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MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline.

Zeiser E, Frøkjær-Jensen C, Jorgensen E, Ahringer J - PLoS ONE (2011)

Bottom Line: These vectors allow tagged transgene constructs to be inserted as single copies into known sites in the C. elegans genome using MosSCI.We also show that two C. elegans heat shock promoters (Phsp-16.2 and Phsp-16.41) can be used to induce transgene expression in the germline when inserted via MosSCI transformation.This flexible set of new vectors, available to the research community in a plasmid repository, should facilitate research focused on the C. elegans germline and early embryo.

View Article: PubMed Central - PubMed

Affiliation: The Gurdon Institute and Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Here we describe a toolkit for the production of fluorescently tagged proteins in the C. elegans germline and early embryo using Mos1-mediated single copy insertion (MosSCI) transformation. We have generated promoter and 3'UTR fusions to sequences of different fluorescent proteins yielding constructs for germline expression that are compatible with MosSCI MultiSite Gateway vectors. These vectors allow tagged transgene constructs to be inserted as single copies into known sites in the C. elegans genome using MosSCI. We also show that two C. elegans heat shock promoters (Phsp-16.2 and Phsp-16.41) can be used to induce transgene expression in the germline when inserted via MosSCI transformation. This flexible set of new vectors, available to the research community in a plasmid repository, should facilitate research focused on the C. elegans germline and early embryo.

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Expression of transgenes generated using toolbox plasmids.(A–C) Pmex-5/his-58/egfp::tbb-2 3′UTR                            expression produced signal marking chromatin in embryos of strain                            JA1522. (D–F) Pmex-5/manS/citrine::tbb-2                                3′UTR expression produced signal marking the Golgi                            apparatus in embryos of strain JA1534. (G–I)                                Pmex-5::mCherry/his-58/tbb-2 3′UTR (strain                            JA1527) (G) late embryo and (H) L1 animals showing high signal in                            germline precursors Z2 and Z3 (arrows), lower signal in somatic nuclei                            (I) fluorescence signal in the germline of L4 stage. (J)                                Pmex-5/his-58/egfp::tbb-2 3′UTR JA1522 adult,                            HIS-58-EGFP can be detected in the gonad, oocytes, sperm and embryos. In                            general, signals were brighter at 25°C than at 15°C, and the                            signal produced by GFP S65C seems to have a better photostability than                            EGFP F64LS65T (not shown) [14].
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pone-0020082-g002: Expression of transgenes generated using toolbox plasmids.(A–C) Pmex-5/his-58/egfp::tbb-2 3′UTR expression produced signal marking chromatin in embryos of strain JA1522. (D–F) Pmex-5/manS/citrine::tbb-2 3′UTR expression produced signal marking the Golgi apparatus in embryos of strain JA1534. (G–I) Pmex-5::mCherry/his-58/tbb-2 3′UTR (strain JA1527) (G) late embryo and (H) L1 animals showing high signal in germline precursors Z2 and Z3 (arrows), lower signal in somatic nuclei (I) fluorescence signal in the germline of L4 stage. (J) Pmex-5/his-58/egfp::tbb-2 3′UTR JA1522 adult, HIS-58-EGFP can be detected in the gonad, oocytes, sperm and embryos. In general, signals were brighter at 25°C than at 15°C, and the signal produced by GFP S65C seems to have a better photostability than EGFP F64LS65T (not shown) [14].

Mentions: In order to validate the 5′ and 3′ entry clones of the toolkit for germline expression, we generated and integrated a series of transgenes fusing GFP, EGFP, Citrine, or mCherry as N-terminal or C-terminal fusions (see methods); representative examples for the histone HIS-58 and a portion of the Golgi enzyme AMAN-2, are shown in Figure 2. All fusion proteins were visible in all regions of the hermaphrodite germline and in embryos (Figure 2 and data not shown). Fluorescence was high in early embryos and then declined in most cells during embryogenesis, presumably through degradation. In the hermaphrodite germline, fluorescence remained continuously high throughout development (Figure 2G, H, I). We also observed mex-5 promoter driven transgene expression in the male germline (data not shown).


MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline.

