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Transgenic methods for increasing Drosophila life span
Mechanisms of Ageing and Development 118 (2000) 1 – 14 www.elsevier.com/locate/mechagedev
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Transgenic methods for increasing Drosophila life span John Tower * Department of Biological Sciences, SHS172, Uni6ersity of Southern California, Los Angeles, CA 90089 -1340, USA Received 17 March 2000; received in revised form 2 June 2000; accepted 20 June 2000
Abstract At least five different transgenic approaches have been applied to the study of Drosophila aging. There are two single component systems: transgenes with native (normal) promoters and transgenes with heterologous promoters; as well as three binary systems: ‘GAL4/UAS’, ‘FLP-out’ and ‘tet-on’. These approaches vary in ability to meet several technical challenges, and the relative advantages and disadvantages of each are discussed. Using these techniques, over-expression of the hsp70, Cu/ZnSOD and MnSOD genes has each been demonstrated to increase Drosophila life span. © 2000 Published by Elsevier Science Ireland Ltd.
1. Introduction Current theory suggests that aging exists due to the decreasing force of natural selection as a function of age, and has an underlying genetic basis (Rose, 1991; Partridge and Barton, 1993; Charlesworth, 1994; Kirkwood, 1995). However the proximal causes of aging and the genes involved remain to be determined. Testing hypothesized mechanisms of aging requires experimentally altering an organism’s genotype and assaying for effects on rate of aging. Creation of transgenic organisms is potentially an ideal approach, and complements more traditional genetics. * Tel.: + 1-213-7405384; fax: +1-213-7408631. E-mail address: [email protected] (J. Tower). 0047-6374/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 1 5 2 - 4
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Drosophila melanogaster is a popular model because of the extensive transgenic and other genetic and molecular biological tools available, as well as it’s short life span (1 – 2 months) and ease of culture (Tower, 1996; Tatar, 1999; Sohal et al., 2000). In small model organisms such as Drosophila it is difficult to study pathology, and life span is usually the most direct measure of aging rate. Decreased life span is problematic, as it is likely to result from novel pathologies that do not normally limit life span. In contrast, increased life span can only result from alterations in limiting processes, and is more likely to identify genes directly related to aging. Life span is a quantitative trait, and this presents several challenges for the design of transgenic experiments. This review focuses on the transgenic technologies available in Drosophila and their application to the goal of increasing life span. Extensive correlative evidence suggests that for most organisms oxidative damage may be a primary cause of aging and functional decline (Harman, 1956; Stadtman, 1992; Sohal and Weindruch, 1996). Reactive oxygen species (ROS) are generated as a byproduct of normal metabolism, and oxidatively damaged molecules and organelles have been found to accumulate in all aging organisms examined. Not surprisingly, the transgenes tested for effects on life span in Drosophila have been ones linked to oxidative stress resistance or response. hsp70 was originally identified as a gene induced in response to heat and oxidative stress (Lindquist and Craig, 1988). Hsp70 family proteins can prevent protein aggregation, facilitate protein re-folding, and facilitate entry of damaged proteins into proteolytic pathways (Parsell and Lindquist, 1993). Hsp70 protein is also induced during normal Drosophila aging, where it appears to be a response to age-associated oxidative damage (Wheeler et al., 1995, 1999). The enzymes superoxide dismutase (SOD) and catalase are primary defenses against reactive oxygen species (ROS) in all cells. SOD exists in two forms: cytoplasmic (Cu/ ZnSOD or SOD1) and mitochondrial (MnSOD or SOD2). SOD converts superoxide to H2O2, and catalase converts H2O2 to H2O and O2. Another important defense against ROS involves the enzyme glutathione reductase. This enzyme generates reduced glutathione, which is an abundant low molecular weight antioxidant. At least five different transgenic approaches have been applied to the study of Drosophila aging, and each has particular advantages and disadvantages. There are two single component systems: transgenes with native (normal) promoters and transgenes with heterologous promoters; as well as three binary systems: ‘GAL4/ UAS’, ‘FLP-out’ and ‘tet-on’. The approaches vary in ability to meet several technical challenges, including: (1) The ability to control for the variability in life span caused by unavoidable differences in genetic background between different transgenic lines. (2) The ability to reproduce the normal regulation of expression of the transgene. (3) The ability to generate novel expression patterns, including tissue and temporal specificity, and stress inducibility. (4) True inducibility — experimenter control of transgene expression, allowing choice of phase of life cycle and ability to avoid toxic or other effects of the transgene during development.
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2. The genetic background problem Controlling for genetic background variation and its effects on life span is a particularly important challenge (Tower, 1996; Kaiser et al., 1997; Tatar, 1999). While several studies have stated that different transgenic lines are in ‘isogenic’ or ‘essentially isogenic’ backgrounds, it is essentially impossible to create multiple transgenic Drosophila lines with completely isogenic backgrounds, and has not been accomplished in the published literature. There are two sources of genetic variation between transgenic lines. First is differences in genetic background at sites other than the P element insertion. All Drosophila transgenics are created by transposon insertion, by far the most common being the P element vector (Rubin and Spradling, 1982; Spradling, 1986). Making transgenics requires identification of relatively rare P element insertion events and then creating a stable line where each individual carries the insertion. These steps require crosses between different strains, and multiple Drosophila generations. Unavoidably, each resultant transgenic strain differs in the genetic background inherited from the strains used in construction. Using strains for construction with similar genetic backgrounds makes the resultant transgenic lines more similar, but it is impossible to create multiple transgenic lines with truly isogenic backgrounds. The genome-wide mutation frequency in Drosophila is great enough (:1%) (Ashburner, 1989), so that by the time the many required crosses or backcrosses have been performed the different lines have diverged due to the accumulation of different mutations. The second source of genetic variation between different transgenic lines is the site of P element insertion. While some sites are preferred relative to others, P elements insert essentially at random into Drosophila chromosomes (Spradling et al., 1995). In each transgenic line the P element construct will be inserted at a different site, and the location of insertion can have various significant effects on the expression of the construct, collectively called chromosomal position effects, or
Fig. 1. Chromosomal position effects (PE).
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‘PE’ (Fig. 1). PE can be positive or negative effects on the magnitude of transgene expression caused by flanking chromosomal sequences or regions (Spradling and Rubin, 1983). Genomic transcriptional regulatory elements such as enhancers often function to control the tissue and temporal specificity of gene expression (Fig. 1A). Enhancers that happen to be located in the regions flanking the P element insertion can impose tissue and temporal specific expression patterns on the promoter(s) in the element (Fig. 1B) (O’Kane and Gehring, 1987; Bellen, 1989). If the transgene has an effect on life span, PE will then tend to cause this effect to vary between different transgenic lines. Finally, the P element insertion is itself a mutation that will vary in each transgenic line. Depending on the site of insertion the P element may disrupt a gene, and the effects can vary from negligible to severe, and may be recessive or in rare instances dominant. The size and location of P element inserts have been reported to have significant effects on life span (Kaiser et al., 1997). In summary, there are multiple, unavoidable sources of genetic variation between different transgenic lines, and these differences in genotype cause variation in life span. Transgenic experiments must be designed so that any effect of the transgene on life span can be identified above this background.
