Stable germline transformation of the malaria mosquito Anopheles stephensi

Catteruccia, Flaminia; Nolan, Tony; Loukeris, Thanasis G.; Blass, Claudia; Savakis, Charalambos; Kafatos, Fotis C.; Crisanti, Andrea

doi:10.1038/35016096

Cited by 354 publications

(246 citation statements)

References 14 publications

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“…Indeed, several of these cut-and-paste transposons were shown to be active in a wide variety of species other than their original hosts, including bacteria [Himar1 (3)], malaria mosquito [Minos (4)], zebrafish [Tc3 (5) and Mos1 (6)], chicken [Mos1 (7)], and human cells [Tc1 (8), Sleeping Beauty (SB) (9), Himar1 (10), and Minos (11)]. Although the reconstructed fish transposon SB has been shown be able to jump in mouse embryonic stem (ES) cells (12), and more recently in mouse somatic tissue (13), germ-line transposition-an important and powerful tool-has yet to be demonstrated.…”

Section: T Ransposon Tagging Is a Valuable Tool For Functional Genomimentioning

confidence: 99%

Regulated transposition of a fish transposon in the mouse germ line

Fischer

Wienholds

Plasterk

2001

Proc. Natl. Acad. Sci. U.S.A.

212

187

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Tc1͞mariner elements are able to transpose in species other than the host from which they were isolated. As potential vectors for insertional mutagenesis and transgenesis of the mouse, these cut-and-paste transposons were tested for their ability to transpose in the mouse germ line. First, the levels of activity of several Tc1͞mariner elements in mammalian cells were compared; the reconstructed fish transposon Sleeping Beauty (SB) was found to be an order of magnitude more efficient than the other tested transposons. SB then was introduced into the mouse germ line as a two-component system: one transgene for the expression of the transposase in the male germ line and a second transgene carrying a modified transposon. In 20% of the progeny of double transgenic male mice the transposon had jumped from the original chromosomal position into another locus. Analysis of the integration sites shows that these jumps indeed occurred through the action of SB transposase, and that SB has a strong preference for intrachromosomal transposition. Analysis of the excision sites suggests that double-strand breaks in haploid spermatids are repaired via nonhomologous end joining. The SB system may be a powerful tool for transposon mutagenesis of the mouse germ line.T ransposon tagging is a valuable tool for functional genomics in model organisms such as Drosophila (P-element) and Caenorhabditis elegans (Tc1 transposon). In the mouse germ line, however, efficient in vivo transposon tagging has not yet been achieved.Unlike P-element, members of the Tc1͞mariner superfamily of transposable elements do not require host-specific factors for their activity in vitro (1, 2). Indeed, several of these cut-and-paste transposons were shown to be active in a wide variety of species other than their original hosts, including bacteria [Himar1 (3)], malaria mosquito [Minos (4)], zebrafish [Tc3 (5) and Mos1 (6)], chicken [Mos1 (7)], and human cells [Tc1 (8), Sleeping Beauty (SB) (9), Himar1 (10), and Minos (11)]. Although the reconstructed fish transposon SB has been shown be able to jump in mouse embryonic stem (ES) cells (12), and more recently in mouse somatic tissue (13), germ-line transposition-an important and powerful tool-has yet to be demonstrated.To assess the utility of Tc1͞mariner elements for transposon tagging in the mouse germ line, we first compared Tc1, Tc3, Himar1, Mos1, and SB in an in vitro mammalian cell culture assay and determined that SB is most efficient. Based on these results, we introduced SB in the mouse germ line. We show that SB is able to jump with a high efficiency in the mouse germ line, demonstrating its potential utility for genetic applications. Materials and MethodsPlasmids. PCR fragments of the ORFs encoding the transposase proteins of Tc1, Tc3, Himar1, and Mos1 were cloned into the Klenow-treated, 3.8-kb NotI fragment of pCMV␤ (CLON-TECH), resulting in, respectively, pRP1341, pRP1342, pRP1389, and pRP1353. The template plasmids were, respectively, pRP470 (14), pRP716 (15), pMar27fH (2), and pMos1 (16). The mu...

