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. 2006 Jun 16:6:27.
doi: 10.1186/1472-6750-6-27.

Insulated piggyBac vectors for insect transgenesis

Abhimanyu Sarkar  1 , Asela Atapattu, Esther J Belikoff, Jörg C Heinrich, Xuelei Li, Carsten Horn, Ernst A Wimmer, Maxwell J Scott
Affiliations

Insulated piggyBac vectors for insect transgenesis

Abhimanyu Sarkar et al. BMC Biotechnol. .

Abstract

Background: Germ-line transformation of insects is now a widely used method for analyzing gene function and for the development of genetically modified strains suitable for pest control programs. The most widely used transposable element for the germ-line transformation of insects is piggyBac. The site of integration of the transgene can influence gene expression due to the effects of nearby transcription enhancers or silent heterochromatic regions. Position effects can be minimized by flanking a transgene with insulator elements. The scs/scs' and gypsy insulators from Drosophila melanogaster as well as the chicken beta-globin HS4 insulator function in both Drosophila and mammalian cells.

Results: To minimize position effects we have created a set of piggyBac transformation vectors that contain either the scs/scs', gypsy or chicken beta-globin HS4 insulators. The vectors contain either fluorescent protein or eye color marker genes and have been successfully used for germ-line transformation of Drosophila melanogaster. A set of the scs/scs' vectors contains the coral reef fluorescent protein marker genes AmCyan, ZsGreen and DsRed that have not been optimized for translation in human cells. These marker genes are controlled by a combined GMR-3xP3 enhancer/promoter that gives particularly strong expression in the eyes. This is also the first report of the use of the ZsGreen and AmCyan reef fluorescent proteins as transformation markers in insects.

Conclusion: The insulated piggyBac vectors should protect transgenes against position effects and thus facilitate fine control of gene expression in a wide spectrum of insect species. These vectors may also be used for transgenesis in other invertebrate species.

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Figures

Figure 1
Figure 1
piggyBac vectors containing the scs/scs' insulator elements. The vectors contain paired scs/scs' insulators flanking the DsRed (A), AmCyan (B), ZsGreen (C) and cinnabar (cn) (D) marker genes. The location of unique restriction endonuclease cloning sites are shown. The vectors have 690 bp of 5' terminal piggyBac sequence and either 769 bp (A-C) or 907 bp (D) of 3' terminal sequence.
Figure 2
Figure 2
GMR-3xP3-reef fluorescent protein marker gene expression in transgenic Drosophila. GMR-3xP3-DsRed, GMR-3xP3-ZsGreen and GMR-3xP3-AmCyan marker gene expression in a yellow white (y w) background observed with white light (A) or with red (B), green (C) or cyan (D) filter sets.
Figure 3
Figure 3
Comparison of fluorescence intensities of lines carrying insulated and uninsulated marker genes. A representative fly is shown from each of the lines carrying the insulated GMR-3xP3-AmCyan (A) and GMR-3xP3-ZsGreen (B) marker genes. Little variation in fluorescence intensity is seen between lines suggesting position effects had been minimized by the flanking scs and scs' insulator elements. In contrast lines carrying an uninsulated 3xP3-DsRed1 marker gene (C) show considerable variation in fluorescence intensity. Some lines are barely detectable above background fluorescence (y w) while other lines show very strong fluorescence.
Figure 4
Figure 4
piggyBac vectors containing the β-globin 5' HS4 and gypsy insulator elements. The vectors contain either the ECFP (A), EYFP (B) or DsRed1 (C) marker genes. The β-globin 5' HS4 (A, B) and gypsy (C) insulator elements do not bracket the marker genes but do flank the unique AscI and FseI cloning sites. The vectors have 685 bp of 5' terminal piggyBac sequence and 1051 bp of 3' terminal sequence.
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