Targeted Integration of Foreign DNA Into a Defined Locus on Chromosome 19 in K562 Cells Using AAV-Derived Components View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2001-05-01

AUTHORS

Katsuhiro Kogure, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Yuko Sato, John Monahan, Keiya Ozawa

ABSTRACT

Targeted integration of foreign DNA is ideal for gene therapy, particularly when target cells such as hematopoietic cells actively divide and proliferate. Adeno-associated virus (AAV) has been shown to integrate its genome into a defined locus, AAVS1 (19q13.3-qter). The inverted terminal repeat (ITR) and Rep proteins are responsible for this site-specific integration, and a system has been developed that delivers a gene preferentially into AAVS1 by using these components of AAV. We examined whether this system could be applied to gene transfer into K562 cells. Tworep expression plasmids were tested, 1 driven by the cytomegalovirus (CMV) promoter (pCMVR78) and the other under the translational control of an internal ribosome entry site (pMGiR78) with mouse mammary tumor virus promoter. K562 cells were cotransfected with arep plasmid and a plasmid containing aneo gene flanked by the ITRs. G418-resistant clones were isolated and analyzed by Southern blot analysis and fluorescence in situ hybridization (FISH). Southern blot analysis suggested AAVS1-specific integration of theneo gene in 6 (35%) of 17 clones when K562 cells were transfected with pMGiR78 by lipofection. FISH located theneo gene on chromosome 19 in 5 of these 6 clones (29%). Eight (32%) of 25 clones obtained by electroporation with pCMVR78 had theneo gene at AAVS1, according to Southern blot analysis, and 4 of these 8 clones (16%) were positive according to FISH analysis. These results suggest that site-specific integration of foreign DNA can be achieved at a significantly high rate in human hematopoietic cells using the A AV components. More... »

PAGES

469-475

References to SciGraph publications

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/bf02994009

DOI

http://dx.doi.org/10.1007/bf02994009

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1040745127

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/11503961


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