Linked Life Data

14628682

Source:http://linkedlifedata.com/resource/pubmed/id/14628682

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SubjectPredicateObjectContext
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pubmed-article:14628682pubmed:dateCreated2003-11-21lld:pubmed
pubmed-article:14628682pubmed:abstractTextSystems for introducing DNA genes-of-interest into mammalian cellular genomes have ranged from the use of different physical techniques to viruses including retroviruses. We have developed a microinjection method for an efficient and permanent integration of a DNA transgene into the cell genome by use of the retrovirus integrase. A 3.0-kb linear DNA fragment containing an internal herpes simplex virus thymidine kinase gene (tk) with flanking avian retrovirus U5 and U3 terminal attachment sites (U5-pgk/tk-U3) recognized by the integrase was constructed. The other donor, a 3.3-kb linear DNA fragment containing the same gene (pgk/tk) flanked by ApaL1 restriction sites not recognized by integrase, was also produced. After assembly of integrase-transgene complexes on ice, the complexes were microinjected into the nucleus of human fibroblast cells (143Btk) containing a defective thymidine kinase. The number of hypoxanthine/aminopterin/thymidine (HAT)-resistant colonies produced upon microinjection of either naked DNA or the independently assembled integrase-transgene complexes were determined. Our data suggests that enhanced integration of U5-pgk/tk-U3 required the DNA attachment sites and co-delivery of integrase. The data was consistent with a direct role for both of these elements in producing an approximate 4-fold increase in the number of HAT-resistant colonies observed over microinjection of just naked U5-pgk/tk-U3 (P < 0.0001).lld:pubmed
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pubmed-article:14628682pubmed:volume35lld:pubmed
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pubmed-article:14628682pubmed:pagination1072-6, 1078lld:pubmed
pubmed-article:14628682pubmed:dateRevised2007-11-14lld:pubmed
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pubmed-article:14628682pubmed:year2003lld:pubmed
pubmed-article:14628682pubmed:articleTitleAvian retrovirus integrase-enhanced transgene integration into mammalian cell DNA in vivo.lld:pubmed
pubmed-article:14628682pubmed:affiliationUniversity of Texas Medical Branch, Galveston, TX, USA.lld:pubmed
pubmed-article:14628682pubmed:publicationTypeJournal Articlelld:pubmed
pubmed-article:14628682pubmed:publicationTypeResearch Support, U.S. Gov't, P.H.S.lld:pubmed
pubmed-article:14628682pubmed:publicationTypeEvaluation Studieslld:pubmed
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