Genetic Engineering In Nondrosophilid Insects

Genetic Transformation of Nondrosophilid Insects

The P element paradigm is successful in nondrosophilid insects. Despite many attempts, the P element was found to be unusable as a gene vector in nondrosophilid insect species.

The reason for the narrow host range of P is unknown; however, it has been proposed that P is dependent for its mobility, in part, on the presence of host-encoded factors. These are thought to be absent, or at least sufficiently diverged, to prevent the mobility of P in these species. The P element is, however, not required for insect transformation because of the discovery and performance of four trans-posable elements, each from a separate family of transposable elements. Each of these is endowed with a broad host range, and each can transform D. melanogaster as well as a number of nondrosophilid species. They are briefly described below.

What is conserved between drosophilid and nondrosophilid transformation has been described as the P element paradigm. This refers to the mode of transformation. The P element and the four elements described shortly are class II transposable elements. They all transposase by a "DNA-only" type of mechanism—no production of an RNA intermediate is needed. These elements have an overall structure that is shared between them. They are short (< 4 kb), have inverted terminal repeated sequences, and encode a transposase enzyme that catalyzes the movement of the transposable element from one genomic location to the next. The same methodology is used to introduce these transposable elements regardless of species. Typically two plasmids are coinjected into preblastoderm embryos. One plasmid contains the transposable element, into which has been placed a genetic marker and an effector gene—a gene meant to alter the phenotype of the insect in a desired way. The placement of the marker gene and the effector gene interrupts and inactivates the transposase gene within the element, necessitating the use of a second plasmid containing the corresponding transposase, which is typically placed under the control of an inducible promoter such as the hsp70 promoter of D. melanogaster. This transposase mediates the transposition of the transposable element from the donor plasmid to the genome of the developing germline cells. As for D. melanogaster transformation, the individual arising from the injected embryo is not transformed; rather, it contains genetically transformed gametes. Individuals are mated, and transgenic insects are screened for in the next generation.

transposable elements used for nondrosophilid insect transformation Four transposable elements can be used to genetically transform nondrosophilid insects: piggyBac, Hermes, Mariner, and Minos.

piggyBac The 2.5-kb piggyBac element has 13-bp inverted terminal repeats and 4-bp direct repeats located proximally to these. It contains a 2.1-kb open reading frame that encodes a transposase enzyme. piggyBac was discovered through its ability to transpose from the chromosomes of the Cabbage looper Trichoplusia ni into the genome of a baculovirus that had infected this TN368 cell line. Transposition ofpiggyBac into the baculovirus genome led to a mutation that resulted in few polyhedra being generated, in turn causing a clear change in cell morphology. piggyBac inserts only at TTAA sites and generates duplications of this sequence at the target site. Excision of piggyBac is precise— unlike other class II insect transposable elements, no deletions or additions of DNA remain at the empty excision site. piggyBac has found wide use as a gene vector in insects and has been used to genetically transform the flies C. capitata, Bactrocera dorsalis, Anastrepha suspensa, Musca domestica, L. cuprina, and D. melanogaster; the mosquitoes Anopheles albimanus, An. stephensi, An. gambiae, and Ae. aegypti; the moths Bombyx mori and Pectinphora gossypiella; and the beetle Tribolium castaneum. Little is known about the distribution of piggyBac throughout insects, although highly similar elements have recently been found in three strains of B. dorsalis. Over the 1.5 kb of nucleic acid sequence examined, these B. dorsalis elements are 95 to 98% identical to the element originally isolated from T. ni cells. Two of these B. dorsalis piggyBac-like sequences contain small deletions that interrupt the open reading frame, whereas the third has an intact open reading frame over the region examined. Conceptual translation of this region yields a sequence identity of 92% compared with the corresponding region of the T. ni piggyBac tranposase. The basis of the distribution of piggyBac-like elements combined with the possible effect that incumbent piggyBac-like sequences may have on introduced elements in transgenic lines is a fertile field for investigation.

