild-type sites if Cre protein is expressed by phage P1 during an infection. The finding enabled us to develop a procedure to truncate DNA progressively from both ends of large genomic inserts flanked by these two loxP sites in pBACe3.6 and related vectors with transposons carrying either a wild-type or a loxP511 sequence. Newly constructed loxP511 transposons contained either a kanamycin resistance gene or no marker. Insert DNA ends in deletions were sequenced with primers unique to each transposon-end remaining after the respective recombination. End-sequencing 223 deletions confirmed that the low level of cross-recombination, observed between those sites during the P1 transductions, does not complicate the procedure: truncations from the unintended end of genomic inserts did not occur. Multiple BACs pooled together could also be processed in a single tube to make end-deletions. This deletion technology, utilizing the very minimal cross-recombination between the mutant and wild-type loxP sites of most BAC clones in the public domain and a heterologous one inserted as a transposon, should facilitate functionally mapping long-range gene regulatory sequences and help to isolate genes with defined functional boundaries in numerous projects including those of therapeutic interest.</description><subject>Bacteriophage P1 - genetics</subject><subject>Base Sequence</subject><subject>Chromosomes, Artificial, Bacterial</subject><subject>Deoxyribonucleases, Type II Site-Specific - metabolism</subject><subject>DNA - chemistry</subject><subject>DNA Primers</subject><subject>DNA Transposable Elements</subject><subject>Integrases - metabolism</subject><subject>Methods Online</subject><subject>Mutagenesis, Insertional</subject><subject>Plasmids</subject><subject>Recombination, Genetic</subject><subject>Regulatory Sequences, Nucleic Acid</subject><subject>Sequence Deletion</subject><subject>Transduction, Genetic</subject><subject>Viral Proteins - metabolism</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkc9uEzEQh1eIiobChQdAFkekbT1rrzd7QUoDJZXagPgjIS6W155NXTZ2sJ20fRjeFTeJWjhZ8nzzeTy_ongF9Bhoy06cCicLZwHaJ8UImKhK3orqaTGijNYlUD4-LJ7HeE0pcKj5s-IQBBXQMjYq_lxaZ5dqIDr4GMuA2i8761Sy3pEO0w2iIzd2MGW6WyFRzpDB336uAUi0CSOxjmzsxpNeaTvYpO7vUlg7nRVuQTqfrgg6E4nvyaDCAsn7-SR3RQxp2706nUyRHYutO-CQDYZsUCcf4ovioFdDxJf786j4fvbh23RWXnz6eD6dXJSac0gl6NqghlrQ1jQdaFb3vELT647iWCDHxqDiVauZqQQK3mBbN2ga1mBtjAF2VLzbeVfrbolGo0tBDXIV8mrCnfTKyv8rzl7Jhd9IgHEFTZUFb_aC4H-vMSZ57dfB5ZllRamoRF2NM_R2B213HbB_eACovE9S5iTlLskMv_53pEd0H10Gyh1gY8Lbh7oKv6TIP6vl7MdPefp1Ppt_YUKesb8I7q1y</recordid><startdate>20050101</startdate><enddate>20050101</enddate><creator>Shakes, Leighcraft A.</creator><creator>Garland, Douglas M.</creator><creator>Srivastava, Deepak K.</creator><creator>Harewood, Ken R.</creator><creator>Chatterjee, Pradeep K.</creator><general>Oxford University Press</general><general>Oxford Publishing Limited (England)</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>7QO</scope><scope>7QP</scope><scope>7QR</scope><scope>7SS</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope></search><sort><creationdate>20050101</creationdate><title>Minimal cross-recombination between wild-type and loxP511 sites in vivo facilitates truncating both ends of large DNA inserts in pBACe3.6 and related vectors</title><author>Shakes, Leighcraft A. ; Garland, Douglas M. ; Srivastava, Deepak K. ; Harewood, Ken R. ; Chatterjee, Pradeep K.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c441t-1c5dec15609d7b1c35f42edfcb0e86e4e7dea429c3d26e647e957ed737e5ddd13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Bacteriophage P1 - genetics</topic><topic>Base Sequence</topic><topic>Chromosomes, Artificial, Bacterial</topic><topic>Deoxyribonucleases, Type II Site-Specific - metabolism</topic><topic>DNA - chemistry</topic><topic>DNA Primers</topic><topic>DNA Transposable Elements</topic><topic>Integrases - metabolism</topic><topic>Methods Online</topic><topic>Mutagenesis, Insertional</topic><topic>Plasmids</topic><topic>Recombination, Genetic</topic><topic>Regulatory Sequences, Nucleic Acid</topic><topic>Sequence Deletion</topic><topic>Transduction, Genetic</topic><topic>Viral Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Shakes, Leighcraft A.