3. Transgenes with native promoters Perhaps the most straightforward way to test a gene for effects on life span is to clone the entire gene, including all 5% and 3% regulatory regions into the P element transformation vector and create multiple transgenic lines. One appropriate control is multiple transgenic lines containing the empty transformation vector. However, it could be argued that the ideal control would be the transgene with a small deletion that inactivates the promoter, and/or a translational stop sequence engineered near the 5% end so that no protein is produced. In this way the size and sequence content of the experimental and control constructs would be nearly identical. Strengths of the endogenous promoter approach are that this is the best way to maintain the normal regulation of expression of the gene, including tissue and temporal specificity and stress responsiveness, although this will not be perfect in all lines due to PE. A weakness of this approach is that it is difficult to control for the differences in genetic background in each line. To do so requires generation of a relatively large number of control and experimental transgenic lines, and averaging the life span for each group. This approach was the first applied to Drosophila life span studies, in analyses of the catalase and Cu/ZnSOD genes. Transgenics containing an extra copy of either the catalase gene (Orr and Sohal, 1992; Griswold et al., 1993) or Cu/ZnSOD gene (Seto et al., 1990; Orr and Sohal, 1993) were reported to have no consistent increase in life span. Transgenics containing an extra copy of both genes were reported to have :30% increase in life span (Orr and Sohal, 1994; Sohal et al., 1995). However, subsequent re-examination of these data suggests that there were not a sufficient number of control and experimental transgenic lines to detect a statistically significant effect of the transgenes on life span (Tower, 1996; Tatar, 1999).
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Pioneering studies, including ones involving the native promoter approach, developed the critical concept of ‘metabolic potential’. This is the total lifetime metabolic activity of the organism (Miquel et al., 1976; Sohal et al., 1995, 2000). In poikilothermic animals such as Drosophila, reduced temperature and numerous other environmental and genetic manipulations that decrease metabolic activity will increase life span. For this reason it is essential to determine whether any life span increases associated with transgenes are associated with altered metabolic activity. It seems certain that a transgene that increases life span without lowering metabolic activity is of interest. However, it remains an open question in the field whether life span increases associated with reduced metabolic activity will be informative. Most recently, the native promoter approach has been used in analyses of the glutathione reductase and MnSOD genes (Mockett et al., 1999a,b). Neither gene was found to increase life span under usual culture conditions. However, glutathione reductase overexpression did increase survival in hyperoxic atmosphere, a condition known to increase oxidative stress.
4. Germ line recombination strategies to reduce PE The first convincing demonstration that a transgene could positively affect Drosophila life span used the native promoter in analysis of the hsp70 gene (Tatar et al., 1997). This success was due in part to the use of an elegant means for reducing PE, and by the fact that the hsp70 promoter is inherently inducible by heat stress. Two important correlations had suggested that hsp70 was a good candidate for study: First, hsp70 expression is upregulated during aging, suggesting that hsp70 may have important functions during aging (Wheeler et al., 1995, 1999). Second, mild heat stress of adults induces multiple hsps, including hsp70, and causes small increases in life span (Khazaeli et al., 1997). To study the ability of hsp70 to protect flies from heat stress, Welte et al. created transgenic lines with differing numbers of extra native hsp70 transgenes all at the same location in the genome (Welte et al., 1993). This was done using the FLP/FRT recombination system (Golic and Lindquist, 1989): a transgenic construct was created and transformed that contained three copies of the native hsp70 gene and a white+ marker gene flanked by FRTs (Fig. 2A). FRTs are the target sites for recombination catalyzed by the yeast FLP recombinase. Expression of FLP protein in germ line cells from a different transgenic construct yielded recombination events, including duplications of the hsp70 and white+ sequences between the FRTs (Fig. 2B). In a white mutant background, eye color is dependent on the copy number of the white+ marker, allowing for identification of various recombination products. Repeated rounds of recombination followed by crossing out of the FLP construct yielded transgenic lines with varying numbers of extra native hsp70 genes, all at the same genomic location. While the final lines were not isogenic because of the other unavoidable differences in genetic background described above, variation in transgene expression due to PE was expected to be reduced. Upon mild heat stress the extra-copy transgenic lines expressed higher amounts of hsp70 and
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Fig. 2. Germ line recombination.
exhibited increased survival, at least at one specific stage of embryonic development. Tatar et al. analyzed the same strains for adult life span (Tatar et al., 1997; Tatar, 1999). Mild heat stress of adults at 4 days of age yielded increased hsp70 expression in the extra-copy transgenic lines, and an increase in mean life span of several percent relative to the normal copy-number controls.
5. Transgenes with heterologous promoters The potential advantage of the heterologous promoter approach is that the transgene can be expressed at different levels and in a variety of different tissueand temporal-specific patterns. Tissue general expression or tissue and temporal specific expression patterns can be achieved by selecting the appropriate heterologous promoter. The only limitation being the number of such promoters characterized for Drosophila — a number that is fortunately quite large. The first use of a heterologous promoter in aging studies was a pioneering study in which the Drosophila cytoplasmic actin (actin5c) promoter was used to drive expression of the bovine Cu/ZnSOD gene (Reveillaud et al., 1991). While there may be some variation in the absolute level of expression between cell types and developmental stages, the actin5c promoter appears to drive high-level transgene expression in all cells of Drosophila at all stages. The bovine Cu/ZnSOD produced was functional, as it could partially rescue viability when crossed into a Cu/ZnSOD null mutant background (Reveillaud et al., 1994). When the bovine SOD transgenes were present in wild type background Cu/ZnSOD activity was increased up to 1.6-fold. A limitation of constitutive promoters is that there is expression during development, and SOD overexpression was sometimes associated with toxic effects at the pupal stages. In this case it seems likely that the toxic effects during development helped mask any beneficial effect on life span that might have been observed in the
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adults. While an increased average life span of : 10% was reported for the transgenic lines relative to a single control line, a single control line is not expected to be sufficient to identify a statistically significant effect of the transgene on life span.
