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PMID: 12080094 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Targeted mutagenesis by homologous recombination in D. melanogaster.

Genes & development ·Vol. 16 ·No. 12 ·2002-06-15 ·Pages 1568-81

Rong YS, Titen SW, Xie HB, Golic MM, Bastiani M, Bandyopadhyay P, Olivera BM, Brodsky M, Rubin GM, Golic KG

Abstract

We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced.

MeSH Terms
Alleles Animals Blotting, Southern DNA/metabolism DNA Nucleotidyltransferases/metabolism Drosophila melanogaster Female Genes, p53/genetics Genome Homozygote Male Models, Genetic Mutagenesis Mutagenesis, Site-Directed Mutation Phenotype Plasmids/metabolism Point Mutation Polymerase Chain Reaction Promoter Regions, Genetic Recombination, Genetic
Chemicals
DNA DNA Nucleotidyltransferases FLP recombinase
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Rong Yikang S
Department of Biology, University of Utah, Salt Lake City 84112, USA.
Titen Simon W
Xie Heng B
Golic Mary M
Bastiani Michael
Bandyopadhyay Pradip
Olivera Baldomero M
Brodsky Michael
Rubin Gerald M
Golic Kent G
References (44)
44 references, click to expand
  1. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast.
    Methods Enzymol. 1991;194:281-301 PMID: 2005793
  2. Homologous recombination in mammalian cells.
    Annu Rev Genet. 1989;23:199-225 PMID: 2694931
  3. Targeted gene replacement in Drosophila via P element-induced gap repair.
    Science. 1991 Sep 6;253(5024):1110-7 PMID: 1653452
  4. Vermilion as a small selectable marker gene for Drosophila transformation.
    Nucleic Acids Res. 1991 Sep 25;19(18):5082 PMID: 1923781
  5. Regulation of HSP70 synthesis by messenger RNA degradation.
    Cell Regul. 1989 Nov;1(1):135-49 PMID: 2519613
  6. Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs.
    Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5128-32 PMID: 1594621
  7. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus.
    Mol Cell Biol. 1992 Aug;12(8):3365-71 PMID: 1321331
  8. Cleavage and recognition pattern of a double-strand-specific endonuclease (I-creI) encoded by the chloroplast 23S rRNA intron of Chlamydomonas reinhardtii.
    Gene. 1992 Oct 1;119(2):247-51 PMID: 1398106
  9. Green fluorescent protein as a marker for gene expression.
    Science. 1994 Feb 11;263(5148):802-5 PMID: 8303295
  10. Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis.
    Nature. 1994 Jun 2;369(6479):400-03 PMID: 7910952
  11. Homologous genetic recombination in Xenopus: mechanism and implications for gene manipulation.
    Prog Nucleic Acid Res Mol Biol. 1996;54:101-25 PMID: 8768073
  12. A quantitative measure of the mitotic pairing of alleles in Drosophila melanogaster and the influence of structural heterozygosity.
    Genetics. 1996 May;143(1):385-400 PMID: 8722790
  13. Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania.
    Nucleic Acids Res. 1997 Nov 1;25(21):4278-86 PMID: 9336458
  14. Genetic analysis of the Chlamydomonas reinhardtii I-CreI mobile intron homing system in Escherichia coli.
    Genetics. 1997 Dec;147(4):1653-64 PMID: 9409828
  15. I-PpoI and I-CreI homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment.
    J Mol Biol. 1998 Jul 17;280(3):345-53 PMID: 9665841
  16. DNA end-joining: from yeast to man.
    Trends Biochem Sci. 1998 Oct;23(10):394-8 PMID: 9810228
  17. Dominant defects in Drosophila eye pigmentation resulting from a euchromatin-heterochromatin fusion gene.
    Genetics. 1998 Dec;150(4):1551-66 PMID: 9832531
  18. Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis.
    Mech Dev. 1999 Apr;82(1-2):3-21 PMID: 10354467
  19. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.
    Microbiol Mol Biol Rev. 1999 Jun;63(2):349-404 PMID: 10357855
