Abstract
Bacteriophage phiC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA. Here we show that the coinjection of mRNA encoding phiC31 integrase with plasmid DNA encoding the green fluorescent protein (GFP) can be used to generate transgenic X. laevis embryos. Despite integration into the genome, appropriate promoter expression required modification of the reporter plasmid by bracketing the GFP reporter gene with tandem copies of the chicken beta-globin 5' HS4 insulator to relieve silencing owing to chromatin position effects. These experiments demonstrate that the integration of insulated gene sequences using phiC31 integrase can be used to efficiently create transgenic embryos in X. laevis and may increase the practical use of phiC31 integrase in other systems as well.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Animals
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Animals, Genetically Modified*
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Bacteriophages / enzymology*
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Beta-Globulins / genetics
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Blotting, Southern
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Chromatin / genetics
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Chromatin / metabolism*
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Embryo, Nonmammalian / physiology*
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Female
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Gene Silencing
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Genetic Engineering / methods*
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Green Fluorescent Proteins / metabolism
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Insulator Elements / genetics
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Integrases / genetics*
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Integrases / metabolism
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Male
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Microinjections
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Oocytes / cytology
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Oocytes / metabolism
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Plasmids
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RNA, Messenger / metabolism
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Recombinant Fusion Proteins / genetics
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Xenopus laevis / embryology
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Xenopus laevis / genetics*
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gamma-Crystallins / genetics
Substances
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Beta-Globulins
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Chromatin
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RNA, Messenger
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Recombinant Fusion Proteins
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enhanced green fluorescent protein
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gamma-Crystallins
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Green Fluorescent Proteins
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Integrases