DNA Interactions in Vivo  

  

Gene Therapy

"Gene Therapy." Scientific American, Nov. '90.

  

Chambers '91

Robert W. Chambers. "Site-specific mutagenesis in cells with normal DNA repair systems: Transitions produced from DNA carrying a single O6-alkylguanine." Nucleic Acids Research 19(9):2485--2488, 1991.

  

Banga & Boyd '92

Satnam S. Banga and James B. Boyd. "Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster." Proc. Natl. Acad. Sci. USA 89:1735-1739, March 1992.

Demonstrates that one can perform site-directed mutagenesis in vivo in the fruit fly. Except not really. The mutated sequence is created externally, and brought back into the fly genone using "P-element insertion."

We're still trying to figure out if our style of site-directed mutagenesis could be made to work in vivo.

  

Fisher et al. '93

Tracey L. Fisher, Terry Terhorst, Xiaodong Cao, and Richard Wagner. Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells. Nucleic Acids Research 21(16):3857-3865, 1993.

Abstract: The intracellular distribution and metabolism of micro-injected fluorescently-labeled oligonucleotides (ODNs) have been evaluated using confocal fluorescence microscopy. Fluorescent phosphodiester ODNs, microinjected into the cytoplasm of mammalian cells, rapidly accumulate within the nucleus; the fluorescence disappears with a half-life of 15-20 minutes. Microinjected fluorescent phosphorothioate ODNs remain in the nucleus for more than 24 hours. The persistence of fluorescence depends on the length of the ODN. Modification of the 3' end of phosphodiester ODNs does not significantly slow the rapid disappearance of the fluorescence, although certain 3' modifications localize ODNs into the cytoplasm. Using specially designed ODNs, endonuclease activity is demonstrated to exist in the cytoplasm and nucleus. Modification of the 2' position of the ribose rings of a fluorescent phosphodiester oligodeoxynucleotide with O-methyl or O-allyl does not alter its intracellular distribution; however, the 2'-O-allyl modification stabilizes the persistence of fluorescence more than 60-fold compared to the 2'-deoxy control. Thus, the experiments indicate that somatic cells contain nucleolytic activities which degrade microinjected ODNs; however, chemical modification can dramatically circumvent this process.

  

Wagner et al. '93

Richard W. Wagner, Mark D. Matteucci, Jason G. Lewis, Arnold J. Gutierrez, Courtney Moulds, and Brian C. Froehler. "Antisense Gene Inhibition by Oligonucleotides Containing C-5 Propyne Pyrimidines." Science 260:1510-1513. June 4, 1993.

  

Milligan et al. '93

John F. Milligan, Mark D. Matteucci and John C. Martin. "Current Concepts in Antisense Drug Design." J. of Medicinal Chemistry 36(14):1923-1937, 1993.

  

Wagner '94

Richard W. Wagner. "Gene inhibition using antisense oligodeoxynucleotides." Nature 372:333-335, Nov 24, 1994.

  

Milligan et al. '94

J. F. Milligan, R. J. Jones, B. C. Froehler, and M. D. Matteucci. "Development of Antisense Therapeutics: Implications for Cancer Gene Therapy." Ann. NY Acad. Sci. 716:228-241, 1994.

  

Tonkinson & Stein '94

John L. Tonkinson and C. A. Stein. "Patterns of intracellular compartmentalization, trafficking and acidification of 5'-fluorescein labeled phosphodiester and phosporothioate oligodeoxynucleotides in HL60 cells." Nucleic Acids Research 22(20):4268-4275, 1994.