Selection of Targeted Mutants from a Library of Randomly Mutagenized ES Cells

  • Kyoji Horie
  • George Gaitanaris
  • Alexander GragerovEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 693)


A method for random relatively unbiased mutagenesis of ES cells with a mutagenic retroviral vector is described. An orderly assembly of mutant ES cells in multi-well plates is generated. 3D pooling of the wells of the assembly allows quick PCR search for insertions in genes of interest. Mutant ES cell clones are then isolated from the positive wells and used to produce mutant animals using conventional techniques.

Key words

Retroviral insertion Random mutagenesis ES cell Knock-out mice 3D pooling Gene targeting 


  1. 1.
    Thomas, K. R., Folger, K. R. & Capecchi, M. R. (1986) High frequency targeting of genes to specific sites in the mammalian genome. Cell 44, 419–428.PubMedCrossRefGoogle Scholar
  2. 2.
    Friedrich, G. & Soriano, P. (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 5, 1513–1523.PubMedCrossRefGoogle Scholar
  3. 3.
    The International Mouse Knockout Consor­tium. (2007) A mouse for all reasons. Cell 128, 9–13.CrossRefGoogle Scholar
  4. 4.
    Carninci, P. (2008) Hunting hidden transcripts. Nat Methods 5, 587–589.PubMedCrossRefGoogle Scholar
  5. 5.
    Gragerov, A., Horie, K., Pavlova, M., Madisen, L., Zeng, H., Vassilatis, D., Bergmann, J., Gragerova, G., Rohde, A., Dolka, I., Ebbert, A., Moe, S., Navas, C., Finn, E. & Gaitanaris, G. (2007) Large-scale, saturating insertional mutagenesis of the mouse genome. Proc Natl Acad Sci U S A 104, 14406–14411.PubMedCrossRefGoogle Scholar
  6. 6.
    Ivics, Z., Hackett, P. B., Plasterk, R. H. & Izsvák, Z. (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501–510.PubMedCrossRefGoogle Scholar
  7. 7.
    Wang, W., Bradley, A. & Huang, Y. (2009) A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells. Genome Res 19, 667–673.PubMedCrossRefGoogle Scholar
  8. 8.
    Kawakami, K. & Noda, T. (2004) Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells. Genetics 166, 895–899.PubMedCrossRefGoogle Scholar
  9. 9.
    Liang, Q., Kong, J., Stalker, J. & Bradley, A. (2009) Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac transposons. Genesis 47, 404–408.PubMedCrossRefGoogle Scholar
  10. 10.
    Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. (2003) Manipulating the mouse embryo. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp. 371–375.Google Scholar
  11. 11.
    Zeng, H., Horie, K., Madisen, L., Pavlova, M. N., Gragerova, G., Rohde, A. D., Schimpf, B. A., Liang, Y., Ojala, E., Kramer, F., Roth, P., Slobodskaya, O., Dolka, I., Southon, E. A., Tessarollo, L., Bornfeldt, K. E., Gragerov, A., Pavlakis, G. N. & Gaitanaris, G. A. (2008) An inducible and reversible mouse genetic rescue system. PLoS Genet 4, e1000069.PubMedCrossRefGoogle Scholar
  12. 12.
    Markowitz, D., Goff, S. & Bank, A. (1988) A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol 62, 1120–1124.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Kyoji Horie
    • 1
  • George Gaitanaris
    • 1
  • Alexander Gragerov
    • 1
    Email author
  1. 1.Omeros CorporationSeattleUSA

Personalised recommendations