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Keywords:

  • Caenorhabditis elegans ;
  • gene induction;
  • heat shock promoter;
  • infrared laser;
  • infrared laser-evoked gene operator

Abstract

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

Since the dawn of transgenic technology some 40 years ago, biologists have sought ways to manipulate, at their discretion, the expression of particular genes of interest in living organisms. The infrared laser-evoked gene operator (IR-LEGO) is a recently developed system for inducing gene expression in living organisms in a targeted fashion. It exploits the highly efficient capacity of an infrared laser for heating cells, to provide a high level of gene expression driven by heat-inducible promoters. By irradiating living specimens with a laser under a microscope, heat shock responses can be induced in individual cells, thereby inducing a particular gene, under the control of a heat shock promoter, in specifically targeted cells. In this review we first summarize previous attempts to drive transgene expression in organisms by using heat shock promoters, and then introduce the basic principle of the IR-LEGO system, and its applications.


Induction of gene expression in targeted cells

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

For studying the development of multicellular organisms, methods for turning on or off the expression a particular gene in any target cell at the discretion of experimenters would be very useful. Methods for induction of gene expression in targeted cells at a desired time would also offer possible benefits for clinical applications such as gene therapy, in which tight control of gene expression in terms of location, timing, and degree is required.

One method of increasing specificity when targeting gene expression is to spatially and temporally control the delivery of transgenes. Various efforts have been made to accomplish such control, including chemical modification of vehicles for gene transfer (Nishikawa 2005), elaboration of electroporation methods (Fukuichi-Shimogori & Grove 2001; Nakamura 2009), and localized perforation of target cells with femtosecond laser pulses for transfection (Tirlapur & Konig 2002). Another method for achieving gene expression in a desired pattern is to control the expression of transgenes by using defined promoter or enhancer sequences. A variety of specific promoters for different cell-types is now available, enabling us to express transgenes in various spatio-temporal patterns. Promoters suited for a particular research purpose, however, may not be always available. In addition, since such promoters drive gene expression in multiple cells of the same cell type, they cannot be used for gene expression in single targeted cells.

Inducible promoters, which enable temporal control of gene expression, are also used to drive transgene expression. Small molecules that can permeate cells are sometimes used as inducers. Heavy metal ions stimulate gene expression under the control of the metallothionine gene (Palmiter et al. 1983; Palmiter 1998). Tetracycline or its derivatives are used as inducers for Tet-On/Tet-Off gene expression system (Clackson 1997; Roman et al. 2001). A light-switchable gene promoter system has been also reported (Shimizu-Sato et al. 2002). Among the most frequently used inducible promoters in a wide variety of organisms are those of heat shock genes (Fig. 1a).

image

Figure 1. Laser-mediated gene induction (a) A heat shock promoter-driven transgene. (b) Scheme of laser-mediated gene induction in a single targeted cell.

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Provided that targetable external stimuli are available to drive an inducible promoter, transgene expression in selected cells at a desired time can be achieved. With heat shock promoters, attempts have been made to deposit heat locally to induce gene expression in selected tissues or cells in vivo. Various non-invasive methods have been reported, including heated needle tips (Monsma et al. 1988), focused ultrasound (Rome et al. 2005), and microwave radiation (Daniells et al. 1998). Irradiation with a laser microbeam is an attractive means for delivering heat locally, since the high spatial resolution in targeting allows single cells to be selectively heated in living organisms (Fig. 1b). In the following sections, we briefly review the heat shock response and previous studies on laser-mediated heat shock response for inducing gene expression.

Heat shock response

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

Heat shock response is a widely conserved cellular defense mechanism. When cells are challenged by exposure to extreme conditions causing acute or chronic stress, proteins can be denatured, as they unfold, misfold, or aggregate, and thereby be made to fail to properly execute their functions. Molecular chaperones and proteases encoded by conserved heat shock genes assist in the refolding and repairing of damaged proteins (Morimoto 1998; Feder & Hofmann 1999). When cells are subjected to harmful environmental changes such as extreme heat, the transcription of heat shock genes is initiated to mitigate the effects. The heat shock response can be induced by a variety of environmental stresses other than heat shock, such as the presence of oxidants, transition heavy metals, and energy metabolism inhibitors. Pathophysiological states, that is, fever, inflammation, hypertrophy, ischemia, and hypoxia can also act as heat shock response inducers.

