Efficient in vitro and in vivo expression of covalently modified plasmid DNA.
The tracking of plasmid DNA (pDNA) movement within cells requires the attachment of labels to the DNA in a manner such that: (a) the pDNA remains intact during the labeling process and (b) the labels remain stably attached to the DNA. Keeping these two criteria in mind, we have recently developed a series of alkylating reagents that facilitate the one-step, covalent attachment of compounds directly onto nucleic acids in a nondestructive manner. Using these DNA-alkylating reagents, we have attached a wide range of both fluorescent and nonfluorescent reporter molecules onto pDNAs. We now show that even with the covalent attachment of various marker compounds, the pDNA remains expression competent. The ability to create labeled, expression-competent DNA allows for the simultaneous tracking of both pDNA location and reporter gene expression within living or fixed cells.
Slattum
Paul S
PS
Mirus Corporation, 505 S. Rosa Road, Madison, Wisconsin 53719, USA. jimh@genetransfer.com
Loomis
Aaron G
AG
Machnik
Kira J
KJ
Watt
Mary-Anne
MA
Duzeski
Jennifer L
JL
Budker
Vladimir G
VG
Wolff
Jon A
JA
Hagstrom
James E
JE
D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D022781:Hepatocytes / Q000378:metabolism; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D005456:Fluorescent Dyes; D001710:Biotin; D008156:Luciferases