Using a novel approach — recombineering — to generate odf2 null alleles

Abstract

This article uses a real-life example to illustrate the concept and methodology of recombineering, a revolutionary genetic engineering technique based on phage-mediated homologous recombination. A step-by-step approach is presented along with a flow diagram, from obtaining gene-harboring BACs to the in vitro generation of a conditional null allele. This method can be used to target any gene at any position, without the knowledge or use of any restriction site. The extensive applicability of recombineering to gene manipulation is discussed.

Key words： gene manipulation; recombineering; odf2

Acknowledgments

This work was carried out in the laboratory of Dr. Jonathan Eggenschwiler at the Department of Molecular Biology at Princeton University, whose expertise and support continue to be invaluable and are much appreciated.

References

1 Brohmann H, Pinnecke S, Hoyer-Fender S (1997). Identification and characterization of new cDNAs encoding outer dense fiber proteins of rat sperm. J Biol Chem, 272(15): 10327-10332

PMID

2 Copeland N G, Jenkins N A, Court D L (2001). Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet, 2(10): 769-779

PMID

3 Cox M M, Goodman M F, Kreuzer K N, Sherratt D J, Sandler S J, Marians K J (2000). The importance of repairing stalled replication forks. Nature, 404(6773): 37-41

PMID

4 Eggenschwiler J T, Anderson K V (2007). Cilia and developmental signaling. Annu Rev Cell Dev Biol, 23(1): 345-373

PMID

5 Ellis H M, Yu D, DiTizio T, Court D L (2001). High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci USA, 98(12): 6742-6746

PMID

6 Gene Bridges (2007). Quick & Easy Conditional Knockout Kit (loxP/Cre) by Red/ET Recombination. http://www.genebridges.com/gb/pdf/K005%20KO%20Kit%20loxP-version%201.1-2007a-screen.pdf.

7 Haber J E (1999). DNA repair. Gatekeepers of recombination. Nature, 398(6729): 665-667, 667

PMID

8 Ishikawa H, Kubo A, Tsukita S, Tsukita S (2005). Odf2-deficient mother centrioles lack distal/subdistal appendages and the ability to generate primary cilia. Nat Cell Biol, 7(5): 517-524

PMID

9 Kogoma T (1996). Recombination by replication. Cell, 85(5): 625-627

PMID

10 Kolodner R, Hall S D, Luisi-DeLuca C (1994). Homologous pairing proteins encoded by the Escherichia coli recE and recT genes. Mol Microbiol, 11(1): 23-30

PMID

11 Muyrers J P, Zhang Y, Buchholz F, Stewart A F (2000). RecE/RecT and Redalpha/Redbeta initiate double-stranded break repair by specifically interacting with their respective partners. Genes Dev, 14(15): 1971-1982

PMID

12 Muyrers J P P, Zhang Y, Stewart A F (2001). Techniques: Recombinogenic Engineering: new options for manipulating DNA. Trends Biochem Sci, 26: 325-331

PMID

13 Nakagawa Y, Yamane Y, Okanoue T, Tsukita S, Tsukita S (2001). Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from isolated centrosomes. Mol Biol Cell, 12(6): 1687-1697

PMID

14 Rosales J L, Rattner J B, Lee K Y (2010). Cdk5 in the centriolar appendages mediates cenexin1 localization and primary cilia formation. Cell Cycle, 9(10): 2037-2039

PMID

15 Schweizer S, Hoyer-Fender S (2009). Mouse Odf2 localizes to centrosomes and basal bodies in adult tissues and to the photoreceptor primary cilium. Cell Tissue Res, 338(2): 295-301

PMID

16 Zhang Y, Buchholz F, Muyrers J P, Stewart A F (1998). A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet, 20(2): 123-128

PMID

17 Zhang Y, Muyrers J P P, Testa G, Stewart A F (2000). DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol, 18(12): 1314-1317

PMID