Transcription of heat shock genes requires activation of a heat shock factor (HSF) that binds to the heat shock promoter element (HSE) in DNA. Stress exposure activates HSF, forming a homotrimer that has DNA-binding activity, which then acquires transcription activity through a sequence of steps. HSF first undergoes relocalization to the nucleus, where it acquires DNA-binding competence but remains transcriptionally inert until it is endowed with transcription activity by stress-inducible phosphorylation.

A heat shock response is triggered quickly and persists for hours. In a nematode, Caenorhabditis elegans, the level of mRNA of the heat shock gene hsp16-2 reached a maximum 1 h after the onset of heat shock, and then decreased to a normal level within 2 h after heat shock was terminated (Jones et al. 1989). In a study using transgenic worms carrying the mab-5/Hox gene, driven by the promoter of the heat shock gene hsp16-1, MAB-5 protein was detected by immunohistological staining immediately following a 15-min heat induction (Salser & Kenyon 1996). The durability of transgene products induced by a heat shock treatment pulse in organisms differs for each protein. This most likely reflects the stability of the product rather than the nature of heat shock response. In the Salser and Kenyon report on MAB-5, high levels of staining persisted for about 4 h after a pulsed heat shock treatment of 15–30 min. We have observed that the fluorescence signal of green fluorescent protein (GFP) in C. elegans carrying hsp16-2::gfp disappears 24 h after heat shock, whereas that of GFP fused to the Arch proton pump can be detected up to 72 h after heat shock (Okazaki et al. 2012).

When performing heat shock-mediated gene induction experiments, we need to bear in mind the fact that many physiological processes are temperature dependent. In addition, heat shock proteins are known to play roles in signal transduction through association with signaling molecules such as v-Src, Raf1, Akt, and steroid receptors (Nollen & Morimoto 2002). Consequently, the induction of heat shock proteins by themselves may affect the behavior of induced cells by activating signal transduction pathways. In fact, too much or too little Hsp70 or Hsp60 in cells can result in aberrant growth control, developmental malformation, and cell death under certain circumstances (Feder et al. 1992; Rutherford & Lindquist 1998; Elefant & Palter 1999). It is known that heat shock activates several signaling pathways regulating a cell death program (Gabai & Sherman 2002). Therefore, when transgene induction experiments are carried out using heat shock promoters, it is important to check whether or not a phenotypic response of cells is due to the expression of a given transgene, by performing appropriate control experiments. Fortunately, previous experiments have shown that, in many cases, heat shock treatment itself does not affect the normal growth and development of cells. Thus, heat shock-induced gene expression is useful for examining the function of genes and for manipulation of cell behavior in developmental biology studies.

Laser-mediated gene induction with visible light

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

Previous attempts at laser-mediated induction of gene expression in living organisms have mainly been carried out using a microscope equipped with a Coumarin 440 dye laser, which was originally used for cell ablation experiments. The first tested organism was C. elegans (Stringham & Candido 1993). A block of glass microscope slides was placed in the laser path to reduce the beam intensity to sublethal levels, but energy output was not precisely determined. Irradiation was performed for a period of 3–10 min (Table 1). Under optimal conditions, induction of the E. coli lacZ gene, driven by the hsp16-1 promoter, was detected at a frequency of approximately 50% in single epidermal, intestinal, neuronal, pharyngeal, and muscle cells in larvae and adults. Subsequently, single cells in Drosophila embryos were targeted using a 0.02 μJ/pulse laser at 4–10 Hz, and marker gene expression occurred in nearly 50% of cases (Halfon et al. 1997). In their report, the authors also described a potential problem: background expression of marker genes that can occur without heat shock treatment, in fly strains carrying heat shock promoter-driven transgenes. The third tested organism was zebrafish (Halloran et al. 2000), where successful induction of the enhanced GFP (EGFP) marker was reported in 24–94% of irradiated cells, depending on cell type. Sema3A1, a repulsive type of guidance molecule, was ectopically induced in single muscle cells, thereby retarding the extension of motor axons. The authors described that targeted cells were undamaged and appeared to develop normally.