6. The GAL4/UAS binary system Gal4 is a yeast DNA binding transcription factor, and ‘upstream activating sequence’ (UAS) is it’s DNA binding site. Gal4 protein is not normally present in Drosophila, but when expressed in transgenic flies it will efficiently drive transcription of synthetic Drosophila promoters containing UAS sequences (Fischer et al., 1988). An elegant system was created by Brand and Perrimon (Brand and Perrimon, 1993) to provide a variety of different patterns of Gal4 expression, and thus a variety of expression patterns for any sequences cloned downstream of a synthetic promoter containing a UAS (Fig. 3). In the GAL4/UAS system one transgenic construct is an ‘enhancer trap’: a P element containing a weak promoter driving expression of Gal4 (Fig. 3A). The weak promoter does not produce significant Gal4 expression on its own. However, when the P element inserts into the genome it becomes subject to PE as described above. In this case PE is a useful tool and depending on the specific insertion site, PE will cause specific tissue and temporal expression patterns for Gal4. These lines are sometimes called Gal4 ‘drivers’. The second transgenic construct is a synthetic promoter containing the UAS driving expression of the transgene of interest (Fig. 3B). When the two constructs are introduced into the same flies by appropriate crosses, the transgene of interest is now expressed in the tissue- and temporal-specific pattern dictated by the Gal4 driver. Advantages of the GAL4/UAS system for aging studies are many: A number of Gal4 drivers have been generated and their expression patterns characterized — so a relatively large and diverse number of tissue and temporal expression patterns are available. This allows investigators to determine in which tissues a transgene exerts positive effects on life span, and potentially to avoid
Fig. 3. The GAL4/UAS system.
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confounding toxic or negative effects the transgene might have in other tissues. The binary nature of the system also provides certain controls for genetic background variation in life span, as is also true for the FLP-out and tet-on binary systems described below: life span extension should be present only in flies containing both constructs, and not in progeny of control crosses containing only one construct or the other. However, the different transgenic lines will always have differences in genetic background that can affect life span, as discussed above, and therefor the increase in life span must be demonstrated to be larger than could be caused by this variation. One disadvantage for aging studies is that none of the GAL4/UAS expression patterns is specific to the adult stage — in each case there is also expression during development. This means that the investigator cannot distinguish whether the effect of a transgene on life span is due to its expression during aging, or due to expression during development, which in turn affects aging, or both. Using the GAL4/UAS system, Parkes et al. (1998) demonstrated that overexpression of human Cu/ZnSOD in Drosophila could increase mean life span up to 40%. While the lines were carefully constructed to have as similar genetic backgrounds as possible, they were not isogenic for the reasons discussed above. However, the magnitude of increase in life span was sufficiently large relative to the possible variation in life span due to genetic background differences to be convincing, and increased life span was not observed in control crosses containing only one of the constructs. Importantly, the increased life span was not associated with any decrease in metabolism as assayed by O2 consumption, and therefor both life span and metabolic potential were increased. The pattern of expression produced by the D42-Gal4 ‘driver’ used was as follows: broad expression during embryogenesis; motorneurons, interneurons, some peripheral glial cells, and low level in fat body in larvae; a small number of cells within the central brain and motorneurons within the ventral ganglia in adults. Expression in any or all of these tissues and stages could be contributing to the extended life span observed in the adult. However, since in the adult where aging occurs expression was primarily in motor neurons, the data suggest that motor neurons are a particularly important site of transgene action.
7. The FLP-out system The FLP-out system was originally developed to create clones of mutant cells during Drosophila development (Struhl and Basler, 1993; Basler and Struhl, 1994), however FLP-out was recently demonstrated to work efficiently in the post-mitotic cells of the adult fly (Sun and Tower, 1999). In the FLP-out approach, the yeast FLP recombinase is expressed under the control of the hsp70 heat-inducible promoter in one transgenic construct, called ‘FLP’ (Fig. 4A). A brief heat stress causes tissue-general expression of FLP recombinase. A second transgenic construct (the ‘target’ or ‘reporter’ construct, Fig. 4B) contains the gene of interest downstream of the constitutive, tissue-general Drosophila actin5C promoter. Transcripts initiating at the actin5C promoter are prevented from reaching the gene of interest
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Fig. 4. The ‘FLP-out’ system.
by a transcriptional ‘stop’ sequence. This transcriptional stop sequence is itself flanked by FRTs, which are the target sites for FLP recombinase. After FLP expression is induced by the heat pulse, the FLP recombinase protein causes the precise excision of the transcriptional stop sequence in the target construct (Fig. 4C). This results in constitutive expression of the gene of interest from that point in time onwards, in all tissues of the fly. One advantage of FLP-out is that it is truly inducible: This allows control of the phase of life cycle for transgene expression, and any toxic effects during development can be reduced or avoided. Induction in adults allows identification of effects specific to aging without altering development. Another advantage is that inducible systems such as FLP-out provide powerful controls for genetic background. Control (no heat pulse) and overexpressing (heat pulsed) populations have identical genetic backgrounds, so any difference in life span is due to the heat pulse and its consequences. Even though each transgenic line for the target construct will have differences in genetic background and differences in starting life span, the system controls for this variation: The transgene is induced in each line/background and any consistent effects such as increased life span can be readily identified. The most significant weakness of the current FLP-out system is that a heat pulse is the trigger for induction. Experiments must control for any effects of the heat pulse in order to identify effects specific to the transgene. The heat pulse regime used for FLP-out had no detectable positive effects on life span on its own (Sun and Tower, 1999). Positive effects on life span have been observed with mild heat pulses of young Drosophila, however this requires treatment at an earlier age, and the magnitude of the effect is too small to be detected with samples less than thousands of animals (Khazaeli et al., 1997). As currently designed, FLP-out is not tissue specific, but it could be modified to yield tissue specificity, as discussed below.
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Using FLP-out, catalase enzyme was over-expressed up to 2.5-fold (Sun and Tower, 1999). Catalase over-expression significantly increased resistance to hydrogen peroxide toxicity, but had neutral or slight negative effects on mean life span. In contrast, Cu/ZnSOD over-expression extended mean life span up to 48%. Simultaneous over-expression of catalase with Cu/ZnSOD had no added benefit, presumably due to a pre-existing excess of catalase. Finally, experimental manipulation of genetic background demonstrated that life span is affected by epistatic interactions between the transgene and allele(s) at other loci: The ability of a given transgenic insertion to affect life span was dependent upon the genetic background in which it was analyzed. Cu/ZnSOD over-expression increased life span in both a longer-lived and a shorter-lived genetic background, however higher levels of enzyme over-expression were required to observed life span increases in the longer-lived genetic background. FLP-out has recently been used to demonstrate that over-expression of the mitochondrial mnSOD can also increase life span (Sun et al., 2000). Moreover, the increased mean and maximum life span caused by either Cu/ZnSOD or MnSOD over-expression was not correlated with decreased metabolic activity, and therefore metabolic potential is increased in each case.
8. The ‘tet-on’ system Bujjard and coworkers developed both tet-off and tet-on system for mammalian cells (Gossen and Bujard, 1992; Gossen et al., 1995). Both tet-on (Bieschke et al., 1998) and tet-off (Bello et al., 1998) have been adapted to transgenic Drosophila, however only tet-on has so far been applied to aging studies. In the tet-on system, the ‘reverse tetracycline repressor’ (rtR) binds a specific DNA element, the ‘tetracycline operator’ (tetO), only in the presence of tetracycline, or derivatives such as doxycycline (DOX). Fusion of rtR to the transcriptional activation domain of herpes virus protein VP16 produced a hybrid eukaryotic transactivator protein,
Fig. 5. The ‘tet-on’ system.
