Using ΦC31 integrase to mediate insertion of DNA in Xenopus embryos

You E Li; Bryan G Allen; Daniel L Weeks

doi:10.1007/978-1-61779-992-1_13

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Using ΦC31 integrase to mediate insertion of DNA in Xenopus embryos

Book chapter

Open access

Using ΦC31 integrase to mediate insertion of DNA in Xenopus embryos

You E Li, Bryan G Allen and Daniel L Weeks

Xenopus Protocols, Vol.917, pp.219-230

Methods in molecular biology (Clifton, N.J.), 917, Humana Press

2012

DOI: 10.1007/978-1-61779-992-1_13

PMCID: PMC3551469

PMID: 22956091

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https://doi.org/10.1007/978-1-61779-992-1_13View

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Abstract

The two most common methods used to generate transgenic Xenopus embryos, restriction enzyme-mediated insertion, and I-SceI meganuclease take advantage of relatively common but spatially unpredictable double-stranded breaks in sperm, egg, or early embryo genomes. These methods also tend to insert multimeric copies of the transgene. An alternative is to use bacteriophage- or transposon-derived integrase or recombinase to mediate more site-specific insertion of the transgene. The use of phiC31 integrase requires a defined sequence for insertion and is compatible with insertion of a single copy of the transgene. We describe the protocol we use to facilitate phiC31 integrase transgene insertion including the use of insulator sequences to reduce position effect disruption of transgene activity.

Plasmids

Gene Transfer Techniques

Embryo, Nonmammalian - cytology

Microinjections

RNA, Messenger - isolation & purification

Viral Proteins - chemistry

RNA, Messenger - genetics

Viral Proteins - genetics

Male

Animals

Chorionic Gonadotropin - administration & dosage

Animals, Genetically Modified - genetics

Base Sequence

Reproductive Control Agents - administration & dosage

Bacteriophages - genetics

Bacteriophages - enzymology

Female

Mutagenesis, Insertional

Xenopus laevis - genetics

Integrases - genetics

Integrases - chemistry

Details

Title: Subtitle: Using ΦC31 integrase to mediate insertion of DNA in Xenopus embryos
Creators: You E Li - Department of Biochemistry, The University of Iowa, Iowa City, IA, USA
Bryan G Allen
Daniel L Weeks
Resource Type: Book chapter
Publication Details: Xenopus Protocols, Vol.917, pp.219-230
Series: Methods in molecular biology (Clifton, N.J.); 917
DOI: 10.1007/978-1-61779-992-1_13
PMID: 22956091
PMCID: PMC3551469
NLM abbreviation: Methods Mol Biol
eISBN: 1617799920; 9781617799921
ISSN: 1064-3745
eISSN: 1940-6029
Publisher: Humana Press; Totowa, NJ
Grant note: R01 GM069944 / NIGMS NIH HHS GM069944 / NIGMS NIH HHS DC007481 / NIDCD NIH HHS R01 DC007481 / NIDCD NIH HHS T32 GM007337 / NIGMS NIH HHS
Language: English
Date published: 2012
Academic Unit: Stead Family Department of Pediatrics; Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024404902771

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