Table 1. Single cell gene induction with 440 nm laser irradiation
SpeciesPromoterInduced geneTargeted tissue/cellDurationSingle cell induction (%)References
Caenorhabditis elegans hsp16-1 lacZ Epidermal cell, intestinal cell, neuron, pharyngeal cell, muscle cell3–10 min50Stringham and Candido (1993)
hsp16-1 mab-5/Hox Q neuroblast ndHarris et al. (1996)
Drosophila hsp26 lacZ

Embryoic cells

(mucle fiber, central nervous system cell, aCC neuron)

75–120 s50Halfon et al. (1997)
Gal4hs lacZ, GFP Embryonic heart, somatic musculature 50 
Zebrafish hsp70-4 GFP, sema3A1 Tail bud progenitors, spinal neurons, muscle pioneers2 min24–94Halloran et al. (2000)

Despite the promising outcomes in these reports, there have been surprisingly few follow-up studies using dye-laser-mediated heat shock induction. A major problem when using Coumarin 440 dye lasers for gene induction is that the irradiation has apparently harmful effects on cells even when the laser intensity is reduced to sublethal levels. Another issue is that relatively long irradiation times are required. Harris et al. (1996) succeeded in rescuing cell migration defects in C. elegans mab-5 mutants by targeted induction of MAB-5 in single cells. The authors, however, mentioned that the laser intensity necessary for rescuing the defects had the side-effect of delaying or blocking the division of the irradiated cells. We attempted irradiation of C. elegans carrying hsp16-2::gfp with a Coumarin 440 dye laser, but could not determine suitable conditions in which gene expression could be sufficiently induced while avoiding cell damage (Kamei et al. 2009).

How visible laser light induces heat shock response

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

In their review of the Coumarin dye laser-mediated cell ablation technique, Bargmann and Avery (1995) wrote that “almost nothing is known about how laser energy destroys a C. elegans nucleus,” but they stated that “energy deposited in the irradiated nucleoplasm eventually takes the form of increased temperature and pressure, either of which can denature proteins [and] break DNA.” Laser light, however, can damage cells through not only thermal, but also mechanical and/or photochemical effects. Any of these three effects, or factors, can cause cellular stress leading to apoptotic death. Activation of a particular factor depends on the wavelength of the light and duration of exposure.

It was originally assumed that the use of Coumarin dye lasers induces a heat shock response by heating cells to a sublethal level. As described above, however, the heat shock response can be induced by a variety of stresses. Moreover, thermal effects may not play a major role in heat shock induction mediated by visible lasers, a possibility supported by a study by Leitz et al. (2002). To investigate potential risks when applying an optical tweezers technique in biology, they monitored heat shock response induced by near-infrared continuous-wave laser light (700, 760, 810 and 850 nm), using excretory cells of C. elegans carrying hsp16-1::lacZ as irradiation targets. They found no correlation between the frequency of marker gene expression and the degree of expected temperature rise associated with different wavelengths, and argued that the laser-mediated heat shock response is caused by photochemical processes when using light in the 700–760 nm range, whereas thermal effects predominate at 810 nm.

The precise amount of energy required for inducing heat shock response in C. elegans with 440-nm laser light is unknown. However, given that the absorption coefficient of water at 440 nm is about 100 times lower than that at 810 nm, it seems plausible that the Coumarin 440 dye laser induces a heat shock response mainly via photochemical effects.

IR- LEGO: a novel approach for laser-mediated gene induction

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

Water, a major constituent of cells, is almost perfectly transparent to light in the visible range, and thus is minimally heated when irradiated with light at visible wavelengths. In contrast, water shows strong absorption in the infrared region. Therefore, infrared light can raise the temperature of water, and thereby heat cells, much more efficiently than 440-nm radiation, for which water has a very low absorption coefficient (3 × 10−4). As described above, Leitz et al. (2002) suggested that the near infrared laser light of 810 nm induces a heat shock response via photothermal effects.