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called ‘reverse tetracycline trans activator’ (rtTA). rtTA has previously been shown to allow DOX dependent transcription of transgenes linked to tetO sequences in mammals. To adapt tet-on to Drosophila (Bieschke et al., 1998), the actin5C promoter was used to drive constitutive expression of rtTA in transgenic flies (Fig. 5A). Reporter constructs encoding E. coli b-galactosidase (b-gal), were also introduced into transgenic flies. The ‘7T40:lacZ’ reporter is diagrammed (Fig. 5B), in which seven tetO sequences are fused to the hsp70 core promoter, consisting of the ‘TATAA’ box plus the transcription initiation site. In transgenic Drosophila containing both the rtTA construct and a 7T40:lacZ reporter construct, DOX feeding produced up to 100-fold induction of b-gal. DOX induced b-gal expression in all tissues, in larvae and in young and senescent adults. As discussed above for FLP-out, a primary advantage of tet-on is that it is truly inducible, allowing temporal control of transgene expression and powerful controls for genetic background. The trigger for tet-on induction, DOX has no detectable effect on life span on its own. Importantly, there was no detectable effect on life span of abundant over-expression of E. coli b-gal. These results suggest that tet-on should be ideal for detecting specific effects of transgenes on aging, even quite small effects. Disadvantages of tet-on are that it has not yet been used to increase life span, so its utility remains theoretical. The published tet-on system does not provide tissue specificity, however this has recently been achieved as described below.
9. The future As discussed above, each of the transgenic systems has distinct advantages and disadvantages that will be important in the design of future experiments, and it is likely that each of these systems will continue to be useful. Certain modifications or combinations of the systems are certain to be useful, and are underway in several laboratories. For example the FLP-out system could be made tissue-specific by using tissue-specific promoters to drive expression of the target construct (Fig. 4B and C). A combination of the UAS/Gal4 and tet-on systems should be particularly powerful and provide the advantages of each: creation of transgenic constructs with UAS sequences regulating expression of rtTA would create a three-component system. GAL4 drivers (Fig. 3A) would drive expression of rtTA (Fig. 3B) in desired patterns. In the presence of DOX the rtTA would be activated and drive expression of transgenes of interest, as in Fig. 5B. In this way truly inducible, tissue and temporal specific expression would be achieved, providing the advantages of each system. Such a system has recently been shown to work as expected (M. Stebbins and J. Yin, MS in preparation). The successful extension of life span with transgenes in Drosophila and in C. elegans (Murakami and Johnson, 1998) represents a breakthrough in aging research. This success presents numerous new questions and challenges for the design of transgenic experiments. For example, how do Drosophila hsp70 and SOD
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increase life span? Hsp70 has multiple functions, which is relevant? SOD over-expression could be decreasing oxidative damage as expected, but could also be altering signal transduction by altering levels of ROS signalling molecules. Are there trade-offs with life span extension, such as reduced reproductive capacity? Recent studies suggest that increasing life span with extra copies of the hsp70 gene is associated with reduced fecundity (Silbermann and Tatar, 2000). Will there be additional genes that can increase life span when over-expressed? It seems likely that the increasingly sophisticated transgenic systems and experimental designs available for Drosophila will allow these questions to be answered in the near future.
Acknowledgements Thank you to Raj Sohal, Bill Orr, Mark Tatar, Nadege Minois, and Michael Stebbins for helpful comments and for communicating unpublished results. Aging research in the author’s laboratory is supported by grants from the Department of Health and Human Services (AG11 833 and AG11 644).
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Kaiser, M., Gasser, M., Ackermann, R., Stearns, S.C., 1997. P element inserts in transgenic flies: a cautionary tale. Heredity 78, 1–11. Khazaeli, A.A., Tatar, M., Pletcher, S.D., Curtsinger, J.W., 1997. Heat-induced longevity extension in Drosophila. I. Heat treatment, mortality, and thermotolerance. J. Gerontol. Biol. Sci. 52A, B48 – B52. Kirkwood, T.B.L., 1995. The evolution of aging. Rev. Clin. Gerontol. 5, 3 – 9. Lindquist, S., Craig, E.A., 1988. The heat-shock proteins. Annu. Rev. Genet. 22, 631 – 677. Miquel, J., Lundgren, P.R., Bensch, K.G., Atlan, H., 1976. Effects of temperature on the life span, vitality and fine structure of Drosophila melanogaster. Mech. Ageing Dev. 5, 347 – 370. Mockett, R.J., Orr, W.C., Rahmandar, J.J., Benes, J.J., Radyuk, S.N., Klichko, V.I., Sohal, R.S., 1999a. Overexpression of Mn-containing superoxide dismutase in transgenic Drosophila melanogaster. Arch. Biochem. Biophys. 371, 260–269. Mockett, R.J., Sohal, R.S., Orr, W.C., 1999b. Overexpression of glutathione reductase extends survival in transgenic Drosophila melanogaster under hyperoxia but not normoxia. FASEB J. 13, 1734 – 1742. Murakami, S., Johnson, T.E., 1998. Life extension and stress resistance in Caenorhabditis elegans modulated by the tkr-1 gene. Curr. Biol. 8, 1091 – 1094. O’Kane, C.J., Gehring, W.J., 1987. Detection in situ of genomic regulatory elements in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 84, 9123–9127. Orr, W.C., Sohal, R.S., 1992. The effects of catalase gene overexpression on life span and resistance to oxidative stress in transgenic Drosophila melanogaster. Arch. Biochem. Biophys. 297, 35 – 41. Orr, W.C., Sohal, R.J., 1993. Effects of Cu – Zn superoxide dismutase overexpression on life span and resistance to oxidative stress in transgenic Drosophila melanogaster. Arch. Biochem. Biophys. 301, 34–40. Orr, W., Sohal, R.S., 1994. Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 263, 1128 – 1130. Parkes, T.L., Elia, A.J., Dickson, D., Hilliker, A.J., Phillips, J.P., Boulianne, G.L., 1998. Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nat. Genet. 19, 171 – 174. Parsell, D.A., Lindquist, S., 1993. The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27, 437 – 456. Partridge, L., Barton, N.H., 1993. Optimality, mutation and the evolution of aging. Nature 362, 305–311. Reveillaud, I., Niedzwiecki, A., Bensch, K.G., Fleming, J.E., 1991. Expression of bovine superoxide dismutase in Drosophila melanogaster augments resistance to oxidative stress. Mol. Cell. Biol. 11, 632–640. Reveillaud, I., Phillips, J., Duyf, B., Hilliker, A., Kongpachith, A., Fleming, J.E., 1994. Phenotypic rescue by a bovine transgene in a Cu/Zn superoxide dismutase-null mutant of Drosophila melanogaster. Mol. Cell. Biol. 14, 1302 – 1307. Rose, M.R., 1991. Evolutionary Biology of Aging. Oxford University Press, New York. Rubin, G.M., Spradling, A.C., 1982. Genetic transformation of Drosophila with transposable element vectors. Science 218, 348–353. Seto, N.O.L., Hayashi, S., Tener, G.M., 1990. Overexpression of Cu – Zn superoxide dismutase in Drosophila does not affect life span. Proc. Natl. Acad. Sci. U.S.A. 87, 4270 – 4274. Silbermann, R., Tatar, M., 2000. Reproductive costs of hsp70 expression in transgenic Drosophila melanogaster. Evolution, in press. Sohal, R.S., Weindruch, R., 1996. Oxidative stress, caloric restriction, and aging. Science 273, 59 – 63. Sohal, R., Agarwal, A., Agarwal, S., Orr, W., 1995. Simultaneous overexpression of copper- and zinc-containing superoxide dismutase and catalase retards age-related oxidative damage and increases metabolic potential in Drosophila melanogaster. J. Biol. Chem. 270, 15671 – 15674. Sohal, R.S., Mockett, R.J., Orr, W.C., 2000. Current issues concerning the role of oxidative stress in aging: a perspective. In: Hekimi (Ed.), Results and Problems in Cell Differentiation. Springer, Berlin. Spradling, A.C., Rubin, G.M., 1983. The effect of chromosomal position on the expression of the Drosophila xanthine dehydrogenase gene. Cell 34, 47 – 57. Spradling, A.C., Stern, D.M., Kiss, I., Roote, J., Laverty, T., Rubin, G.M., 1995. Gene disruptions using P transposable elements: an integral component of the Drosophila genome project. Proc. Natl. Acad. Sci. U.S.A. 92, 10824–10830.