Zeiser E, Frøkjær-Jensen C, Jorgensen E, Ahringer J - PLoS ONE (2011)

Expression of transgenes generated using toolbox plasmids.(A–C) Pmex-5/his-58/egfp::tbb-2 3′UTR                            expression produced signal marking chromatin in embryos of strain                            JA1522. (D–F) Pmex-5/manS/citrine::tbb-2                                3′UTR expression produced signal marking the Golgi                            apparatus in embryos of strain JA1534. (G–I)                                Pmex-5::mCherry/his-58/tbb-2 3′UTR (strain                            JA1527) (G) late embryo and (H) L1 animals showing high signal in                            germline precursors Z2 and Z3 (arrows), lower signal in somatic nuclei                            (I) fluorescence signal in the germline of L4 stage. (J)                                Pmex-5/his-58/egfp::tbb-2 3′UTR JA1522 adult,                            HIS-58-EGFP can be detected in the gonad, oocytes, sperm and embryos. In                            general, signals were brighter at 25°C than at 15°C, and the                            signal produced by GFP S65C seems to have a better photostability than                            EGFP F64LS65T (not shown) [14].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102689&req=5

pone-0020082-g002: Expression of transgenes generated using toolbox plasmids.(A–C) Pmex-5/his-58/egfp::tbb-2 3′UTR expression produced signal marking chromatin in embryos of strain JA1522. (D–F) Pmex-5/manS/citrine::tbb-2 3′UTR expression produced signal marking the Golgi apparatus in embryos of strain JA1534. (G–I) Pmex-5::mCherry/his-58/tbb-2 3′UTR (strain JA1527) (G) late embryo and (H) L1 animals showing high signal in germline precursors Z2 and Z3 (arrows), lower signal in somatic nuclei (I) fluorescence signal in the germline of L4 stage. (J) Pmex-5/his-58/egfp::tbb-2 3′UTR JA1522 adult, HIS-58-EGFP can be detected in the gonad, oocytes, sperm and embryos. In general, signals were brighter at 25°C than at 15°C, and the signal produced by GFP S65C seems to have a better photostability than EGFP F64LS65T (not shown) [14].
Mentions: In order to validate the 5′ and 3′ entry clones of the toolkit for germline expression, we generated and integrated a series of transgenes fusing GFP, EGFP, Citrine, or mCherry as N-terminal or C-terminal fusions (see methods); representative examples for the histone HIS-58 and a portion of the Golgi enzyme AMAN-2, are shown in Figure 2. All fusion proteins were visible in all regions of the hermaphrodite germline and in embryos (Figure 2 and data not shown). Fluorescence was high in early embryos and then declined in most cells during embryogenesis, presumably through degradation. In the hermaphrodite germline, fluorescence remained continuously high throughout development (Figure 2G, H, I). We also observed mex-5 promoter driven transgene expression in the male germline (data not shown).

Bottom Line: These vectors allow tagged transgene constructs to be inserted as single copies into known sites in the C. elegans genome using MosSCI.We also show that two C. elegans heat shock promoters (Phsp-16.2 and Phsp-16.41) can be used to induce transgene expression in the germline when inserted via MosSCI transformation.This flexible set of new vectors, available to the research community in a plasmid repository, should facilitate research focused on the C. elegans germline and early embryo.

View Article: PubMed Central - PubMed

Affiliation: The Gurdon Institute and Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Here we describe a toolkit for the production of fluorescently tagged proteins in the C. elegans germline and early embryo using Mos1-mediated single copy insertion (MosSCI) transformation. We have generated promoter and 3'UTR fusions to sequences of different fluorescent proteins yielding constructs for germline expression that are compatible with MosSCI MultiSite Gateway vectors. These vectors allow tagged transgene constructs to be inserted as single copies into known sites in the C. elegans genome using MosSCI. We also show that two C. elegans heat shock promoters (Phsp-16.2 and Phsp-16.41) can be used to induce transgene expression in the germline when inserted via MosSCI transformation. This flexible set of new vectors, available to the research community in a plasmid repository, should facilitate research focused on the C. elegans germline and early embryo.

Show MeSH
Related in: MedlinePlus