The absorption spectrum of liquid water has a notable peak at 1480 nm (with an absorption coefficient of 21), due to the combination of symmetric and antisymmetric O-H stretching modes (first overtone) of the water molecule. Taking advantage of this, Dr Shunsuke Yuba and his colleagues at the National Institute of Advanced Industrial Science and Technology (AIST) in Japan developed a microscope equipped with an infrared laser (1480 nm) irradiation system, called infrared laser-evoked gene operator (IR-LEGO), whose purpose is to heat individual cells in living organisms (Kamei et al. 2009; Fig. 2).

image

Figure 2. The infrared laser-evoked gene operator (IR-LEGO) system. (a) Our IR-LEGO system (a) consisting of a microscope (Olympus IX70, Japan) equipped with the infra red IR-laser (Model FRL-DC:1480 nm IRE-Polus, Russia). LD: laser driver, ShC: shutter controller. IR-LEGO is now commercially available as an add-on to existing microscopes (Sigma Koki, Saitama, Japan). (b) The optical system in the box. Sh: shutter, M: mirror, NDF: natural density filters. (c) The scheme of the optical path of the IR-LEGO system.

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The IR-LEGO heating profile, which was determined using a polyacrylamide gel tissue model, showed that the temperature at the irradiation focus changes very quickly in response to on and off switching of the IR light, and that the extent of temperature change is nearly proportional to the laser power, with a relationship of 1°C/mW (Kamei et al. 2009). Temperature shifts of approximately 30°C can be achieved at the irradiation focus, with negligible temperature changes at points 20 μm further away. In this research, the area undergoing a temperature shift in excess of 20°C was confined to a 7 μm-diameter area (xy axes) of the focal plane, sufficiently small that single cells in most organisms could be individually heated.

Application of IR-LEGO to C. elegans

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

We used the C. elegans organism to assess the utility of IR-LEGO in vivo (Kamei et al. 2009). This organism has several advantages for this purpose: the worm has a transparent body, well-characterized heat shock promoters are available, and efficient protocols for producing transgenic lines have been devised. Importantly, during development, it consists solely of identifiable somatic cells with stereotyped lineages, which enables accurate targeting and tracing of cells during, as well as following, irradiation.

To determine the optimum irradiation conditions for gene induction, we generated transgenic lines carrying hsp16-2::gfp and monitored GFP expression as an index of heat shock-induced gene expression. A standard heat shock treatment, bathing the transgenic worms in water at 30°C, caused GFP expression in nearly all cells, while the GFP expression was essentially nil in the absence of heat shock treatment (see also Stringham et al. 1992). Because transgenic lines with high background GFP expression could obscure the effect of irradiation, practical application of IR-LEGO depends on the appropriate choice of promoters and transgenic strains.

The effects of different irradiation conditions were examined by varying the irradiation intensity and duration. Epidermal seam cells, which are arranged in a row along the anterior-posterior axis on the lateral side of the worm body, were first used as irradiation targets (Fig. 3). GFP expression was not induced when the IR irradiation was either too weak or strong, or the duration too short or long. In the latter situations, the failure to induce GFP expression appeared to be due to cellular damage cause by heat, and some irradiated cells subsequently failed to undergo normal development. A surprising finding is that a heat shock response was induced by IR laser irradiation of <1 s, a much shorter duration compared with those reported in gene induction experiments when using a Coumarin 440 dye laser. Concerning laser power and irradiation duration, we observed that there is a range in which GFP expression can be induced without apparent damage to cells, with single targeted cells induced at a success rate of 40% (Table 2). Since seam cells next to the target are located on the order of 5–10 μm away from the focus, the induction of individual target cells confirms that the spread of heat is limited, at least in the xy directions.

Table 2. Gene induction by IR-LEGO
SpeciesPromoterInduced geneTargeted tissue/cellPower (mW)Duration (s)Repetition of irradiationSingle cell inductionReferences
  1. IR-LEGO, infrared laser-evoked gene operator; nd, not determined.

Caenorhabditis elegans hsp16-2 GFP Seam cell (epidermal cell)111 40%Kamei et al. (2009)

GFP

mab-5/Hox

V5/V6 descendant (epidermal cell)111 nd 
GFP ALM neuron120.5  nd 

GFP

unc-6/netrin

Body wall muscle cell110.25×459% 

GFP

mig-23/bHLH

Distal tip cells of gonad110.25×456% 

GFP

smp-1/semaphorin1

plx-1/plexinA

Vulva precursor cell (epidermal cell)130.25×440%Suzuki et al. (submitted)
Medaka hsp70.1 Venus Teeth, pineal gland, heart, skeletal muscles20–250.5–1 ndDeguchi et al. (2009)
Zebrafish hsp70 GFP, Kaede Single muscle, pronephric duct, notochord, retina201×3ndDeguchi et al. (2009)
A. thaliana hsp18.2 β-glucuronidase Singel cells in lateral root tips of seedling11–151–2 ndDeguchi et al. (2009)
image

Figure 3. Induction of GFP expression in single targeted cells of Caenorhabditis elegans by infrared laser-evoked gene operator (IR-LEGO). A larval seam cell (a), a body wall muscle cell (b) and distal tip cells (white triangle) (c) were targeted to induce expression of GFP. The insets show schematically the position of targeted cells in a worm. In (a), boundaries of seam cells are also visualized with AJM-1:GFP. Scale bar, 100 μm.

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Unfortunately, temperature shifts caused by IR-irradiation could not be determined directly in the cells. Using the frequency of marker gene induction as an index of temperature, we estimate a rate of 3°C/mW for the temperature shift in irradiated seam cells, three times higher than that determined using the acrylamide gel in vitro model. This discrepancy may stem from different heat dissipation conditions; in the acrylamide gel, where water can move freely, irradiation may generate strong convective currents, whereas in cells, where membrane systems restrict water movement, dissipation of heat by convective flow would be highly limited.

Induction of functional genes in C. elegans with IR-LEGO

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

The transient nature of heat shock promoter-mediated gene expression enables us to examine the timing that is critical for the action of a given gene. In C. elegans worms, mutants for the mig-24 gene encoding a bHLH-type transcription factor, migration of distal tip cells (DTC) of the gonad is arrested at the halfway point. IR-LEGO can be used to rescue this defect, by inducing wild-type MIG-24 in single DTCs in the early stage of migration, but not in the later stages (Kamei et al. 2009). In mab-5 mutants, where cell fate decision of larval epidermal cells is defective, IR-LEGO-mediated induction of wild-type mab-5 in single epidermal cells at the middle larval stage 2 (L2), but not at late L2, rescued the defect in a lineage-specific fashion (Kamei et al. 2009).

When studying the development of multicellular organisms, it is often necessary to determine whether a given gene acts in cells autonomously or not. Analysis of genetically mosaic organisms, in which mutant cells and wild-type cells are present in a mosaic arrangement, is a powerful method for investigating this question (Yochem & Herman 2005). Mosaic analysis, however, is often laborious and time consuming, because appropriate individuals must be selected for the analysis from haphazardly generated mosaic individuals. IR-LEGO can complement the conventional genetic technique in such cases, since it can efficiently generate a mosaic gene expression arrangement. IR-LEGO is also particularly useful when gene expression needs to be induced in specific targets among a population of homologous cells, which cannot be achieved by using cell-type-specific promoters. We have used IR-LEGO to analyze intricate interactions between epidermal cells mediated by the semaphorin-plexin signaling system in vivo (Suzuki, unpubl. data, 2010).

Application of IR-LEGO with organisms other than C. elegans

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

Infrared laser-evoked gene operator can be applied in studies of various transparent organisms for which transgenic technology is available and has already been used with certain species other than C. elegans (Deguchi et al. 2009; Table 2). The teeth, pineal gland, skeletal muscle, and heart cells of medaka fish (Oryzias latipes) larvae have been induced, as have single muscle cells, pronephric duct cells, notochord cells, and retinal cells in zebrafish (Danio rerio) embryos. In a higher plant (Arabidopsis thalina), single cells in the lateral root tips of seedlings have been induced. The authors of this paper have also reported that cells at a depth of 150 μm can be induced with the IR-LEGO system.