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Spradling, A.C., 1986. P element-mediated transformation. In: Roberts, D.B. (Ed.), Drosophila a Practical Approach. IRL, Oxford, pp. 175 – 197. Stadtman, E.R., 1992. Protein oxidation and aging. Science 257, 1220 – 1224. Struhl, G., Basler, K., 1993. Organizing activity of wingless protein in Drosophila. Cell 72, 527 – 540. Sun, J., Tower, J., 1999. FLP recombinase-mediated induction of Cu/Zn-superoxide dismutase transgene expression can extend the life span of adult Drosophila melanogaster flies. Mol. Cell. Biol. 19, 216–228. Sun, J., Folk, D., Bradley, T.J., Tower, J., 2000. Induced over-expression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster without changing metabolic rate, submitted for publication. Tatar, M., Khazaeli, A.A., Curtsinger, J.W., 1997. Chaperoning extended life. Nature 390, 30. Tatar, M., 1999. Transgenes in the analysis of life span and fitness. Am. Nat. 154 (Suppl.), S67 – S81. Tower, J., 1996. Aging mechanisms in fruit flies. Bioessays 18, 799 – 807. Welte, M.A., Tetrault, J.M., Dellavalle, R.P., Lindquist, S.L., 1993. A new method for manipulating transgenes: engineering heat tolerance in a complex, multicellular organism. Curr. Biol. 3, 842 – 853. Wheeler, J.C., Bieschke, E.T., Tower, J., 1995. Muscle-specific expression of Drosophila hsp70 in response to aging and oxidative stress. Proc. Natl. Acad. Sci. USA 92, 10408 – 10412. Wheeler, J.C., King, V., Tower, J., 1999. Sequence requirements for upregulated expression of Drosophila hsp70 transgenes during aging. Neurobiol. Aging 20, 545 – 553.
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Progress
Transgenic methods for increasing Drosophila life span John Tower * Department of Biological Sciences, SHS172, Uni6ersity of Southern California, Los Angeles, CA 90089 -1340, USA Received 17 March 2000; received in revised form 2 June 2000; accepted 20 June 2000
Abstract At least five different transgenic approaches have been applied to the study of Drosophila aging. There are two single component systems: transgenes with native (normal) promoters and transgenes with heterologous promoters; as well as three binary systems: ‘GAL4/UAS’, ‘FLP-out’ and ‘tet-on’. These approaches vary in ability to meet several technical challenges, and the relative advantages and disadvantages of each are discussed. Using these techniques, over-expression of the hsp70, Cu/ZnSOD and MnSOD genes has each been demonstrated to increase Drosophila life span. © 2000 Published by Elsevier Science Ireland Ltd.
1. Introduction Current theory suggests that aging exists due to the decreasing force of natural selection as a function of age, and has an underlying genetic basis (Rose, 1991; Partridge and Barton, 1993; Charlesworth, 1994; Kirkwood, 1995). However the proximal causes of aging and the genes involved remain to be determined. Testing hypothesized mechanisms of aging requires experimentally altering an organism’s genotype and assaying for effects on rate of aging. Creation of transgenic organisms is potentially an ideal approach, and complements more traditional genetics. * Tel.: + 1-213-7405384; fax: +1-213-7408631. E-mail address: [email protected] (J. Tower). 0047-6374/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 1 5 2 - 4
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Drosophila melanogaster is a popular model because of the extensive transgenic and other genetic and molecular biological tools available, as well as it’s short life span (1 – 2 months) and ease of culture (Tower, 1996; Tatar, 1999; Sohal et al., 2000). In small model organisms such as Drosophila it is difficult to study pathology, and life span is usually the most direct measure of aging rate. Decreased life span is problematic, as it is likely to result from novel pathologies that do not normally limit life span. In contrast, increased life span can only result from alterations in limiting processes, and is more likely to identify genes directly related to aging. Life span is a quantitative trait, and this presents several challenges for the design of transgenic experiments. This review focuses on the transgenic technologies available in Drosophila and their application to the goal of increasing life span. Extensive correlative evidence suggests that for most organisms oxidative damage may be a primary cause of aging and functional decline (Harman, 1956; Stadtman, 1992; Sohal and Weindruch, 1996). Reactive oxygen species (ROS) are generated as a byproduct of normal metabolism, and oxidatively damaged molecules and organelles have been found to accumulate in all aging organisms examined. Not surprisingly, the transgenes tested for effects on life span in Drosophila have been ones linked to oxidative stress resistance or response. hsp70 was originally identified as a gene induced in response to heat and oxidative stress (Lindquist and Craig, 1988). Hsp70 family proteins can prevent protein aggregation, facilitate protein re-folding, and facilitate entry of damaged proteins into proteolytic pathways (Parsell and Lindquist, 1993). Hsp70 protein is also induced during normal Drosophila aging, where it appears to be a response to age-associated oxidative damage (Wheeler et al., 1995, 1999). The enzymes superoxide dismutase (SOD) and catalase are primary defenses against reactive oxygen species (ROS) in all cells. SOD exists in two forms: cytoplasmic (Cu/ ZnSOD or SOD1) and mitochondrial (MnSOD or SOD2). SOD converts superoxide to H2O2, and catalase converts H2O2 to H2O and O2. Another important defense against ROS involves the enzyme glutathione reductase. This enzyme generates reduced glutathione, which is an abundant low molecular weight antioxidant. At least five different transgenic approaches have been applied to the study of Drosophila aging, and each has particular advantages and disadvantages. There are two single component systems: transgenes with native (normal) promoters and transgenes with heterologous promoters; as well as three binary systems: ‘GAL4/ UAS’, ‘FLP-out’ and ‘tet-on’. The approaches vary in ability to meet several technical challenges, including: (1) The ability to control for the variability in life span caused by unavoidable differences in genetic background between different transgenic lines. (2) The ability to reproduce the normal regulation of expression of the transgene. (3) The ability to generate novel expression patterns, including tissue and temporal specificity, and stress inducibility. (4) True inducibility — experimenter control of transgene expression, allowing choice of phase of life cycle and ability to avoid toxic or other effects of the transgene during development.
