Genetic Engineering In Drosophila Melanogaster

Genetic Technologies Are More Advanced in Drosophila Than in Other Insect Species

One cannot discuss the genetic manipulation of insects without describing the molecular genetic tools that are available in D. melanogaster. Traditionally, a gulf has existed between entomologists who view the harmless vinegar fly as being distant to the problems of insect control and Drosophila geneticists who utilize the many biological attributes of Drosophila to understand the basis of gene action. This gulf will close as comparative genomics reveals similarities and differences in the conservation of many genes and molecular pathways between Drosophila and other insect species. The power of this comparative approach to modern biology will offer insect scientists and traditional entomologists exciting opportunities to bring the power of genetics and molecular biology to the control of insects. The development and application of these tools is what insect scientists seek to achieve in pestiferous and beneficial insects.

Genetic engineering in D. melanogaster is an extremely mature technology. It is founded on several independent phenomena:

1. The presence of a transposable element, called the P element, which is an efficient genetic transformation vector. This vector has been available and exploited since the early 1980s.

2. The ability to create and maintain genetic mutants by traditional techniques such as chemical- or radiation-induced mutagenesis or by transposon insertion mutagenesis, and the construction and availability of balancer chromosomes to maintain many of these mutants.

3. The presence of strains that lack the P element, thus providing recipient strains suitable for P element transformation.

4. The completion of the Drosophila genome project and the public availability of the data generated.

These planks of achievement are a consequence of the intense and sustained research that has been invested into Drosophila over the course of the last 90 years. The picture in all other insect species is, by comparison, sparse. For example, transposable elements capable of transforming nondrosophilid species have been available only since 1996.

Also, traditional mutagenesis approaches have been used to generate mutations in a handful of insect species. Many of these have been lost because of problems arising from the rearing of these species (it should be noted that what attracted T. H. Morgan to Drosophila was the ease with which it could be reared and mated in the laboratory) and, often, because it had been necessary to depend on a handful of dedicated workers to maintain these strains. (In Drosophila, by contrast, there are central repositories for strain maintenance as well as hundreds, if not thousands, of researchers who maintain even the most problematic genetic stocks.) Except for medfly, balancer chromosomes have not been constructed in nondrososphild insects.

Two other factors are important. The interactions, if any, of the transposable elements so far known to transform non-drosophilids with components of the insect genome remain unknown, as do the molecular mechanisms by which these elements move both within and between insect genomes. Second, to date, no insect species other than Drosophila has had its entire genome sequenced. Some mosquito genomes are the target for current and future genomic projects.

Transformation Technologies in D. melanogaster

P element transformation Population geneticists in the 1970s had observed that when males from certain strains of flies recently established from wild populations were mated to females from long-established laboratory populations, a number of abnormal traits were observed in the progeny. These traits included high rates of mutation, sterility, and recombination in males, traits that are not usually seen in this species. Collectively the traits that arose only when specific hybrid insects were created were thought to be manifestations of a single syndrome that became known as "hybrid dysgenesis." Because the factors responsible for this syndrome were transmitted by males, they were referred to as paternal or "P" factors. Those working on P-factor-mediated hybrid dysgenesis quickly realized that there were multiple factors that mapped to many different locations within the genome. Some of the mutations that were induced during hybrid dysgenesis were very unstable and were themselves capable of mutating further to result in more extreme phenotypes or to revert to wild type. This instability as well as other genetic observations suggested that the P factors were mobile genetic elements or transposons. As long ago as 1989, Engels gave a comprehensive description of the P element, P factors, and their use in genetic transformation.

Concurrent with the efforts of population geneticists to understand the phenomenon of hybrid dysgenesis were efforts of molecular geneticists to clone genes from D. melanogaster. The eye color gene known as white was one of the first genes to be cloned from this species, largely owing to the great amount of genetic analysis that had been done on this locus. Having the white gene cloned provided a unique opportunity to isolate P factors. Because P factors were responsible for causing mutations, a genetic screen was performed to seek mutations induced by hybrid dysgenesis of the white gene. The reasoning behind this experiment was that once a P-factor-induced mutation of the white gene was obtained, it should, in theory, be readily cloned by conventional genomic DNA library screening using the wild-type allele of the white gene as a probe. By comparing the mutant allele with the wild-type allele, the nature of the P factor might be deduced. As expected, mutations of the white gene induced by hybrid dysgenesis contained insertions, and the insertion sequences had all the characteristics of a transposable element. In fact, P factors were transposable elements and became known as P elements. Complete P elements were about 3 kb in length and contained four open reading frames encoding for a protein essential for P element movement. The terminal sequences of P elements consisted of inverted repeat sequences of 31 bp. In structure, the P elements generally resembled other trans-posable elements that had been isolated from bacteria and were referred to as short, inverted repeat—type transposable elements.

The physical isolation of an active transposable element from D. melanogaster provided researchers with a unique opportunity to integrate foreign DNA into the chromosomes of this species. Efforts to integrate exogenous DNA into the chromosomes of insects can be traced back to the late 1960s. While there was an interest in genetically transforming insects and a few reports of minor successes, there was no reliable method for creating transgenic D. melanogaster. The P element solved that problem. It was reasoned that if the terminal, noncoding sequences of the element, which serve as signal sequences directing the cutting and pasting of the element, were attached to any piece of DNA, that piece of DNA would acquire the mobility properties of a P element. Furthermore, if this altered transposable element could be introduced into a cell that was going to form gametes and the element jumped (i.e., transposed) onto one of the chromosomes of this cell, then the gametes arising from this cell would be transgenic and would give rise to transgenic progeny. This reasoning proved to be precisely correct. Under the appropriate conditions, genes to which the terminal noncoding sequences of a P element have been attached can readily integrate into the chromosomes of presumptive germ cells and lead to the efficient creation of transgenic insects (Fig. 1).

This relatively simple technology helped fuel a revolution in the study of this model organism. Today, this transposable element forms the basis for a suite of technologies that allow researchers to identify and analyze genes in a variety of ways. The P element gene transformation system has also served as a paradigm for the development of similar technologies applicable to other species of insects.

gene tagging with transposable elements

The key to the identification and isolation of P elements was the availability of the cloned white gene. The white locus was used as a trap; once the P element had been identified and cloned, it could be used as a way of identifying and isolating

FIGURE 1 P element transformation of D. melanogaster. Two plasmids, one containing a P element into which has been cloned a genetic marker (green) and a helper plasmid containing the P element transposase (white) placed under the control of an inducible promoter (blue) are coinjected into embryos. The P element inverted terminal repeats are shown as black arrowheads. G0 adults arising from injected embryos are not transgenic but some will contain a percentage of gametes containing the P transposable element. These adults are outcrossed and G1 progeny are examined for the presence of transgenic individuals (green fly).

FIGURE 1 P element transformation of D. melanogaster. Two plasmids, one containing a P element into which has been cloned a genetic marker (green) and a helper plasmid containing the P element transposase (white) placed under the control of an inducible promoter (blue) are coinjected into embryos. The P element inverted terminal repeats are shown as black arrowheads. G0 adults arising from injected embryos are not transgenic but some will contain a percentage of gametes containing the P transposable element. These adults are outcrossed and G1 progeny are examined for the presence of transgenic individuals (green fly).

genes. As already discussed, one of the prominent features of P element movement (as revealed by the phenomenon of hybrid dysgenesis) was the creation of mutations. These mutations are caused by the insertion of the P element into an essential region of a gene, thereby altering its level or pattern of expression.

Mutations and their associated phenotypes define genetic loci. The existence of a mutant insect with an altered eye color defines a locus that plays some role in eye pigmentation. Although the existence of a mutant reveals the presence of a gene and its location, it does not provide researchers with a means of readily isolating the DNA containing the gene. If, however, the mutation is caused by the insertion of a sequence, such as a P element, and we know the sequence of the insertion sequence, we can use this information to isolate the DNA of the gene that was mutated. By making a genomic DNA library from the mutant insect, one can use conventional DNA hybridization techniques to identify sequences in the library that contain the P element. Because the mutation was caused by the insertion of the P element into a gene, the DNA adjacent to the P element is likely to be the gene responsible for the mutant phenotype. This methodology of transposon tagging is very powerful and has been used not only in D. melanogaster but in a number of other organisms as well. The requirements for an effective transposon-tagging system that ensures unambiguous gene identification are an active transposon that has little integration site specificity and insect strains that contain few or only one transposon-tagging transposable element. Roberts has described the use of the P element for gene tagging and enhancer trapping.

enhancer trapping Transposable element-based mutagenesis or transposon tagging is a powerful technology with one limitation: it can identify only genes that have a recognizable mutant phenotype following element integration. Many of the genes that one mutates either do not result in a visible phenotype or cause the death of the organism. Such genes will never be recovered from a screen based on transposon tagging.

A complementary methodology that does not rely on mutagenesis for gene identification is called enhancer trapping. Enhancers are gene expression regulatory elements, and they function to fine-tune the control of gene expression, temporally and spatially. They are quite distinct from gene promoters in that enhancers are not sites of RNA polymerase binding but are instead sites for protein binding that influence when and how often RNA polymerase will associate with a promoter. A remarkable and useful feature of enhancers is their ability to act over long distances by mechanisms that are not entirely clear. That is, an enhancer may be located hundreds or even thousands of bases away from its target promoter. If a new promoter is inserted near the enhancer, it too will become regulated by that enhancer. This phenomenon provides a clever, nonmutagenic method for gene identification based on patterns of gene expression called enhancer trapping.

Like transposon tagging, enhancer trapping relies on the movement of a transposable element. The element in this case has been engineered to contain a gene whose expression is readily detected. Today the green fluorescent protein from the jellyfish is a common choice. The reporter gene has been engineered to contain a minimal basal promoter, meaning that it contains an RNA polymerase binding site but no associated enhancers. Consequently, this enhancerless gene construct does not result in reporter gene expression unless the transposon in which it is contained integrates near an active enhancer. The presence of enhancers can be detected by moving the transposon around the genome and looking for expression of the reporter gene. By identifying enhancers with particular properties, one then has indirectly identified the genes controlled by these enhancers. Often, the genes regulated by enhancers identified by using this method are located in the proximity of the enhancer. The significant difference between this method of gene identification and transposon tagging is that enhancer detection does not require mutating the enhancer or its associated gene. Consequently, genes that may not have been detected by a transposon tagging screen might be detected using an enhancer trap (Fig. 2).

FIGURE 2 Example of enhancer trapping in insects: three pairs of chromosome with their centromeres (purple). One strain contains a MOS element into which the Hermes transposase has been cloned (orange). The MOS inverted terminal repeats are shown as pink arrowheads. A second strain contains a Hermes element containing a genetic marker (blue) placed under the control of a weak promoter. The two strains are crossed, whereupon the Hermes transposase causes the Hermes elements to move to new regions of the insect genome. Should a Hermes element insert near an enhancer element (black box), the genetic marker in the Hermes element would show the same tissue-and stage-specific expression of the gene controlled by the enhancer. The gene and the enhancer can then be cloned by standard gene tagging techniques.

FIGURE 2 Example of enhancer trapping in insects: three pairs of chromosome with their centromeres (purple). One strain contains a MOS element into which the Hermes transposase has been cloned (orange). The MOS inverted terminal repeats are shown as pink arrowheads. A second strain contains a Hermes element containing a genetic marker (blue) placed under the control of a weak promoter. The two strains are crossed, whereupon the Hermes transposase causes the Hermes elements to move to new regions of the insect genome. Should a Hermes element insert near an enhancer element (black box), the genetic marker in the Hermes element would show the same tissue-and stage-specific expression of the gene controlled by the enhancer. The gene and the enhancer can then be cloned by standard gene tagging techniques.

Transposon tagging and enhancer trapping are rather intense genetic methods for gene identification. Such methods require the ability to efficiently perform genetic crosses, to recognize mutants or desirable reporter gene expression patterns, and then to maintain large numbers of distinct genetic lines of insects. Although Drosophila is readily amenable to such manipulations, other insects may be less so. Nevertheless these methods will be of great value to those entomologists working on a variety of insect species.

homologous recombination Transposon tagging and enhancer trapping are methods of identifying genes based on a phenotype: a mutant phenotype in transposon tagging, an expression phenotype in enhancer trapping. The availability of essentially the entire DNA sequence of the genome of D. melanogaster has permitted the identification of genes based entirely on DNA sequence patterns. Often the role of these genes is completely unknown because flies with mutations in these genes have not been identified. Without the ability to examine the phenotypes of flies with mutant alleles of the gene, gene function must be deduced entirely by other means, such as patterns of expression or analysis of the protein gene product. Today, however, it is possible for researchers who know the DNA sequence of a specific gene to create D. melanogaster with mutations in that gene. This method of targeted mutagenesis relies on the process of homologous recombination.

Homologous recombination, the process of gene exchange that typically occurs during meiosis, depends on the association of DNA sequences that are identical or nearly identical. Breaks in one of the strands of a DNA duplex can result in this strand becoming associated with its homologue on another chromosome, leading to gene exchange. It is now possible to exchange a gene located on a chromosome of a fly with a nearly identical gene created in the laboratory. This somewhat involved process relies on the use of a site-specific recombinase and a site-specific endonuclease, but it is potentially a method that will be generally applicable to any insect. Rong and Golic have described this technology in D. melanogaster.

The strategy behind using homologous recombination takes advantage of the high recombinogenicity of linear molecules of DNA. Such molecules will preferentially recombine with sequences homologous to the sequence at the end of the linear molecule. Gene targeting by homologous recombination in D. melanogaster is based on a clever method for generating the highly recombinogenic targeting molecule in vivo. The process begins by creating a transgenic insect using, for example, a P element gene vector that contains the targeting sequences flanked by site-specific recombination sites such as the FRT sites of the FLP recombinase system. When FLP recombinase is expressed (from a previously integrated transgene) in the insect, the FRT sites will recombine causing the targeting gene to be excised from the integrated gene vector. This recombination event results in the creation of extrachromosomal circular molecules in the nuclei of the insect. These extrachromosomal circles are then linearized by expressing a site-specific endonuclease (from a previously integrated transgene) that recognizes a DNA sequence that has been placed in the targeting gene in such a way that digestion results in the target gene sequences being located at the ends of the linearized circle. This highly recombinogenic molecule will then recombine with the chromosomal homologue, resulting in gene disruption.

Homologous gene replacement has been achieved for two Drosophila genes, the yellow gene and the pugilist gene, and most likely will be applicable to a large number of D. melanogaster genes. In particular it should enable gene function to be assigned to the thousands of new genes identified in the Drosophila genome project through replacing the wild-type forms with nonfunctional mutations that have been created in vitro.

A prerequisite for targeted gene replacement is a set of transgenic insects that can express the appropriate restriction enzyme and the FLP recombinase. This is readily achieved in D. melanogaster and now can also be accomplished, in principle, in other insect species because transposable elements exist that can be used to genetically transform them. The FLP recombinase system has been shown to function correctly in the yellow fever mosquito, Aedes aegypti, and most likely will function in all insects into which it is placed. Similarly, the ability of a yeast restriction enzyme to function in Drosophila suggests that it should also function correctly in a range of insect species into which it is placed.

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