Open AccessStable transformation of a mosquito cell line results in extraordinarily high copy numbers of the plasmid.
Author(s) -
Thomas J. Monroe,
María C. Mühlmann-Díaz,
Margaret J. Kovach,
Jonathan O. Carlson,
Joel S. Bedford,
Barry J. Beaty
Publication year - 1992
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.89.13.5725
Subject(s) - plasmid , biology , microbiology and biotechnology , transformation (genetics) , plasmid preparation , restriction enzyme , southern blot , agarose gel electrophoresis , ethidium bromide , chromosome , dna , low copy number , puc19 , pbr322 , genetics , gene
Stable incorporation of high copy numbers (greater than 10,000 per cell) of a plasmid vector containing a gene conferring resistance to the antibiotic hygromycin was achieved in a cell line derived from the Aedes albopictus mosquito. Plasmid sequences were readily observed by ethidium bromide staining of cellular DNA after restriction endonuclease digestion and agarose gel electrophoresis. The plasmid was demonstrated by in situ hybridization to be present in large arrays integrated in metaphase chromosomes and in minute and double-minute replicating elements. In one subclone, approximately 60,000 copies of the plasmid were organized in a large array that resembles a chromosome, morphologically and in the segregation of its chromatids during anaphase. The original as well as modified versions of the plasmid were rescued by transformation of Escherichia coli using total cellular DNA. Southern blot analyses of recovered plasmids indicate the presence of mosquito-derived sequences.