Abstract
Bacterially propagated plasmid DNA can be transfected into established eukaryotic cell lines or primary cell cultures by a variety of techniques, such as electroporation (see Chapter 5, this vol) (1), scrape-loading (2), and DEAE dextran (see Chapter 3) or calcium phosphate mediated gene transfer (see Chapter 2) (3–5). At least some of the DNA introduced into the cells enters into the nucleus, where it is thought to be assembled into chromatin (6), and is maintained extrachromosomally for at least 48 h. During this time, the cellular chromosomal DNA may have undergone one or more rounds of DNA replication. However, the extrachromosomal transfected DNA will not replicate unless the DNA sequences contained in the plasmid include a DNA origin of replication recognized by the host cell. Origin sequences have so far proved difficult to identify in eukaryotic chromosomes. In contrast, viral genomes, such as SV40 and polyoma, have well-characterized origins of replication (7), which, when included on DNA
Keywords
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Andreason, G. L and Evans, G. A. (1988) Introduction and expression of DNA molecules in eukaryotic cells by electroporation. BioTechniques 6, 650–660.
Wilson, A. C. and Patient, R. K. Scrapefection-A rapid and simple method for transfecting adherent cells. Manuscript in prep
McCutchan, J. H. and Pagano, J. S. (1968) Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethyl-ammo-ethyl-dextran. J. Natl Cancer Inst.41, 35l–356.
Lopata, M., Cleveland, D., and Soller-Webb, B. (1984) High level transient expression of chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment, Nucleic Acids Res. 12, 5707–5717.
Graham, F. L. and van der Eb, A. J, (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–467.
Reeves, R., Gorman, C. M., and Howard, B. (1985) Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells. Nucleic Acids Res. 13, 3599–3615.
Hay, R. T. and Russell, W. C. (1989) Recognition mechanisms in the synthesis of animal virus DNA. Biochem.J. 258, 3–16.
Enver, T., Brewer, A. C., and Patient, R. K. (1988) Role for DNA replication in β-globin gene activation. Mol. Cell. Biol. 8, 1301–1308.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1991 The Humana Press Inc., Clifton, NJ
About this protocol
Cite this protocol
Brewer, A.C., Patient, R.K. (1991). Use of Dpn I Restriction Enzyme to Assess Newly Replicated Gene Copies in Amplifiable Vector Systems. In: Murray, E.J. (eds) Gene Transfer and Expression Protocols. Methods in Molecular Biology, vol 7. Humana Press. https://doi.org/10.1385/0-89603-178-0:405
Download citation
DOI: https://doi.org/10.1385/0-89603-178-0:405
Publisher Name: Humana Press
Print ISBN: 978-0-89603-178-4
Online ISBN: 978-1-59259-494-8
eBook Packages: Springer Protocols