NDLI: Construction of transformed, cultured silkworm cells and transgenic silkworm using the site-specific integrase system from phage φC31

Content Provider	Springer Nature Link
Author	Yin, Yajuan Cao, Guangli Xue, Renyu Gong, Chengliang
Copyright Year	2014
Abstract	The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp-zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the egfp-zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp-zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals.
Starting Page	6449
Ending Page	6456
Page Count	8
File Format	PDF
ISSN	03014851
Journal	Molecular Biology Reports
Volume Number	41
Issue Number	10
e-ISSN	15734978
Language	English
Publisher	Springer Netherlands
Publisher Date	2014-07-03
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	φC31 integrase Site-specific recombination attB attP Bombyx mori Exogenous Animal Biochemistry Animal Anatomy / Morphology / Histology
Content Type	Text
Resource Type	Article
Subject	Genetics Medicine Molecular Biology

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