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      MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

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          Abstract

          We demonstrate the versatility of a collection of insertions of the transposon Minos mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow + marker flanked by two inverted bacteriophage ΦC31 attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp system. Insertions within coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the Drosophila melanogaster toolkit.

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          Most cited references77

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          An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.

          Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a phiC31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the phiC31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the phiC31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, high-throughput screening of large cDNA sets and regulatory elements.
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            Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.

            The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.
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              P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.

              We describe a transgenesis platform for Drosophila melanogaster that integrates three recently developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and bacteriophage PhiC31-mediated transgenesis. The BAC is maintained at low copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. PhiC31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure/function analyses of most Drosophila genes.
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nature methods
                1548-7091
                1548-7105
                8 July 2011
                2011
                12 October 2011
                : 8
                : 9
                : 737-743
                Affiliations
                [1 ]Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
                [2 ]Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA
                [3 ]Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94702, USA
                [4 ]Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, MD 21218, USA
                [5 ]Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA
                [6 ]Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA
                Author notes

                AUTHOR CONTRIBUTIONS K.J.T.V. designed the MiMIC technique.

                R.W.L., A.C.S., R.A.H. and H.J.B. conceived the application of MiMIC to the Gene Disruption Project.

                K.J.T.V. designed vectors and performed all molecular biology, except for mapping of insertions.

                H.P. and Y.H. performed microinjections.

                K.J.T.V., H.P. and Y.H. performed fly genetics.

                M.E.-H. and R.A.H. performed inverse PCR and DNA sequencing to map insertions.

                K.J.T.V., Y.H., M.E.-H., J.W.C., R.W.L. and R.A.H. analyzed insertion data, annotated insertions, and prepared public database submissions.

                J.W.C. performed bioinformatic analysis.

                K.J.T.V., N.A.H, and H.P. verified RMCE events by PCR.

                K.J.T.V and K.L.S. did stainings of gene trap events.

                K.L.S. and N.A.H. did stainings of protein trap events.

                K.J.T.V., K.L.S., N.A.H. and H.J.B. analyzed expression patterns.

                K.J.T.V. and H.J.B. wrote the paper.

                R.A.H. and R.W.L. edited the paper.

                [* ]Correspondence: kv134369@ 123456bcm.edu (K.J.T.V) and hbellen@ 123456bcm.tmc.edu (H.J.B)
                Article
                nihpa309875
                10.1038/nmeth.1662
                3191940
                21985007
                25a22d72-86bf-4329-a9a9-79c7b1272ae5

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