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2. The genetic background problem Controlling for genetic background variation and its effects on life span is a particularly important challenge (Tower, 1996; Kaiser et al., 1997; Tatar, 1999). While several studies have stated that different transgenic lines are in ‘isogenic’ or ‘essentially isogenic’ backgrounds, it is essentially impossible to create multiple transgenic Drosophila lines with completely isogenic backgrounds, and has not been accomplished in the published literature. There are two sources of genetic variation between transgenic lines. First is differences in genetic background at sites other than the P element insertion. All Drosophila transgenics are created by transposon insertion, by far the most common being the P element vector (Rubin and Spradling, 1982; Spradling, 1986). Making transgenics requires identification of relatively rare P element insertion events and then creating a stable line where each individual carries the insertion. These steps require crosses between different strains, and multiple Drosophila generations. Unavoidably, each resultant transgenic strain differs in the genetic background inherited from the strains used in construction. Using strains for construction with similar genetic backgrounds makes the resultant transgenic lines more similar, but it is impossible to create multiple transgenic lines with truly isogenic backgrounds. The genome-wide mutation frequency in Drosophila is great enough (:1%) (Ashburner, 1989), so that by the time the many required crosses or backcrosses have been performed the different lines have diverged due to the accumulation of different mutations. The second source of genetic variation between different transgenic lines is the site of P element insertion. While some sites are preferred relative to others, P elements insert essentially at random into Drosophila chromosomes (Spradling et al., 1995). In each transgenic line the P element construct will be inserted at a different site, and the location of insertion can have various significant effects on the expression of the construct, collectively called chromosomal position effects, or
Fig. 1. Chromosomal position effects (PE).
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‘PE’ (Fig. 1). PE can be positive or negative effects on the magnitude of transgene expression caused by flanking chromosomal sequences or regions (Spradling and Rubin, 1983). Genomic transcriptional regulatory elements such as enhancers often function to control the tissue and temporal specificity of gene expression (Fig. 1A). Enhancers that happen to be located in the regions flanking the P element insertion can impose tissue and temporal specific expression patterns on the promoter(s) in the element (Fig. 1B) (O’Kane and Gehring, 1987; Bellen, 1989). If the transgene has an effect on life span, PE will then tend to cause this effect to vary between different transgenic lines. Finally, the P element insertion is itself a mutation that will vary in each transgenic line. Depending on the site of insertion the P element may disrupt a gene, and the effects can vary from negligible to severe, and may be recessive or in rare instances dominant. The size and location of P element inserts have been reported to have significant effects on life span (Kaiser et al., 1997). In summary, there are multiple, unavoidable sources of genetic variation between different transgenic lines, and these differences in genotype cause variation in life span. Transgenic experiments must be designed so that any effect of the transgene on life span can be identified above this background.
3. Transgenes with native promoters Perhaps the most straightforward way to test a gene for effects on life span is to clone the entire gene, including all 5% and 3% regulatory regions into the P element transformation vector and create multiple transgenic lines. One appropriate control is multiple transgenic lines containing the empty transformation vector. However, it could be argued that the ideal control would be the transgene with a small deletion that inactivates the promoter, and/or a translational stop sequence engineered near the 5% end so that no protein is produced. In this way the size and sequence content of the experimental and control constructs would be nearly identical. Strengths of the endogenous promoter approach are that this is the best way to maintain the normal regulation of expression of the gene, including tissue and temporal specificity and stress responsiveness, although this will not be perfect in all lines due to PE. A weakness of this approach is that it is difficult to control for the differences in genetic background in each line. To do so requires generation of a relatively large number of control and experimental transgenic lines, and averaging the life span for each group. This approach was the first applied to Drosophila life span studies, in analyses of the catalase and Cu/ZnSOD genes. Transgenics containing an extra copy of either the catalase gene (Orr and Sohal, 1992; Griswold et al., 1993) or Cu/ZnSOD gene (Seto et al., 1990; Orr and Sohal, 1993) were reported to have no consistent increase in life span. Transgenics containing an extra copy of both genes were reported to have :30% increase in life span (Orr and Sohal, 1994; Sohal et al., 1995). However, subsequent re-examination of these data suggests that there were not a sufficient number of control and experimental transgenic lines to detect a statistically significant effect of the transgenes on life span (Tower, 1996; Tatar, 1999).
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Pioneering studies, including ones involving the native promoter approach, developed the critical concept of ‘metabolic potential’. This is the total lifetime metabolic activity of the organism (Miquel et al., 1976; Sohal et al., 1995, 2000). In poikilothermic animals such as Drosophila, reduced temperature and numerous other environmental and genetic manipulations that decrease metabolic activity will increase life span. For this reason it is essential to determine whether any life span increases associated with transgenes are associated with altered metabolic activity. It seems certain that a transgene that increases life span without lowering metabolic activity is of interest. However, it remains an open question in the field whether life span increases associated with reduced metabolic activity will be informative. Most recently, the native promoter approach has been used in analyses of the glutathione reductase and MnSOD genes (Mockett et al., 1999a,b). Neither gene was found to increase life span under usual culture conditions. However, glutathione reductase overexpression did increase survival in hyperoxic atmosphere, a condition known to increase oxidative stress.
