New Technology | Published:

Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step

Nature Genetics volume 30, pages 3139 (2002) | Download Citation

Subjects

This article has been updated

Abstract

We have developed technologies that simplify genomic library construction and screening, substantially reducing both the time and the cost associated with traditional library screening methods and facilitating the generation of gene-targeting constructs. By taking advantage of homologous recombination in Escherichia coli, we were able to use as little as 80 bp of total sequence homology to screen for a specific gene from a genomic library in plasmid or phage form. This method, called recombination cloning (REC), takes only a few days instead of the several weeks required for traditional plaque-lift methods. In addition, because every clone in the mouse genomic library we have constructed has a negative selection marker adjacent to the genomic insert, REC screening can generate gene-targeting vectors in one step, from library screening to finished construct. Conditional targeting constructs can be generated easily with minimal additional manipulation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Change history

  • 27 March 2018

    This article was initially published with an incorrect DOI that did not match the registered version at Crossref. The DOI has been corrected in the article.

References

  1. 1.

    & Gene targeting vectors for mammalian cells. in Gene Targeting: A Practical Approach (ed. Joyner, A.L.) 1–31 (IRL Press, Oxford, 1993).

  2. 2.

    , , & The initiation and control of homologous recombination in Escherichia coli. Philos. Trans. R. Soc. Lond. B Biol. Sci. 347, 13–20 (1995).

  3. 3.

    , & Isolation of exonuclease VIII: the enzyme associated with sbcA indirect suppressor. Proc. Natl Acad. Sci. USA 71, 3593–3597 (1974).

  4. 4.

    The segregation of the SbcA and Rac phenotypes in an Escherichia coli recB mutant. Mol. Gen. Genet. 134, 249–259 (1974).

  5. 5.

    Recombination deficient mutants of E. coli and other bacteria. Annu. Rev. Genet. 7, 67–86 (1973).

  6. 6.

    , , & Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J. Bacteriol. 161, 1219–1221 (1985).

  7. 7.

    Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180, 2063–2071 (1998).

  8. 8.

    , & Roles of RuvC and RecG in phage λ red-mediated recombination. J. Bacteriol. 181, 5402–5408 (1999).

  9. 9.

    & Phasmid vectors for identification of genes by complementation of Escherichia coli mutants. J. Bacteriol. 162, 777–783 (1985).

  10. 10.

    , , , & λ YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations. Proc. Natl Acad. Sci. USA 88, 1731–1735 (1991).

  11. 11.

    & Position and density effects on repression by stationary and mobile DNA-binding proteins. Genes Dev. 3, 185–197 (1989).

  12. 12.

    , & A complex control circuit. Regulation of immunity in temperate bacteriophages. Eur. J. Biochem. 71, 211–227 (1976).

  13. 13.

    , , , & The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr. Biol. 8, 1300–1309 (1998).

  14. 14.

    & Structural and functional analysis of cloned DNA segments containing the replication and incompatibility regions of a miniplasmid derived from a copy number mutant of NR1. J. Bacteriol. 137, 92–104 (1979).

  15. 15.

    , , & The TGV transgenic vectors for single-copy gene expression from the Escherichia coli chromosome. Gene 273, 97–104 (2001).

  16. 16.

    & A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila. Cell 101, 523–531 (2000).

  17. 17.

    , , & Tribbles coordinates mitosis and morphogenesis in Drosophila by regulating string/CDC25 proteolysis. Cell 101, 511–522 (2000).

  18. 18.

    et al. Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase. Science 294, 173–177 (2001).

  19. 19.

    et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).

  20. 20.

    et al. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature 392, 608–611 (1998).

  21. 21.

    , , & A new logic for DNA engineering using recombination in Escherichia coli. Nature Genet. 20, 123–128 (1998).

  22. 22.

    et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl Acad. Sci. USA 97, 5978–5983 (2000).

  23. 23.

    , , van der , & Simplified generation of targeting constructs using ET recombination. Nucleic Acids Res. 27, e16 (1999).

  24. 24.

    , , , & Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse. Nucleic Acids Res. 24, 4594–4596 (1996).

  25. 25.

    , & Construction of gene targeting vectors from lambda KOS genomic libraries. Biotechniques 26, 1150–1156, 1158, 1160 (1999).

  26. 26.

    , , & DNA cloning by homologous recombination in Escherichia coli. Nature Biotechnol. 18, 1314–1317 (2000).

  27. 27.

    , , & Novel bacteriophage λ cloning vector. Proc. Natl Acad. Sci. USA 77, 5172–5176 (1980).

  28. 28.

    , & Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352 (1988).

  29. 29.

    , , & Positive selection for loss of tetracycline resistance. J. Bacteriol. 143, 926–933 (1980).

Download references

Acknowledgements

We thank M.J. Lombardo, R. Ponder, S.M. Rosenberg and K. Murphy for bacterial strains. This work was supported by NIH and ATP grants to S.J.E. and by a HHMI grant to P.Z. S.J.E. is an Investigator with the Howard Hughes Medical Institute.

Author information

Author notes

    • Pumin Zhang
    •  & Mamei Z. Li

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.

    • Pumin Zhang
  2. Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

    • Pumin Zhang
    • , Mamei Z. Li
    •  & Stephen J. Elledge
  3. Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030, USA.

    • Mamei Z. Li
    •  & Stephen J. Elledge

Authors

  1. Search for Pumin Zhang in:

  2. Search for Mamei Z. Li in:

  3. Search for Stephen J. Elledge in:

Corresponding author

Correspondence to Stephen J. Elledge.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ng797

Further reading