Production of single-stranded DNA in mammalian cells by means of a bacterial retron.
msDNA-Ec67, a peculiar multicopy single-stranded DNA of a specific sequence was produced in NIH3T3 mouse cells. Retron-Ec67, a retroelement from Escherichia coli, was introduced under the T7 polymerase promoter and the non-translated 5'-region of the encephalomyocarditis virus. The construct was then transfected into the NIH3T3 cells constitutively producing T7 RNA polymerase. Forty-eight hours after transfection, msDNA-Ec67 was detected in the cells by means of Southern blot hybridization and reverse transcriptase extension assay. The potential use of bacterial retrons as a vector for single-stranded DNA production in mammalian cells is discussed.
Mirochnitchenko
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Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Inouye
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Inouye
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D014764:Viral Proteins
D017931:DNA Primers; D004277:DNA, Single-Stranded; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases