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Introduction to Enhancer Trap Analysis

In this laboratory Drosophila melanogaster is used as the model organism and a set of techniques known as enhancer trap analysis is employed to begin the experiments. Drosophila is one of the favored models systems for molecular and genetic studies for a number of reasons. Drosophila are easy to keep in the laboratory and their generation time is short. In addition, they survive well with both chemically induced and X-ray-induced mutations and a large number of mutants have been characterized. Drosophila is an ideal model species for genetic studies, as it possesses a relatively simple genome that has been characterized extensively. Moreover, many of the genes studied in Drosophila have provided new information about their vertebrate counterparts due to a high level of evolutionary conservation of the encoded proteins. Finally, the entire genome of Drosophila melanogaster has now been sequenced.

The popularity of Drosophila was increased dramatically in the 1980s and 1990s, first by the creation and molecular genetic analysis of a battery of early developmental mutants followed by the development of a set of techniques, known as enhancer trap analysis, which makes gene mutagenesis and the initial cloning of genes relatively easy. Once genes are cloned, common genetic and recombinant DNA strategies can be used in combination with standard cell biological, histological and physiological techniques to study the functions of the encoded proteins.

The basis of enhancer trap techniques is the insertion of a P-transposable element (or P-elements) into the Drosophila genome. Transposable elements are discrete sequences of DNA that are mobile. They can "jump" into a chromosome, "jump" out again, and reinsert randomly into the genome. At each step they are capable of causing a mutation.

Transposable elements as they appear in nature contain the coding region for the enzyme, transposase, which is responsible for the removal and reinsertion of DNA. The P-elements used in enhancer trap analysis have had the transposase gene completely removed. This means that once the P-element is inserted it is stable and cannot jump around useless it is caused to do so by the reintroduction of transposase. The P-elements that will be used in this laboratory have been engineered not only to remove the transposase but also to add several useful features. The features that have been added are the lacZ reporter gene, the mini-white+ or rosy+ eye color gene as a genetic marker and a plasmid cloning vector. Figure 1 depicts the P-element that was introduced into the mutant that is the basis of this laboratory. Figures 2 and 3 are other P-element constructs that you also may encounter in this class. Lines of flies that have these modified P-elements inserted somewhere in the genome are called Enhancer Trap Lines.

 

Figure 1. P-element Construct found in the Mhc mutant

 

Figure 2. P(lArB)

 

Figure 3. P(lwB)

Let us consider each of the features of the P-element in turn starting with the mini-white+ or rosy+ gene. white+ codes for red eye color. rosy+is a modifier of the normal eye color; null mutants have brick colored eyes rather than red eyes. The inclusion of an eye color gene in the P-element allows us to trace the presence of the P-element during genetic crosses. Before the entire Drosophila genome was sequenced, this gene was also used frequently for mapping of the P-element to a physical location on the chromosome.

The P-element found in the genome of the flies that will be used in this laboratory was initially inserted by injection into embryos at the syncytial blastoderm stage when nuclei are dividing but cellularization has not yet occurred. Since this P-element does not have an active transposase enzyme, a separate transposon containing the transposase gene was inserted at the same time. The transposon containing the transposase has been engineered so that it produces the enzyme but cannot incorporate into the genome (the inverted repeats have been removed). White-eyed, but otherwise wild-type flies are chosen as the host strain for P-element insertion lines that carry the white+ genetic marker and rosy mutants are used when rosy+ is the genetic marker so that the P-element can be traced through genetic crosses. During cellularization of the blastoderm, both the P-element and the transposase-containing transposon can be incorporated into any of the newly formed cells. Due to defective splicing of the transposase gene in somatic cells, however, the P-element is incorporated into the genomic DNA of germline cells only. Therefore, all of the injected embryos will grow into flies with mutant eye color. Some of the germline cells of these flies will contain the P-element with the wild type eye color gene. Therefore, in the next generation, some of the flies will have red eyes indicative of the presence of the P-element. A tutorial of the scheme used to generate the first generation P-element lines is available at this web site.

The initial insertion of the P-element into the genome may result in the mutation of a gene. In many cases, however, the initial insertion of the P-element does not cause an observable mutant phenotype. This usually occurs because the P-element has inserted into non-coding and non-regulatory regions of DNA ("junk DNA"). The availability of mutants is, of course, important because they provide insight into the function of novel genes and their encoded proteins. In the cases where the initial insertion of the P-element does not cause an observable mutation, mutations can be induced by the subsequent reintroduction of transposase and genetic remobilization of the P-element. When a P-element is re-mobilized it will sometimes take flanking DNA with it, thus causing a deletion type mutation. These mutants are known as "jump-out" mutants. The P-element may also jump-out without removing flanking DNA and without leaving a footprint (evidence of the repair of overhang DNA during the initial insertion of the transposon). This is called a "clean jump". P-elements from a jump can re-insert at some other location in the genome. A mutation caused by reinsertion is known as "jump-start" mutation. In both jump-out and jump-start mutagenesis, the P-element is genetically removed from the chromosome by crossing an enhancer trap line that is homozygous or heterozygous for the insertion of a P-element, with a line of flies that contains a functional gene for the transposase enzyme. Several crosses are required for "jump-out" and "jump-start" schemes. It is important to remember that the "jump out" flies will revert to white or brick eyes since the P-element has been removed. "Jump-start" flies, on the other hand, will continue to exhibit the red eye color. Tutorials of the schemes used to generate "jump-out" and "jump-start" lines are available at this web site.

The second useful feature of the P-element is the lacZ gene. It is the presence of this gene that gives these techniques their name. The presence of this gene in the P-element allows us to know where and when genes affected by the P-element insertion are expressed. The lacZ gene codes for ß- galactosidase, an enzyme that is produced in E. coli but not Drosophila. ß-galactosidase, in the presence of the correct substrate and the indicator chemical, X-gal, produces a blue color in tissues where it is expressed. The lacZ gene on the P-element is expressed only weakly unless it is activated by enhancers in the vicinity of the P-element. Nearby enhancers that normally activate Drosophila genes in the vicinity of the P-element insertion will also activate the lacZ gene on the P-element. The lacZ gene will be expressed with the same tissue specificity and temporal pattern as the nearby Drosophila gene that normally is regulated by these enhancers. Therefore, tissues that express the Drosophila genes driven by these enhancer elements will turn blue in the presence of X-gal. The Drosophila enhancer has in effect been trapped into telling us something about the tissue specificity and temporal expression pattern of genes in the vicinity of the P-element insertion. The genes driven by these enhancer elements are also likely to be the genes mutated by the insertion or remobilization of the P-element. This reporter gene system is not always accurate. Sometimes a nearby gene with a strong enhancer will drive the lac Z expression while the insertion of the P-element causes the disruption of a different adjacent gene.

The final feature of the P-element that we need to consider is the plasmid cloning vector. The vectors incorporated into the P-elements discussed here all include: 1) an origin of replication that allows many copies of the vector plus any attached DNA to be made when the vector is inserted into bacteria, 2) two polycloning sites, each of which contains a number of unique restriction endonuclease recognition sites and 3) an ampicillin resistance gene. It is the presence of this vector that allows us to clone genomic DNA in the vicinity of the P-element insertion. The recovery of this fragment of genomic DNA is known as a plasmid rescue.

The power of enhancer trap techniques is that the simple insertion of a P-element into the genome of a fruitfly creates mutants and allows us to obtain genomic clones in the area of the mutation, to map the mutated gene to a physical location on the chromosome and to know the tissue specificity and expression pattern of the gene of interest.

References

Hoy, Majorie A. (1994) Insect Molecular Genetics: An Introduction to Principles and Applications. Academic Press, San Diego.