4. Germ line recombination strategies to reduce PE The first convincing demonstration that a transgene could positively affect Drosophila life span used the native promoter in analysis of the hsp70 gene (Tatar et al., 1997). This success was due in part to the use of an elegant means for reducing PE, and by the fact that the hsp70 promoter is inherently inducible by heat stress. Two important correlations had suggested that hsp70 was a good candidate for study: First, hsp70 expression is upregulated during aging, suggesting that hsp70 may have important functions during aging (Wheeler et al., 1995, 1999). Second, mild heat stress of adults induces multiple hsps, including hsp70, and causes small increases in life span (Khazaeli et al., 1997). To study the ability of hsp70 to protect flies from heat stress, Welte et al. created transgenic lines with differing numbers of extra native hsp70 transgenes all at the same location in the genome (Welte et al., 1993). This was done using the FLP/FRT recombination system (Golic and Lindquist, 1989): a transgenic construct was created and transformed that contained three copies of the native hsp70 gene and a white+ marker gene flanked by FRTs (Fig. 2A). FRTs are the target sites for recombination catalyzed by the yeast FLP recombinase. Expression of FLP protein in germ line cells from a different transgenic construct yielded recombination events, including duplications of the hsp70 and white+ sequences between the FRTs (Fig. 2B). In a white mutant background, eye color is dependent on the copy number of the white+ marker, allowing for identification of various recombination products. Repeated rounds of recombination followed by crossing out of the FLP construct yielded transgenic lines with varying numbers of extra native hsp70 genes, all at the same genomic location. While the final lines were not isogenic because of the other unavoidable differences in genetic background described above, variation in transgene expression due to PE was expected to be reduced. Upon mild heat stress the extra-copy transgenic lines expressed higher amounts of hsp70 and
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Fig. 2. Germ line recombination.
exhibited increased survival, at least at one specific stage of embryonic development. Tatar et al. analyzed the same strains for adult life span (Tatar et al., 1997; Tatar, 1999). Mild heat stress of adults at 4 days of age yielded increased hsp70 expression in the extra-copy transgenic lines, and an increase in mean life span of several percent relative to the normal copy-number controls.
5. Transgenes with heterologous promoters The potential advantage of the heterologous promoter approach is that the transgene can be expressed at different levels and in a variety of different tissueand temporal-specific patterns. Tissue general expression or tissue and temporal specific expression patterns can be achieved by selecting the appropriate heterologous promoter. The only limitation being the number of such promoters characterized for Drosophila — a number that is fortunately quite large. The first use of a heterologous promoter in aging studies was a pioneering study in which the Drosophila cytoplasmic actin (actin5c) promoter was used to drive expression of the bovine Cu/ZnSOD gene (Reveillaud et al., 1991). While there may be some variation in the absolute level of expression between cell types and developmental stages, the actin5c promoter appears to drive high-level transgene expression in all cells of Drosophila at all stages. The bovine Cu/ZnSOD produced was functional, as it could partially rescue viability when crossed into a Cu/ZnSOD null mutant background (Reveillaud et al., 1994). When the bovine SOD transgenes were present in wild type background Cu/ZnSOD activity was increased up to 1.6-fold. A limitation of constitutive promoters is that there is expression during development, and SOD overexpression was sometimes associated with toxic effects at the pupal stages. In this case it seems likely that the toxic effects during development helped mask any beneficial effect on life span that might have been observed in the
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adults. While an increased average life span of : 10% was reported for the transgenic lines relative to a single control line, a single control line is not expected to be sufficient to identify a statistically significant effect of the transgene on life span.
6. The GAL4/UAS binary system Gal4 is a yeast DNA binding transcription factor, and ‘upstream activating sequence’ (UAS) is it’s DNA binding site. Gal4 protein is not normally present in Drosophila, but when expressed in transgenic flies it will efficiently drive transcription of synthetic Drosophila promoters containing UAS sequences (Fischer et al., 1988). An elegant system was created by Brand and Perrimon (Brand and Perrimon, 1993) to provide a variety of different patterns of Gal4 expression, and thus a variety of expression patterns for any sequences cloned downstream of a synthetic promoter containing a UAS (Fig. 3). In the GAL4/UAS system one transgenic construct is an ‘enhancer trap’: a P element containing a weak promoter driving expression of Gal4 (Fig. 3A). The weak promoter does not produce significant Gal4 expression on its own. However, when the P element inserts into the genome it becomes subject to PE as described above. In this case PE is a useful tool and depending on the specific insertion site, PE will cause specific tissue and temporal expression patterns for Gal4. These lines are sometimes called Gal4 ‘drivers’. The second transgenic construct is a synthetic promoter containing the UAS driving expression of the transgene of interest (Fig. 3B). When the two constructs are introduced into the same flies by appropriate crosses, the transgene of interest is now expressed in the tissue- and temporal-specific pattern dictated by the Gal4 driver. Advantages of the GAL4/UAS system for aging studies are many: A number of Gal4 drivers have been generated and their expression patterns characterized — so a relatively large and diverse number of tissue and temporal expression patterns are available. This allows investigators to determine in which tissues a transgene exerts positive effects on life span, and potentially to avoid
Fig. 3. The GAL4/UAS system.
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confounding toxic or negative effects the transgene might have in other tissues. The binary nature of the system also provides certain controls for genetic background variation in life span, as is also true for the FLP-out and tet-on binary systems described below: life span extension should be present only in flies containing both constructs, and not in progeny of control crosses containing only one construct or the other. However, the different transgenic lines will always have differences in genetic background that can affect life span, as discussed above, and therefor the increase in life span must be demonstrated to be larger than could be caused by this variation. One disadvantage for aging studies is that none of the GAL4/UAS expression patterns is specific to the adult stage — in each case there is also expression during development. This means that the investigator cannot distinguish whether the effect of a transgene on life span is due to its expression during aging, or due to expression during development, which in turn affects aging, or both. Using the GAL4/UAS system, Parkes et al. (1998) demonstrated that overexpression of human Cu/ZnSOD in Drosophila could increase mean life span up to 40%. While the lines were carefully constructed to have as similar genetic backgrounds as possible, they were not isogenic for the reasons discussed above. However, the magnitude of increase in life span was sufficiently large relative to the possible variation in life span due to genetic background differences to be convincing, and increased life span was not observed in control crosses containing only one of the constructs. Importantly, the increased life span was not associated with any decrease in metabolism as assayed by O2 consumption, and therefor both life span and metabolic potential were increased. The pattern of expression produced by the D42-Gal4 ‘driver’ used was as follows: broad expression during embryogenesis; motorneurons, interneurons, some peripheral glial cells, and low level in fat body in larvae; a small number of cells within the central brain and motorneurons within the ventral ganglia in adults. Expression in any or all of these tissues and stages could be contributing to the extended life span observed in the adult. However, since in the adult where aging occurs expression was primarily in motor neurons, the data suggest that motor neurons are a particularly important site of transgene action.