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Section: T Ransposon Tagging Is a Valuable Tool For Functional Genomimentioning

confidence: 99%

Regulated transposition of a fish transposon in the mouse germ line

Fischer

Wienholds

Plasterk

2001

Proc. Natl. Acad. Sci. U.S.A.

212

187

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“…Although EGFP has proven to be an invaluable visible marker for identifying transformed individuals in different insect species (1,(5)(6)(7), the availability of only this selectable marker limits the range of applications of a gene transfer technology in malaria vectors. New molecular tools are now needed to exploit fully the potential of germline transformation for Anopheles mosquitoes, with the aim of unraveling the interactions between host molecules and Plasmodium parasites, as well as performing functional studies such as transposon tagging and enhancer trapping.…”

mentioning

confidence: 99%

“…The recent development of an efficient gene transfer technology for Anopheles stephensi mosquitoes, achieved by using a Minos-based transposon (4) loaded with the EGFP 1 selectable marker (1), has expanded the possibility of studying the genetics of human malaria vectors at the functional level. Although EGFP has proven to be an invaluable visible marker for identifying transformed individuals in different insect species (1,(5)(6)(7), the availability of only this selectable marker limits the range of applications of a gene transfer technology in malaria vectors.…”

mentioning

confidence: 99%

piggyBac-mediated Germline Transformation of the Malaria Mosquito Anopheles stephensi Using the Red Fluorescent Protein dsRED as a Selectable Marker

Nolan¹,

Bower²,

Brown

et al. 2002

Journal of Biological Chemistry

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It is estimated that every year malaria infects ϳ300 million people and accounts for the death of 2 million individuals. The Plasmodium parasites that cause malaria in humans are transmitted exclusively by mosquito species belonging to the Anopheles genus. The recent development of a gene transfer technology for Anopheles stephensi mosquitoes, using the Minos transposable element marked with the enhanced green fluorescent protein EGFP (Catteruccia, F., Nolan, T., Loukeris, T. G., Blass, C., Savakis, C., Kafatos, F. C., and Crisanti, A. (2000) Nature 405, 959 -962), provides now a powerful tool to investigate the role of mosquito molecules involved in the interaction with the malaria parasite. Such technology, when further developed with additional markers and transposable elements, will be invaluable for analyzing the biology of the vector and for developing malaria-resistant mosquitoes to be used as a tool to control malaria transmission in the field. We report here the germline transformation of A. stephensi mosquitoes using a piggyBac-based transposon to drive integration of the gene encoding for the red fluorescent protein dsRED. A. stephensi embryos were injected with transformation vector pPBRED containing the dsRED marker cloned within the arms of piggyBac. Microscopic analysis of G 1 larvae revealed the presence of seven fluorescent phenotypes whose different molecular origins were confirmed by Southern blotting analysis. Sequencing of the insertion sites in two lines demonstrated that integrations had occurred at TTAA nucleotides in accordance with piggyBac-mediated transpositions.The recent development of an efficient gene transfer technology for Anopheles stephensi mosquitoes, achieved by using a Minos-based transposon (4) loaded with the EGFP 1 selectable marker (1), has expanded the possibility of studying the genetics of human malaria vectors at the functional level. Although EGFP has proven to be an invaluable visible marker for identifying transformed individuals in different insect species (1, 5-7), the availability of only this selectable marker limits the range of applications of a gene transfer technology in malaria vectors. New molecular tools are now needed to exploit fully the potential of germline transformation for Anopheles mosquitoes, with the aim of unraveling the interactions between host molecules and Plasmodium parasites, as well as performing functional studies such as transposon tagging and enhancer trapping. Ultimately this technology could be used to develop transgenic mosquitoes with a nonpermissive phenotype for parasite development. These mosquitoes could be used in malaria control programs with the aim to replace the permissive wild type vectors.As shown in Drosophila melanogaster, the availability of various molecular and genetic tools to achieve germline transformation has contributed tremendously to our understanding of the fruit fly biology, leading to the identification of hundreds of genes involved in development, immunity, tissue modeling, and embryogenesis. The tran...

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“…stephensi 21 and the discovery that the piggyBac transposable element could mediate germ-line transformation in many different insect species, Grossman et al 22 succeeded in producing An. gambiae transgenic mosquitoes.…”

Section: Transposon Mediated Germ-line Transformation Of An Gambiaementioning

confidence: 99%