Plasmid rescue from transgenic mouse DNA using LacI repressor protein conjugated to magnetic beads.
A method for the efficient rescue of lac operator containing plasmids from transgenic mouse genomic DNA is described. The method is based on the high affinity of the LacI repressor protein for the lac operator sequence. Using the LacI repressor protein conjugated to magnetic beads, more than 95% of plasmid sequences could be purified from restriction enzyme digested genomic DNA. After circularization, the plasmids were introduced into Escherichia coli by means of electroporation. Since the plasmid was cloned into a bacteriophage lambda vector, the efficiency of plasmid rescue could easily be compared with in vitro packaging. Our results indicate that plasmid rescue is about 25 times more efficient. Application of this method should be especially useful with transgenic mouse models harboring LacZ plasmid shuttle vectors for studying spontaneous or induced mutations in vivo.
Gossen
J A
JA
Ingeny B.V., The Netherlands.
de Leeuw
W J
WJ
Molijn
A C
AC
Vijg
J
J
D000818:Animals; D001426:Bacterial Proteins; D010582:Bacteriophage lambda / Q000235:genetics; D001709:Biotechnology; D004274:DNA, Recombinant / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005069:Evaluation Studies as Topic; D005821:Genetic Techniques; D005822:Genetic Vectors; D007763:Lac Operon; D056968:Lac Repressors; D008280:Magnetics; D051379:Mice; D008822:Mice, Transgenic; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*; D012097:Repressor Proteins
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins