DOI: 10.1073/pnas.0611511104. An optimized transgenesis system for <i>Drosophila</i> using germ-line-specific φC31 integrases

An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases

10.1073/pnas.0611511104

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster . However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a φC31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the φC31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ , extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the φC31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, high-throughput screening of large cDNA sets and regulatory elements.

Bibliography

Bischof, J., Maeda, R. K., Hediger, M., Karch, F., & Basler, K. (2007). An optimized transgenesis system for Drosophila using germ-line-specific ÏC31 integrases. Proceedings of the National Academy of Sciences, 104(9), 3312â3317.

Authors 5

Johannes Bischof (first)
Robert K. Maeda (additional)
Monika Hediger (additional)
François Karch (additional)
Konrad Basler (additional)

References 32 Referenced 1,799

10.1038/nrg751
10.1126/science.6289436
10.1126/science.6289435
10.1242/dev.118.2.401
10.1126/science.2830671
10.1093/genetics/118.3.461
10.1093/nar/25.18.3665
10.1073/pnas.0504305102
10.1038/nmeth775
10.1242/dev.00774
10.1073/pnas.95.10.5505
10.1093/genetics/166.4.1775
10.1046/j.1365-2958.2000.02142.x
10.1007/s004270000111
10.1093/nar/30.11.2299
10.1016/S0925-4773(01)00654-2
10.1016/0092-8674(91)90110-K
10.1038/369315a0
10.1101/gad.10.20.2610
10.1101/gad.10.20.2600
10.1093/emboj/18.9.2610
10.1016/S1097-2765(00)80368-8
10.1146/annurev.genet.35.102401.090756
10.1038/sj.gt.3302789
10.1073/pnas.090527097
10.1093/nar/gni192
10.1534/genetics.106.056945
10.1073/pnas.93.22.12418
10.1007/s004380050377
10.1101/gad.4.9.1573
M Pardue Drosophila: A Practical Approach, ed D Roberts (IRL, Oxford), pp. 111–137 (1986). / Drosophila: A Practical Approach by Pardue M (1986)
10.1093/genetics/144.2.715

Dates

Type	When
Created	18 years, 6 months ago (Feb. 22, 2007, 8:41 p.m.)
Deposited	8 months ago (Dec. 27, 2024, 10:53 a.m.)
Indexed	1 hour, 7 minutes ago (Sept. 3, 2025, 5:50 a.m.)
Issued	18 years, 6 months ago (Feb. 27, 2007)
Published	18 years, 6 months ago (Feb. 27, 2007)
Published Online	18 years, 6 months ago (Feb. 27, 2007)
Published Print	18 years, 6 months ago (Feb. 27, 2007)

Funders 0

None

BibTeX

@article{Bischof_2007, title={An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases}, volume={104}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.0611511104}, DOI={10.1073/pnas.0611511104}, number={9}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Bischof, Johannes and Maeda, Robert K. and Hediger, Monika and Karch, François and Basler, Konrad}, year={2007}, month=feb, pages={3312–3317} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        9,
        3
      ]
    ],
    "date-time": "2025-09-03T09:50:21Z",
    "timestamp": 1756893021117,
    "version": "3.32.0"
  },
  "reference-count": 32,
  "publisher": "Proceedings of the National Academy of Sciences",
  "issue": "9",
  "content-domain": {
    "domain": [
      "www.pnas.org"
    ],
    "crossmark-restriction": true
  },
  "published-print": {
    "date-parts": [
      [
        2007,
        2,
        27
      ]
    ]
  },
  "abstract": "<jats:p>\n            Germ-line transformation via transposable elements is a powerful tool to study gene function in\n            <jats:italic>Drosophila melanogaster</jats:italic>\n            . However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a \u03c6C31-based integration system. We generated a collection of lines with precisely mapped\n            <jats:italic>attP</jats:italic>\n            sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the\n            <jats:italic>nanos</jats:italic>\n            and\n            <jats:italic>vasa</jats:italic>\n            genes, we established endogenous sources of the \u03c6C31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a\n            <jats:italic>white</jats:italic>\n            gene-based reconstitution system was generated that enables visual selection for precise\n            <jats:italic>attP</jats:italic>\n            targeting. Finally, we demonstrate that our chromosomal\n            <jats:italic>attP</jats:italic>\n            sites can be modified\n            <jats:italic>in situ</jats:italic>\n            , extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the \u03c6C31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, high-throughput screening of large cDNA sets and regulatory elements.\n          </jats:p>",
  "DOI": "10.1073/pnas.0611511104",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2007,
        2,
        23
      ]
    ],
    "date-time": "2007-02-23T01:41:40Z",
    "timestamp": 1172194900000
  },
  "page": "3312-3317",
  "update-policy": "https://doi.org/10.1073/pnas.cm10313",
  "source": "Crossref",
  "is-referenced-by-count": 1799,
  "title": "An optimized transgenesis system for\n            <i>Drosophila</i>\n            using germ-line-specific \u03c6C31 integrases",
  "prefix": "10.1073",
  "volume": "104",
  "author": [
    {
      "given": "Johannes",
      "family": "Bischof",
      "sequence": "first",
      "affiliation": [
        {
          "name": "*Frontiers in Genetics, National Center of Competence in Research, Institute of Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; and"
        }
      ]
    },
    {
      "given": "Robert K.",
      "family": "Maeda",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Frontiers in Genetics, National Center of Competence in Research, Department of Zoology and Animal Biology, University of Geneva, 30 quai E. Ansermet, CH-1211 Geneva, Switzerland"
        }
      ]
    },
    {
      "given": "Monika",
      "family": "Hediger",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "*Frontiers in Genetics, National Center of Competence in Research, Institute of Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; and"
        }
      ]
    },
    {
      "given": "Fran\u00e7ois",
      "family": "Karch",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Frontiers in Genetics, National Center of Competence in Research, Department of Zoology and Animal Biology, University of Geneva, 30 quai E. Ansermet, CH-1211 Geneva, Switzerland"
        }
      ]
    },
    {
      "given": "Konrad",
      "family": "Basler",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "*Frontiers in Genetics, National Center of Competence in Research, Institute of Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; and"
        }
      ]
    }
  ],
  "member": "341",
  "published-online": {
    "date-parts": [
      [
        2007,
        2,
        27
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_3_5_1_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/nrg751"
    },
    {
      "key": "e_1_3_5_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.6289436"
    },
    {
      "key": "e_1_3_5_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.6289435"
    },
    {
      "key": "e_1_3_5_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1242/dev.118.2.401"
    },
    {
      "key": "e_1_3_5_5_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.2830671"
    },
    {
      "key": "e_1_3_5_6_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/genetics/118.3.461"
    },
    {
      "key": "e_1_3_5_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/25.18.3665"
    },
    {
      "key": "e_1_3_5_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.0504305102"
    },
    {
      "key": "e_1_3_5_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/nmeth775"
    },
    {
      "key": "e_1_3_5_10_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1242/dev.00774"
    },
    {
      "key": "e_1_3_5_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.95.10.5505"
    },
    {
      "key": "e_1_3_5_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/genetics/166.4.1775"
    },
    {
      "key": "e_1_3_5_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1365-2958.2000.02142.x"
    },
    {
      "key": "e_1_3_5_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1007/s004270000111"
    },
    {
      "key": "e_1_3_5_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/30.11.2299"
    },
    {
      "key": "e_1_3_5_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0925-4773(01)00654-2"
    },
    {
      "key": "e_1_3_5_17_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(91)90110-K"
    },
    {
      "key": "e_1_3_5_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/369315a0"
    },
    {
      "key": "e_1_3_5_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1101/gad.10.20.2610"
    },
    {
      "key": "e_1_3_5_20_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1101/gad.10.20.2600"
    },
    {
      "key": "e_1_3_5_21_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/emboj/18.9.2610"
    },
    {
      "key": "e_1_3_5_22_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S1097-2765(00)80368-8"
    },
    {
      "key": "e_1_3_5_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.genet.35.102401.090756"
    },
    {
      "key": "e_1_3_5_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/sj.gt.3302789"
    },
    {
      "key": "e_1_3_5_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.090527097"
    },
    {
      "key": "e_1_3_5_26_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/gni192"
    },
    {
      "key": "e_1_3_5_27_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1534/genetics.106.056945"
    },
    {
      "key": "e_1_3_5_28_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.93.22.12418"
    },
    {
      "key": "e_1_3_5_29_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1007/s004380050377"
    },
    {
      "key": "e_1_3_5_30_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1101/gad.4.9.1573"
    },
    {
      "key": "e_1_3_5_31_2",
      "first-page": "111",
      "volume-title": "Drosophila: A Practical Approach",
      "author": "Pardue M",
      "year": "1986",
      "unstructured": "M Pardue Drosophila: A Practical Approach, ed D Roberts (IRL, Oxford), pp. 111\u2013137 (1986)."
    },
    {
      "key": "e_1_3_5_32_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/genetics/144.2.715"
    }
  ],
  "container-title": "Proceedings of the National Academy of Sciences",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://pnas.org/doi/pdf/10.1073/pnas.0611511104",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2024,
        12,
        27
      ]
    ],
    "date-time": "2024-12-27T15:53:24Z",
    "timestamp": 1735314804000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://pnas.org/doi/10.1073/pnas.0611511104"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        2007,
        2,
        27
      ]
    ]
  },
  "references-count": 32,
  "journal-issue": {
    "issue": "9",
    "published-print": {
      "date-parts": [
        [
          2007,
          2,
          27
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1073/pnas.0611511104"
  ],
  "URL": "http://dx.doi.org/10.1073/pnas.0611511104",
  "relation": {},
  "ISSN": [
    "0027-8424",
    "1091-6490"
  ],
  "subject": [],
  "container-title-short": "Proc. Natl. Acad. Sci. U.S.A.",
  "published": {
    "date-parts": [
      [
        2007,
        2,
        27
      ]
    ]
  },
  "assertion": [
    {
      "value": "2006-10-10",
      "order": 0,
      "name": "received",
      "label": "Received",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "2007-02-27",
      "order": 3,
      "name": "published",
      "label": "Published",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    }
  ]
}