Gene targeting in mouse embryonic stem cells with an adenoviral vector |
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| Authors: | Kohnosuke Mitani Maki Wakamiya Paul Hasty Frank L. Graham Allan Bradley C. Thomas Caskey |
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| Affiliation: | (1) Department of Molecular and Human Genetics, Baylor College of Medicine, 77030 Houston, Texas;(2) Howard Hughes Medical Institute, Baylor College of Medicine, 77030 Houston, Texas;(3) Department of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Cancer Center, 77030 Houston, Texas;(4) Departments of Biology and Pathology, McMaster University, L8S 4K1, Ontario, Canada;(5) Present address: Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, 113 Tokyo, Japan |
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| Abstract: | We examined the ability of an E1, E3-defective adenoviral vector to act as a substrate for homologous recombination with chromosomal DNA by including host chromosomal sequence from the mouse Fgr locus that also contained a selectable marker. After infection of mouse embryonic stem cells, stable integration was selected for neomycin resistance and the efficiency of homologous recombination was evaluated. The adenoviral vector was capable of infecting mouse embryonic stem cells efficiently. Between 30–50% of the input virus reached the nuclei after 24 hours of infection. Surprisingly, even without negative selection, 25–40% of the integration resulted from homologous recombination at m.o.i. 10 and 100, although the absolute efficiency of integration was low. Our results suggest that it is possible to modify the structure of an adenoviral vector to achieve a high gene targeting efficiency, resulting in regulated and long-term expression of an introduced gene. |
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