Advertisement

Transgenic Research

, Volume 3, Issue 1, pp 67–69 | Cite as

An improved selection method for λlacZ phages based on galactose sensitivity

  • Edwin J. Mientjes
  • Joost H. M. van Delft
  • Brenda M. op't Hof
  • Jan A. Gossen
  • Jan Vijg
  • Paul H. M. Lohman
  • Robert A. Baan
Short Communication

Abstract

The determination of thelacZ mutant frequency in λgt10lacZ phage vectors isolated from the transgenic mouse strain 40.6 (MutaMouse), requires the screening of large numbers of phages on β-galactosidase activity. Existing methods rely on distinguishing a few white plaques on X-gal containing plates amongst a multide of blue ones which is both time-consuming and expensive. The new screening method described here employs the galactose sensitiveEscherichia coli C lacZ recA galE strain into which a multicopy plasmid has been introduced, which results in over-expression of thegalK andgalT genes. In the presence of phenyl-β-d-galactopyranoside, a substrate for β-galactosidase, this leads to the suppression of λlacZ+ phage propagation without affecting the ability of λlacZ phages to form plaques. With this method it is possible to screen 1.5×106 phages on a single 9-cm Petri dish. Furthermore, the need for blue/white screening has been eliminated.

Key words

40.6 transgenic mouse MutaMouse mutant selection λgt10lacZ phage galE 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Adhya, S. (1987) The galactose operon. In Neidhart, F.C. ed., Escherichia coli and Salmonella typhimuriumCellular and Molecular Biology, Vol. 1, pp. 1503–12. Washington, D.C.: American Society for Microbiology.Google Scholar
  2. Dreyfus, M., Kotlarz, D. and Busby, S. (1985) Point mutations that affect translation initiation in theEscherichia coli galE gene.J. Mol. Biol.,182, 411–7.Google Scholar
  3. Gossen, J.A., de Leeuw, W.J.F., Tan, C.H.T., Lohman, P.H.M., Berends, F., Knook, D.L., Zwarthoff, E.C. and Vijg, J. (1989) Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying gene mutationsin vivo.Proc. Natl Acad. Sci. USA,86, 7971–5.Google Scholar
  4. Gossen, J.A., Molijn, A.C., Douglas, G.R. and Vijg, T. (1992) Application of galactose-sensitiveE. coli strains as selective hosts forlacZ plasmids.Nucl. Acids Res.,20, 3254.Google Scholar
  5. Gossen, J.A., de Leeuw, W.J.F., Molijn, A. and Vijg, J. (1993a) A selective system forlacZ phage using a galactosesensitiveE. coli host.Bio Techniques 14, 324–5.Google Scholar
  6. Gossen, J.A., de Leeuw, W.J.F., Molijn, A. and Vijg, J. (1993b) Plasmid rescue from transgenic mouse DNA usinglacI repressor protein conjugated to magnetic beads.Bio Techniques 14, 624–9.Google Scholar
  7. Myhr, B.C. (1991) Validation studies with MutaMOuse: a transgenic mouse model for detecting mutationsin vivo.Environ. Mol. Mutagenesis,18, 308–15.Google Scholar
  8. Sambrook, J., Fritch, E.F. and Maniatis, T. (1989)Molecular Cloning: a Laboratory Manual. 2nd Ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.Google Scholar

Copyright information

© Chapman & Hall 1994

Authors and Affiliations

  • Edwin J. Mientjes
    • 1
    • 2
  • Joost H. M. van Delft
    • 1
  • Brenda M. op't Hof
    • 1
  • Jan A. Gossen
    • 3
    • 4
  • Jan Vijg
    • 3
    • 4
  • Paul H. M. Lohman
    • 2
  • Robert A. Baan
    • 1
  1. 1.Department of Genetic ToxicologyTNO Medical Biological LaboratoryRijswijkThe Netherlands
  2. 2.MGC, Department of Radiation Genetics and Chemical Mutagenesis, Sylvius LaboratoriesState University of LeidenLeidenThe Netherlands
  3. 3.Ingeny B. V.LeidenThe Netherlands
  4. 4.Harvard Medical SchoolBostonUSA

Personalised recommendations