7. The FLP-out system The FLP-out system was originally developed to create clones of mutant cells during Drosophila development (Struhl and Basler, 1993; Basler and Struhl, 1994), however FLP-out was recently demonstrated to work efficiently in the post-mitotic cells of the adult fly (Sun and Tower, 1999). In the FLP-out approach, the yeast FLP recombinase is expressed under the control of the hsp70 heat-inducible promoter in one transgenic construct, called ‘FLP’ (Fig. 4A). A brief heat stress causes tissue-general expression of FLP recombinase. A second transgenic construct (the ‘target’ or ‘reporter’ construct, Fig. 4B) contains the gene of interest downstream of the constitutive, tissue-general Drosophila actin5C promoter. Transcripts initiating at the actin5C promoter are prevented from reaching the gene of interest
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Fig. 4. The ‘FLP-out’ system.
by a transcriptional ‘stop’ sequence. This transcriptional stop sequence is itself flanked by FRTs, which are the target sites for FLP recombinase. After FLP expression is induced by the heat pulse, the FLP recombinase protein causes the precise excision of the transcriptional stop sequence in the target construct (Fig. 4C). This results in constitutive expression of the gene of interest from that point in time onwards, in all tissues of the fly. One advantage of FLP-out is that it is truly inducible: This allows control of the phase of life cycle for transgene expression, and any toxic effects during development can be reduced or avoided. Induction in adults allows identification of effects specific to aging without altering development. Another advantage is that inducible systems such as FLP-out provide powerful controls for genetic background. Control (no heat pulse) and overexpressing (heat pulsed) populations have identical genetic backgrounds, so any difference in life span is due to the heat pulse and its consequences. Even though each transgenic line for the target construct will have differences in genetic background and differences in starting life span, the system controls for this variation: The transgene is induced in each line/background and any consistent effects such as increased life span can be readily identified. The most significant weakness of the current FLP-out system is that a heat pulse is the trigger for induction. Experiments must control for any effects of the heat pulse in order to identify effects specific to the transgene. The heat pulse regime used for FLP-out had no detectable positive effects on life span on its own (Sun and Tower, 1999). Positive effects on life span have been observed with mild heat pulses of young Drosophila, however this requires treatment at an earlier age, and the magnitude of the effect is too small to be detected with samples less than thousands of animals (Khazaeli et al., 1997). As currently designed, FLP-out is not tissue specific, but it could be modified to yield tissue specificity, as discussed below.
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Using FLP-out, catalase enzyme was over-expressed up to 2.5-fold (Sun and Tower, 1999). Catalase over-expression significantly increased resistance to hydrogen peroxide toxicity, but had neutral or slight negative effects on mean life span. In contrast, Cu/ZnSOD over-expression extended mean life span up to 48%. Simultaneous over-expression of catalase with Cu/ZnSOD had no added benefit, presumably due to a pre-existing excess of catalase. Finally, experimental manipulation of genetic background demonstrated that life span is affected by epistatic interactions between the transgene and allele(s) at other loci: The ability of a given transgenic insertion to affect life span was dependent upon the genetic background in which it was analyzed. Cu/ZnSOD over-expression increased life span in both a longer-lived and a shorter-lived genetic background, however higher levels of enzyme over-expression were required to observed life span increases in the longer-lived genetic background. FLP-out has recently been used to demonstrate that over-expression of the mitochondrial mnSOD can also increase life span (Sun et al., 2000). Moreover, the increased mean and maximum life span caused by either Cu/ZnSOD or MnSOD over-expression was not correlated with decreased metabolic activity, and therefore metabolic potential is increased in each case.
8. The ‘tet-on’ system Bujjard and coworkers developed both tet-off and tet-on system for mammalian cells (Gossen and Bujard, 1992; Gossen et al., 1995). Both tet-on (Bieschke et al., 1998) and tet-off (Bello et al., 1998) have been adapted to transgenic Drosophila, however only tet-on has so far been applied to aging studies. In the tet-on system, the ‘reverse tetracycline repressor’ (rtR) binds a specific DNA element, the ‘tetracycline operator’ (tetO), only in the presence of tetracycline, or derivatives such as doxycycline (DOX). Fusion of rtR to the transcriptional activation domain of herpes virus protein VP16 produced a hybrid eukaryotic transactivator protein,
Fig. 5. The ‘tet-on’ system.
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called ‘reverse tetracycline trans activator’ (rtTA). rtTA has previously been shown to allow DOX dependent transcription of transgenes linked to tetO sequences in mammals. To adapt tet-on to Drosophila (Bieschke et al., 1998), the actin5C promoter was used to drive constitutive expression of rtTA in transgenic flies (Fig. 5A). Reporter constructs encoding E. coli b-galactosidase (b-gal), were also introduced into transgenic flies. The ‘7T40:lacZ’ reporter is diagrammed (Fig. 5B), in which seven tetO sequences are fused to the hsp70 core promoter, consisting of the ‘TATAA’ box plus the transcription initiation site. In transgenic Drosophila containing both the rtTA construct and a 7T40:lacZ reporter construct, DOX feeding produced up to 100-fold induction of b-gal. DOX induced b-gal expression in all tissues, in larvae and in young and senescent adults. As discussed above for FLP-out, a primary advantage of tet-on is that it is truly inducible, allowing temporal control of transgene expression and powerful controls for genetic background. The trigger for tet-on induction, DOX has no detectable effect on life span on its own. Importantly, there was no detectable effect on life span of abundant over-expression of E. coli b-gal. These results suggest that tet-on should be ideal for detecting specific effects of transgenes on aging, even quite small effects. Disadvantages of tet-on are that it has not yet been used to increase life span, so its utility remains theoretical. The published tet-on system does not provide tissue specificity, however this has recently been achieved as described below.
9. The future As discussed above, each of the transgenic systems has distinct advantages and disadvantages that will be important in the design of future experiments, and it is likely that each of these systems will continue to be useful. Certain modifications or combinations of the systems are certain to be useful, and are underway in several laboratories. For example the FLP-out system could be made tissue-specific by using tissue-specific promoters to drive expression of the target construct (Fig. 4B and C). A combination of the UAS/Gal4 and tet-on systems should be particularly powerful and provide the advantages of each: creation of transgenic constructs with UAS sequences regulating expression of rtTA would create a three-component system. GAL4 drivers (Fig. 3A) would drive expression of rtTA (Fig. 3B) in desired patterns. In the presence of DOX the rtTA would be activated and drive expression of transgenes of interest, as in Fig. 5B. In this way truly inducible, tissue and temporal specific expression would be achieved, providing the advantages of each system. Such a system has recently been shown to work as expected (M. Stebbins and J. Yin, MS in preparation). The successful extension of life span with transgenes in Drosophila and in C. elegans (Murakami and Johnson, 1998) represents a breakthrough in aging research. This success presents numerous new questions and challenges for the design of transgenic experiments. For example, how do Drosophila hsp70 and SOD
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increase life span? Hsp70 has multiple functions, which is relevant? SOD over-expression could be decreasing oxidative damage as expected, but could also be altering signal transduction by altering levels of ROS signalling molecules. Are there trade-offs with life span extension, such as reduced reproductive capacity? Recent studies suggest that increasing life span with extra copies of the hsp70 gene is associated with reduced fecundity (Silbermann and Tatar, 2000). Will there be additional genes that can increase life span when over-expressed? It seems likely that the increasingly sophisticated transgenic systems and experimental designs available for Drosophila will allow these questions to be answered in the near future.
Acknowledgements Thank you to Raj Sohal, Bill Orr, Mark Tatar, Nadege Minois, and Michael Stebbins for helpful comments and for communicating unpublished results. Aging research in the author’s laboratory is supported by grants from the Department of Health and Human Services (AG11 833 and AG11 644).
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