Aug 16, 2001

Targeted integration of foreign DNA into a defined locus on chromosome 19 in K562 cells using AAV-derived components

International Journal of Hematology
Katsuhiro KogureKeiya 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. Two rep 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 a rep plasmid and a plasmid containing a neo 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 the neo gene in 6 (35%) of 17 clones when K562 cells were transfected with pMGiR78 by lipofection. FISH located the neo gene on chromosome 19 in 5 of these 6 c...Continue Reading

Mentioned in this Paper

Fluorescent in Situ Hybridization
AAVS1
Shuttle Vectors
Integration, Provirus
Transfection
Colony-Forming Units, Hematopoietic
Virus Diseases
Adeno-Associated Virus
DNA, Viral
Promoter

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