Advertisement

Generating Mouse Lines for Lineage Tracing and Knockout Studies

  • Petra Kraus
  • V. Sivakamasundari
  • Xing Xing
  • Thomas LufkinEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1194)

Abstract

In 2007 Capecchi, Evans, and Smithies received the Nobel Prize in recognition for discovering the principles for introducing specific gene modifications in mice via embryonic stem cells, a technology, which has revolutionized the field of biomedical science allowing for the generation of genetically engineered animals. Here we describe detailed protocols based on and developed from these ground-breaking discoveries, allowing for the modification of genes not only to create mutations to study gene function but additionally to modify genes with fluorescent markers, thus permitting the isolation of specific rare wild-type and mutant cell types for further detailed analysis at the biochemical, pathological, and genomic levels.

Key words

Mouse Gene targeting Embryonic stem cell culture Pluripotent Chimera Microinjection Germline transmission Cre-recombinase 

Notes

Acknowledgements

We are grateful to Siew Lan Lim, Sook Peng Yap, Hsiao Yun Chan, Song Jie, Geraldine Leong, Valerie Tan, Jie Wei Goh, and Manickam Arun Kumar for their helping hands as well as sharing insight and expertise along the way.

References

  1. 1.
    Ghigna C, Goodell J (1999) Mice Are Nice. Random House Children’s Books. ISBN: 9780679989295Google Scholar
  2. 2.
    Skarnes WC, Rosen B, West AP et al (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474:337–342PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Bower DV, Sato Y, Lansford R (2011) Dynamic lineage analysis of embryonic morphogenesis using transgenic quail and 4D multispectral imaging. Genesis 49:619–643PubMedCrossRefGoogle Scholar
  4. 4.
    Day RN, Davidson MW (2009) The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38:2887–2921PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Hadjantonakis AK, Nagy A (2001) The color of mice: in the light of GFP-variant reporters. Histochem Cell Biol 115:49–58PubMedCrossRefGoogle Scholar
  6. 6.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909PubMedCrossRefGoogle Scholar
  7. 7.
    Chan HY, V S, Xing X et al. (2011) Comparison of IRES and F2A-based locus-specific multicistronic expression in stable mouse lines. PLoS One 6:e28885Google Scholar
  8. 8.
    Feil R (2007) Conditional somatic mutagenesis in the mouse using site-specific recombinases. Handb Exp Pharmacol (178) 3–28Google Scholar
  9. 9.
    Kraus P, Sivakamasundari V, Lim SL et al (2013) Making sense of Dlx1 antisense RNA. Dev Biol 376:224–235PubMedCrossRefGoogle Scholar
  10. 10.
    Lee WJ, Kraus P, Lufkin T (2012) Endogenous tagging of the murine transcription factor Sox5 with hemagglutinin for functional studies. Transgenic Res 21:293–301PubMedCrossRefGoogle Scholar
  11. 11.
    Sivakamasundari V, Chan HY, Yap SP et al (2012) New Bapx1(Cre-EGFP) mouse lines for lineage tracing and conditional knockout studies. Genesis 50:375–383PubMedCrossRefGoogle Scholar
  12. 12.
    Sivakamasundari V, Kraus P, Jie S et al (2013) Pax1(EGFP): New wild-type and mutant EGFP mouse lines for molecular and fate mapping studies. Genesis 51(6):420–429PubMedCrossRefGoogle Scholar
  13. 13.
    Yap SP, Xing X, Kraus P et al (2011) Generation of mice with a novel conditional null allele of the Sox9 gene. Biotechnol Lett 33:1551–1558PubMedCrossRefGoogle Scholar
  14. 14.
    Kraus P, Xing X, Lim SL et al (2012) Mouse strain specific gene expression differences for illumina microarray expression profiling in embryos. BMC Res Notes 5:232PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Nagy A, Getsenstein M, Vintersten K et al (2002) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  16. 16.
    Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71PubMedCrossRefGoogle Scholar
  17. 17.
    Metzger D, Chambon P (2001) Site- and time-specific gene targeting in the mouse. Methods 24:71–80PubMedCrossRefGoogle Scholar
  18. 18.
    Metzger D, Clifford J, Chiba H et al (1995) Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc Natl Acad Sci U S A 92:6991–6995PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Zhang J, Zhao J, Jiang WJ et al (2012) Conditional gene manipulation: Cre-ating a new biological era. J Zhejiang Univ Sci B 13:511–524PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Kraus P, Leong G, Tan V et al (2010) A more cost effective and rapid high percentage germ-line transmitting chimeric mouse generation procedure via microinjection of 2-cell, 4-cell, and 8-cell embryos with ES and iPS cells. Genesis 48:394–399PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Petra Kraus
    • 1
  • V. Sivakamasundari
    • 2
  • Xing Xing
    • 2
  • Thomas Lufkin
    • 1
    Email author
  1. 1.Department of BiologyClarkson UniversityPotsdamUSA
  2. 2.Stem Cell and Developmental Biology, Genome InstituteSingaporeSingapore

Personalised recommendations