</creatorcontrib><creatorcontrib>Garland, Douglas M.</creatorcontrib><creatorcontrib>Srivastava, Deepak K.</creatorcontrib><creatorcontrib>Harewood, Ken R.</creatorcontrib><creatorcontrib>Chatterjee, Pradeep K.</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>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Entomology Abstracts (Full archive)</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>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shakes, Leighcraft A.</au><au>Garland, Douglas M.</au><au>Srivastava, Deepak K.</au><au>Harewood, Ken R.</au><au>Chatterjee, Pradeep K.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Minimal cross-recombination between wild-type and loxP511 sites in vivo facilitates truncating both ends of large DNA inserts in pBACe3.6 and related vectors</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucl. Acids Res</addtitle><date>2005-01-01</date><risdate>2005</risdate><volume>33</volume><issue>13</issue><spage>e118</spage><epage>e118</epage><pages>e118-e118</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><coden>NARHAD</coden><abstract>Contrary to several earlier reports, we find that cross-recombination between wild-type and the mutant loxP511 sites is <0.5% of that between two wild-type sites if Cre protein is expressed by phage P1 during an infection. The finding enabled us to develop a procedure to truncate DNA progressively from both ends of large genomic inserts flanked by these two loxP sites in pBACe3.6 and related vectors with transposons carrying either a wild-type or a loxP511 sequence. Newly constructed loxP511 transposons contained either a kanamycin resistance gene or no marker. Insert DNA ends in deletions were sequenced with primers unique to each transposon-end remaining after the respective recombination. End-sequencing 223 deletions confirmed that the low level of cross-recombination, observed between those sites during the P1 transductions, does not complicate the procedure: truncations from the unintended end of genomic inserts did not occur. Multiple BACs pooled together could also be processed in a single tube to make end-deletions. This deletion technology, utilizing the very minimal cross-recombination between the mutant and wild-type loxP sites of most BAC clones in the public domain and a heterologous one inserted as a transposon, should facilitate functionally mapping long-range gene regulatory sequences and help to isolate genes with defined functional boundaries in numerous projects including those of therapeutic interest.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>16061933</pmid><doi>10.1093/nar/gni119</doi><oa>free_for_read</oa></addata></record> |
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| ispartof | Nucleic acids research, 2005-01, Vol.33 (13), p.e118-e118 |
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| source | Oxford Journals Open Access Collection; MEDLINE; DOAJ Directory of Open Access Journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Bacteriophage P1 - genetics Base Sequence Chromosomes, Artificial, Bacterial Deoxyribonucleases, Type II Site-Specific - metabolism DNA - chemistry DNA Primers DNA Transposable Elements Integrases - metabolism Methods Online Mutagenesis, Insertional Plasmids Recombination, Genetic Regulatory Sequences, Nucleic Acid Sequence Deletion Transduction, Genetic Viral Proteins - metabolism |
| title | Minimal cross-recombination between wild-type and loxP511 sites in vivo facilitates truncating both ends of large DNA inserts in pBACe3.6 and related vectors |
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