Hermes Hermes elements are members of the hAT family of transposable elements that are widely dispersed in animals and plants. Some members of this family, such as the Ac element of maize and the Tam3 element of snapdragon, have a broad host range, and this attribute is shared with the Hermes element. Hermes was isolated from the house fly, M. domestica, and was first recognized by its ability to cross-mobilize the related hobo element when this was introduced into house fly embryos by microinjection. The 2.7-kb Hermes elements contain 17-bp inverted terminal repeats and a 1.8-kb open reading frame that encodes a transposase of 70 kDa. Hermes elements exhibit a preference for inserting at 5'-GTnnnnAC 3' sites and create 8-bp duplications of these sites upon insertion. They have been used to genetically transform D. melanogaster, C. capitata, Stomoxys calcitrans, Ae. aegypti, Culex quinquefasciatus, and T. casteneum. Plasmid-based transposition assays have shown that Hermes can transposase in several other insect species as well. Hermes transposes by a cut-and-paste mode of transposition in higher Diptera but seems to integrate by another, transposase-dependent mode in mosquito germlines. The molecular basis of this remains unknown. Hermes elements can interact with the related hobo element (and vice versa) when both are present in the genome of D. melanogaster.

Mariner Mariner elements are widespread among arthropods. They are approximately 1.3 kb with inverted terminal repeats typically around 30 bp long. Mariner elements can be present in an extremely high copy number in some species; however, it seems likely that only a handful (if any) of these may contain a single open reading frame that encodes an active transposase of approximately 33 kDa. Based on DNA sequence comparisons, five different subfamilies of Mariner elements exist in arthropods. The distribution of members of these subfamilies is inconsistent with the established evolutionary histories of their host species and it is now accepted that Mariner elements have been horizontally transferred throughout evolutionary time. At present only one naturally occurring, active Mariner element has been discovered. This is the MOS element from Drosophila mauritiana and has been used to genetically transform D. melanogaster, Ae. aegypti, and M. domestica. Indeed MOS displays a broad host range and has been used to genetically transform Leishmania, chickens, and zebrafish. The mobility characteristics of MOS are preserved in these species; it transposes by a cut-and-paste mechanism and inserts at, and duplicates, TA nucleotides. A second active element, Himar, was constructed based on a consensus of Mariner sequences obtained from the horn fly, Haemotobia irritans. Himar is active in Escherichia coli but so far is inactive in insects.

Minos The Minos element is a member of the Tc1 family of transposable elements. The Tc1 family of elements is related in sequence and mobility properties to the Mariner family of elements, and both are grouped into a single superfamily of elements. Minos elements are approximately 1.8 kb and possess long, 254-bp inverted terminal repeats. Minos contains two long open reading frames that are interrupted by an intron. Conceptual translation of the Minos transposase gene reveals a greater than 40% identity with the Tc1 transposase of Caenorhabditis elegans. Minos has been used to genetically transform C. capitata, D. melanogaster, and An. stephensi.

Transposable Elements in New Hosts

These four transposable elements just discussed provide the means by which genes can be introduced into pest insect species. Although these elements represent four different transposable element families, the transformation frequencies achieved, with some exceptions, are in the range of 1 to 10%. It seems likely that all will enjoy use as gene vectors in a range of insect species, and all may well be subject to interactions with endogenous transposable elements or other host factors present in these species. This is an important point that is not encountered by geneticists working on Drosophila. The recipient strains used for P element transformation are devoid of P elements (and any other related elements) and are deliberately chosen for this reason. This is not possible in other insect species in which the composition of the target genome with respect to transposable elements is unknown.

Whether interactions with endogenous transposable elements and/or host factors occur at levels that detrimentally affect transgenic stability is an issue that must be addressed.

Central to this is development of a complete understanding of how these transposable elements are regulated both in their original host species and in species into which they have been introduced.

Genetic Markers

The development of universal genetic marker genes, together with the identification of promoters to drive their expression in heterologous species, has played a major role in the extension of genetic engineering into nondrosophilid insects. Natural and modified forms of the green fluorescent protein (GFP) gene of the jellyfish, Aequeria victoria, have enabled transgenic insects in several species to be easily identified from nontransgenic siblings at most stages of development. These include D. melanogaster, C. capitata, B. dorsalis, Ae. aegypti, An. stephensi, Cx. quinquefasciatus, P. gossypiella, T. casteneum, and S. calcitrans. In these species, the GFP gene has been placed under the control of a promoter that enables either organelle-specific or tissue-specific expression of the marker gene to occur. Examples of the former are the actin5C and polyubiquitin promoters of D. melanogaster. Examples of the latter are the Pax6 and actin88 promoters. The actin88 promoter is from D. melanogaster and is specifically expressed in the indirect flight muscles of the pharate adult and adults. Pax6 is a member of the Pax family of transcription factors and is specifically involved in the development of the eye and central nervous system.

The enhanced GFP (EGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and Ds Red forms of the fluorescent protein genes can also function as genetic markers in insects.

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