Future perspectives

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

Irradiation conditions

Up to now, a continuous irradiation procedure has been used for IR-LOGO-mediated gene induction. Although a variety of conditions can be tested by changing the duration and power of continuous irradiation, pulsed irradiation enables a much greater number of testable conditions, since more finely tuned heating is possible. More importantly, irradiation in brief pulses with sufficient time for dissipation of heat between pulses allows a more localized deposition of thermal energy. Bargmann and Avery (1995) discussed that, with irradiation times shorter than 70 ns, heat would not dissipate beyond the diameter of an irradiated spot with a radius of 200 nm. Accordingly, in laser killing experiments with C. elegans cells using a dye laser, irradiation with 0.2 ns pulses allowed more localized damage, with lower pulse energy, than when using a laser with a pulse length of 500 ns (Bargmann & Avery 1995). Ramos et al. (2006) also reported that pulsed irradiation with 532 nm laser light using an optical chopper reduced cell damage in the wings of butterfly pupae. In C. elegans, although a continuous IR-laser irradiation procedure can be used for gene induction of single cells with many cell types, it often induces gene expression in multiple cells when the target is in a densely packed cell cluster such as a ganglion. On the other hand, we found that gene expression can be efficiently induced in single neurons in the head ganglion using a pulsed irradiation procedure (Suzuki, Toyoda & Takagi, unpubl. data, 2011).

Combination with other tools

Heat shock promoter-mediated gene expression occurs only transiently. This can be a problem when using IR-LEGO in a situation where sustained expression of a gene is required. Another possible problem when using heat shock promoters is that the level of induced gene expression depends on, and is different for, each heat shock promoter and cannot be controlled at will. Particularly robust promoter activity may cause artificial effects by overexpression of a gene. To circumvent these problems, we developed an inducible sustained gene expression system by combining Cre/loxP in vivo recombination systems (Branda & Dymecki 2004) with IR-LEGO (Suzuki & Takagi, unpubl. data, 2009). In this system, a heat shock-mediated recombination activates gene expression under the control of a chosen promoter. This approach can be used to perform mutant rescue experiments in single cells by using the native promoter of the relevant gene. One application of this system is for lineage tracing of single cells, by triggering the expression of a marker protein driven by a constitutive promoter.

Heat shock promoters and heat shock factors

To regulate gene expression levels, the availability of arrays of heat shock promoters that have various strengths is desirable. Analysis using DNA microarrays has identified a variety of heat-inducible genes whose promoters may be useful (GuhaThakurta et al. 2002). In the same study, the authors identified a novel cis-regulatory element involved in the heat shock response. Such Information may be helpful for designing a cis-regulatory element and modulating the strength of gene induction in predictable ways.

Heat shock response can be also modulated through manipulation of factors involved in the response. Overexpression of a conserved protein called HSF binding protein 1 (HSBP1) can block the activation of a heat shock response in selected tissues in C. elegans (Satyal et al. 1998), and dominant negative forms of HSF (Voellmy 2005) can also be used to block the response. These molecules may be useful for blocking the induction of gene expression in cells where this is not desired.

Conclusions

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

Infrared laser-evoked gene operator has enabled new and powerful approaches in studies of multicellular organisms. It has already proven to be a useful research tool for studying development in animals and plants. In neurobiology, it can provide novel opportunities for analyzing neuron networks by allowing genetic manipulation of individual neurons. In combination with other techniques such as RNA-mediated gene suppression, IR-LEGO has the potential to become an even more powerful tool for gene manipulation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (S.T.). The authors would like to thank Shunsuke Yuba (AIST) and Yasuhiro Kamei (National Institute of Basic Biology, Japan) for generously introducing the IR-LEGO system to us and for our fruitful collaboration. We would also like to thank the members of the Oda Laboratory for their stimulating discussions. Additionally, we are grateful for the invaluable technical support provided by Hiromichi Ohmiya (Sigma Koki, Japan).

References

  1. Top of page
  2. Abstract
  3. Induction of gene expression in targeted cells
  4. Heat shock response
  5. Laser-mediated gene induction with visible light
  6. How visible laser light induces heat shock response
  7. IR- LEGO: a novel approach for laser-mediated gene induction
  8. Application of IR-LEGO to C. elegans
  9. Induction of functional genes in C. elegans with IR-LEGO
  10. Application of IR-LEGO with organisms other than C. elegans
  11. Future perspectives
  12. Conclusions
  13. Acknowledgments
  14. References
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