Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis
Application of the founder principle from population genetics to variant selection after recombineering allows the isolation of rare unselected recombinants. Current methods to isolate rare (1:10,000–1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamle...
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| Veröffentlicht in: | Nature methods 2014-09, Vol.11 (9), p.966-970 |
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| creator | Lyozin, George T Bressloff, Paul C Kumar, Amit Kosaka, Yasuhiro Demarest, Bradley L Yost, H Joseph Kuehn, Michael R Brunelli, Luca |
| description | Application of the founder principle from population genetics to variant selection after recombineering allows the isolation of rare unselected recombinants.
Current methods to isolate rare (1:10,000–1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle–driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof of principle, we isolated 1:100,000 seamless fluorescent protein–modified
Nodal
BACs and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1 phage–derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated with |
| doi_str_mv | 10.1038/nmeth.3030 |
| format | Article |
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Current methods to isolate rare (1:10,000–1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle–driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof of principle, we isolated 1:100,000 seamless fluorescent protein–modified
Nodal
BACs and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1 phage–derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated with <40 PCRs, and we developed a web-based calculator to optimize FPE.</description><identifier>ISSN: 1548-7091</identifier><identifier>EISSN: 1548-7105</identifier><identifier>DOI: 10.1038/nmeth.3030</identifier><identifier>PMID: 25028895</identifier><language>eng</language><publisher>New York: Nature Publishing Group US</publisher><subject>14/19 ; 14/35 ; 38/70 ; 38/77 ; 45/22 ; 45/41 ; 45/44 ; 631/114/2397 ; 631/1647/1511 ; 631/208/457 ; 64/60 ; 96/109 ; Animals ; Bioinformatics ; Biological markers ; Biological Microscopy ; Biological Techniques ; Biomedical Engineering/Biotechnology ; Chromosomes ; Chromosomes, Artificial, Bacterial - genetics ; Genetic markers ; Genetic Markers - genetics ; Genetic recombination ; Genomics ; Life Sciences ; Mice ; Mutagenesis ; Mutagenesis, Site-Directed - methods ; Physiological aspects ; Protein Engineering - methods ; Proteomics ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Scientific method ; Statistical analysis</subject><ispartof>Nature methods, 2014-09, Vol.11 (9), p.966-970</ispartof><rights>Springer Nature America, Inc. 2014</rights><rights>COPYRIGHT 2014 Nature Publishing Group</rights><rights>Copyright Nature Publishing Group Sep 2014</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c608t-9e22fbae498f5266282f6c37a8f7d43cf0fa558835cfcfda50b5449d83dbad403</citedby><cites>FETCH-LOGICAL-c608t-9e22fbae498f5266282f6c37a8f7d43cf0fa558835cfcfda50b5449d83dbad403</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/nmeth.3030$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/nmeth.3030$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,776,780,881,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25028895$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lyozin, George T</creatorcontrib><creatorcontrib>Bressloff, Paul C</creatorcontrib><creatorcontrib>Kumar, Amit</creatorcontrib><creatorcontrib>Kosaka, Yasuhiro</creatorcontrib><creatorcontrib>Demarest, Bradley L</creatorcontrib><creatorcontrib>Yost, H Joseph</creatorcontrib><creatorcontrib>Kuehn, Michael R</creatorcontrib><creatorcontrib>Brunelli, Luca</creatorcontrib><title>Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis</title><title>Nature methods</title><addtitle>Nat Methods</addtitle><addtitle>Nat Methods</addtitle><description>Application of the founder principle from population genetics to variant selection after recombineering allows the isolation of rare unselected recombinants.
Current methods to isolate rare (1:10,000–1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle–driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof of principle, we isolated 1:100,000 seamless fluorescent protein–modified
Nodal
BACs and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1 phage–derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated with <40 PCRs, and we developed a web-based calculator to optimize FPE.</description><subject>14/19</subject><subject>14/35</subject><subject>38/70</subject><subject>38/77</subject><subject>45/22</subject><subject>45/41</subject><subject>45/44</subject><subject>631/114/2397</subject><subject>631/1647/1511</subject><subject>631/208/457</subject><subject>64/60</subject><subject>96/109</subject><subject>Animals</subject><subject>Bioinformatics</subject><subject>Biological markers</subject><subject>Biological Microscopy</subject><subject>Biological Techniques</subject><subject>Biomedical Engineering/Biotechnology</subject><subject>Chromosomes</subject><subject>Chromosomes, Artificial, Bacterial - genetics</subject><subject>Genetic markers</subject><subject>Genetic Markers - genetics</subject><subject>Genetic recombination</subject><subject>Genomics</subject><subject>Life 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Methods</addtitle><date>2014-09-01</date><risdate>2014</risdate><volume>11</volume><issue>9</issue><spage>966</spage><epage>970</epage><pages>966-970</pages><issn>1548-7091</issn><eissn>1548-7105</eissn><abstract>Application of the founder principle from population genetics to variant selection after recombineering allows the isolation of rare unselected recombinants.
Current methods to isolate rare (1:10,000–1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle–driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof of principle, we isolated 1:100,000 seamless fluorescent protein–modified
Nodal
BACs and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1 phage–derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated with <40 PCRs, and we developed a web-based calculator to optimize FPE.</abstract><cop>New York</cop><pub>Nature Publishing Group US</pub><pmid>25028895</pmid><doi>10.1038/nmeth.3030</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | 14/19 14/35 38/70 38/77 45/22 45/41 45/44 631/114/2397 631/1647/1511 631/208/457 64/60 96/109 Animals Bioinformatics Biological markers Biological Microscopy Biological Techniques Biomedical Engineering/Biotechnology Chromosomes Chromosomes, Artificial, Bacterial - genetics Genetic markers Genetic Markers - genetics Genetic recombination Genomics Life Sciences Mice Mutagenesis Mutagenesis, Site-Directed - methods Physiological aspects Protein Engineering - methods Proteomics Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Scientific method Statistical analysis |
| title | Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis |
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