  20. The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.
    Genetics. 1999 Sep;153(1):135-77 PMID: 10471706
  21. The genome sequence of Drosophila melanogaster.
    Science. 2000 Mar 24;287(5461):2185-95 PMID: 10731132
  22. Drosophila p53 is a structural and functional homolog of the tumor suppressor p53.
    Cell. 2000 Mar 31;101(1):91-101 PMID: 10778859
  23. Drosophila p53 binds a damage response element at the reaper locus.
    Cell. 2000 Mar 31;101(1):103-13 PMID: 10778860
  24. Identification of a Drosophila vitamin K-dependent gamma-glutamyl carboxylase.
    J Biol Chem. 2000 Jun 16;275(24):18291-6 PMID: 10748045
  25. Gene targeting by homologous recombination in Drosophila.
    Science. 2000 Jun 16;288(5473):2013-8 PMID: 10856208
  26. Reversal of fortune for Drosophila geneticists?
    Science. 2000 Jun 16;288(5473):1973-5 PMID: 10877715
  27. In search of a function.
    Nat Cell Biol. 2000 Aug;2(8):E137-8 PMID: 10934480
  28. Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system.
    Int J Dev Biol. 2000 Jun;44(4):349-59 PMID: 10949044
  29. Targeted recovery of mutations in Drosophila.
    Genetics. 2000 Nov;156(3):1169-73 PMID: 11063692
  30. A targeted gene knockout in Drosophila.
    Genetics. 2001 Mar;157(3):1307-12 PMID: 11238415
  31. On a potential global role for vitamin K-dependent gamma-carboxylation in animal systems. Evidence for a gamma-glutamyl carboxylase in Drosophila.
    J Biol Chem. 2001 Mar 16;276(11):7769-74 PMID: 11110799
  32. Gene-targeting in Drosophila validated.
    Trends Genet. 2001 Oct;17(10):549-51 PMID: 11585643
  33. The molecular biology of the SIR proteins.
    Gene. 2001 Nov 14;279(1):1-16 PMID: 11722841
  34. Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.
    Genes Dev. 2001 Dec 15;15(24):3237-42 PMID: 11751629
  35. Differential effects of Rad52p overexpression on gene targeting and extrachromosomal homologous recombination in a human cell line.
    Nucleic Acids Res. 2002 Feb 1;30(3):740-8 PMID: 11809887
  36. Effect of chromosomal locus, GC content and length of homology on PCR-mediated targeted gene replacement in Saccharomyces.
    Nucleic Acids Res. 2001 Dec 15;29(24):5156-62 PMID: 11812849
  37. Genetic transformation of Drosophila with transposable element vectors.
    Science. 1982 Oct 22;218(4570):348-53 PMID: 6289436
  38. The double-strand-break repair model for recombination.
    Cell. 1983 May;33(1):25-35 PMID: 6380756
  39. Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae.
    Proc Natl Acad Sci U S A. 1986 Feb;83(3):735-9 PMID: 3003748
  40. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.
    Nature. 1988 Nov 24;336(6197):348-52 PMID: 3194019
  41. Efficient repair of HO-induced chromosomal breaks in Saccharomyces cerevisiae by recombination between flanking homologous sequences.
    Mol Cell Biol. 1988 Sep;8(9):3918-28 PMID: 3065627
  42. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome.
    Cell. 1989 Nov 3;59(3):499-509 PMID: 2509077
  43. Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products.
    Mol Cell Biol. 1990 Jan;10(1):103-12 PMID: 2294396
  44. Involvement of single-stranded tails in homologous recombination of DNA injected into Xenopus laevis oocyte nuclei.
    Mol Cell Biol. 1991 Jun;11(6):3268-77 PMID: 2038330
Article Info
Journal
Genes & development
Abbr.
Genes Dev
ISSN
0890-9369
DOI
10.1101/gad.986602
Published
2002-06-15
Pages
1568-81
Language
English
Region
United States
NLM ID
8711660
PMCID
PMC186348
Subset
IM
Grants
NIGMS NIH HHS · R01 GM065604 · United States
NIGMS NIH HHS · GM60700 · United States
NIGMS NIH HHS · GM65604 · United States
External Links
PubMed source DOI: 10.1101/gad.986602 PMC Full Text View journal details
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