Logo of geneticsGeneticsCurrent IssueInformation for AuthorsEditorial BoardSubscribeSubmit a Manuscript
Genetics. 2004 Apr; 166(4): 1775–1782.
PMCID: PMC1470814

Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.


The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.

Full Text

The Full Text of this article is available as a PDF (169K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bhanot P, Brink M, Samos CH, Hsieh JC, Wang Y, Macke JP, Andrew D, Nathans J, Nusse R. A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature. 1996 Jul 18;382(6588):225–230. [PubMed]
  • Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993 Jun;118(2):401–415. [PubMed]
  • Golic MM, Rong YS, Petersen RB, Lindquist SL, Golic KG. FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes. Nucleic Acids Res. 1997 Sep 15;25(18):3665–3671. [PMC free article] [PubMed]
  • Groth AC, Olivares EC, Thyagarajan B, Calos MP. A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci U S A. 2000 May 23;97(11):5995–6000. [PMC free article] [PubMed]
  • Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. [PubMed]
  • Lebkowski JS, DuBridge RB, Antell EA, Greisen KS, Calos MP. Transfected DNA is mutated in monkey, mouse, and human cells. Mol Cell Biol. 1984 Oct;4(10):1951–1960. [PMC free article] [PubMed]
  • Levis R, Hazelrigg T, Rubin GM. Effects of genomic position on the expression of transduced copies of the white gene of Drosophila. Science. 1985 Aug 9;229(4713):558–561. [PubMed]
  • O'Kane CJ, Gehring WJ. Detection in situ of genomic regulatory elements in Drosophila. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9123–9127. [PMC free article] [PubMed]
  • Olivares EC, Hollis RP, Calos MP. Phage R4 integrase mediates site-specific integration in human cells. Gene. 2001 Oct 31;278(1-2):167–176. [PubMed]
  • Olivares Eric C, Hollis Roger P, Chalberg Thomas W, Meuse Leonard, Kay Mark A, Calos Michele P. Site-specific genomic integration produces therapeutic Factor IX levels in mice. Nat Biotechnol. 2002 Nov;20(11):1124–1128. [PubMed]
  • Ortiz-Urda Susana, Thyagarajan Bhaskar, Keene Douglas R, Lin Qun, Fang Min, Calos Michele P, Khavari Paul A. Stable nonviral genetic correction of inherited human skin disease. Nat Med. 2002 Oct;8(10):1166–1170. [PubMed]
  • Ortiz-Urda Susana, Lin Qun, Green Cheryl L, Keene Douglas R, Marinkovich M Peter, Khavari Paul A. Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue. J Clin Invest. 2003 Jan;111(2):251–255. [PMC free article] [PubMed]
  • Smith JG, Calos MP. Autonomous replication in Drosophila melanogaster tissue culture cells. Chromosoma. 1995 May;103(9):597–605. [PubMed]
  • Ortiz-Urda Susana, Thyagarajan Bhaskar, Keene Douglas R, Lin Qun, Calos Michele P, Khavari Paul A. PhiC31 integrase-mediated nonviral genetic correction of junctional epidermolysis bullosa. Hum Gene Ther. 2003 Jun 10;14(9):923–928. [PubMed]
  • Stoll Stephanie M, Ginsburg Daniel S, Calos Michele P. Phage TP901-1 site-specific integrase functions in human cells. J Bacteriol. 2002 Jul;184(13):3657–3663. [PMC free article] [PubMed]
  • Rong YS, Golic KG. A targeted gene knockout in Drosophila. Genetics. 2001 Mar;157(3):1307–1312. [PMC free article] [PubMed]
  • Thorpe HM, Smith MC. In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A. 1998 May 12;95(10):5505–5510. [PMC free article] [PubMed]
  • Rong Yikang S, Titen Simon W, Xie Heng B, Golic Mary M, Bastiani Michael, Bandyopadhyay Pradip, Olivera Baldomero M, Brodsky Michael, Rubin Gerald M, Golic Kent G. Targeted mutagenesis by homologous recombination in D. melanogaster. Genes Dev. 2002 Jun 15;16(12):1568–1581. [PMC free article] [PubMed]
  • Thyagarajan B, Guimarães MJ, Groth AC, Calos MP. Mammalian genomes contain active recombinase recognition sites. Gene. 2000 Feb 22;244(1-2):47–54. [PubMed]
  • Siegal ML, Hartl DL. Transgene Coplacement and high efficiency site-specific recombination with the Cre/loxP system in Drosophila. Genetics. 1996 Oct;144(2):715–726. [PMC free article] [PubMed]
  • Thyagarajan B, Olivares EC, Hollis RP, Ginsburg DS, Calos MP. Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol Cell Biol. 2001 Jun;21(12):3926–3934. [PMC free article] [PubMed]
  • Siegal ML, Hartl DL. Application of Cre/loxP in Drosophila. Site-specific recombination and transgene coplacement. Methods Mol Biol. 2000;136:487–495. [PubMed]

Articles from Genetics are provided here courtesy of Genetics Society of America


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...