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Short Technical Reports Green Fluorescent Protein Expressed in Living Mosquitoes— Without the Requirement of Transformation BioTechniques 21:660-664 (October 1996)

ABSTRACT Mosquitoes transmit viruses, protozoa and nematodes that are major causes of morbidity and mortality in humans. Details of arthropod anatomy and development, and the replication and development of pathogens in the arthropod vector, have relied upon examination of dissected or histologically processed material. We constructed a double-subgenomic Sindbis (dsSIN) virus expressing green fluorescent protein to demonstrate the potential of this protein for studying pathogen development in living arthropods. We were able to observe dissemination of virus, and furthermore, it was possible to observe components of the nervous system of mosquito larvae in extraordinary detail and record this on video tape. Although green fluorescent protein has been used as a reporter gene in a number of organisms, expression has relied upon transformation of cells or embryos. Transformation technology has limited applicability, thus we have described an alternative system that, due to the broad host range and viral tropisms of dsSIN viruses, may be useful to scientists in a range of disciplines. Green fluorescent protein may also provide a nonlethal selection method for use in transgenic arthropod research.

ploited to identify genes that have the potential to disrupt vector competence in transgenic mosquitoes (4,23,25). Recently, dsSIN viruses have been used to interfere with bunyavirus and flavivirus replication and transmission in vivo (23,25). In these studies, viral replication and gene expression could only be determined in killed and processed mosquitoes. Our knowledge of viral replication and tropisms in arthropod vectors is therefore primarily based upon techniques such as immunofluorescent or immunohistochemical staining of fixed tissues, which can destroy viral epitopes and tissue integrity. To determine if viral replication and protein expression could be visualized in living mosquitoes, the gene encoding green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria (26), was cloned into the infectious SIN expression system (Figure 1). GFP is a 27-kDa monomer of 238 amino acids and emits green light (λmax = 509) when excited with UV light (λmax = 395). Since GFP fluoresces without the necessity of cofactors, sub-

strates or co-expressed genes, it can be visualized in situ in living organisms. GFP has been used as a reporter gene under the control of a neuron-specific promoter in transgenic Caenorhabditis elegans (5) to monitor the formation of neuronal processes in the developing nematode and to locate Exu protein during Drosophila melanogater oogenesis (31). More recently, GFP has been used as a reporter of successful transformation in studies of zebra fish (1) and mice (22). MATERIALS AND METHODS Construction of Expression Vector and Production of Virus Plasmid pTE/3′2J/GFP was constructed from the pTE3′2J (9) using standard procedures (29). Unless otherwise stated, reagents were obtained from Promega (Madison, WI, USA) or Sigma Chemical (St. Louis, MO, USA). An XbaI/SpeI fragment from pGFP encoding the GFP protein (CLONTECH Laboratories, Palo Alto,

INTRODUCTION An infectious expression system based upon the alphavirus Sindbis (7,9) has proven utility in the expression of genes in mosquitoes (12–14,23–25,27). An additional promoter and multiple cloning sites, immediately downstream of the structural genes, facilitate the expression of an introduced gene (9). Genomic RNA and two subgenomic RNAs are therefore produced, rather than genomic and a single-subgenomic RNA, as is typical for a wild-type Sindbis virus (12). This double-subgenomic Sindbis (dsSIN) system may be ex660 BioTechniques

Figure 1. Map of pTE3′′2J/GFP. Vol. 21, No. 4 (1996)

CA, USA) was cloned into the XbaI site of pTE3′2J. pTE3′2J was treated with calf alkaline phosphatase before ligation. pTE/3′2J/GFP was linearized with XhoI to produce DNA templates for run-off transcription with SP6 polymerase. RNA was synthesized for 2 h at 37°C using a mMessage mMachine kit (Ambion, Austin, TX, USA) without additional GTP. The resulting capped RNA was stored at -70°C in transcription buffer until needed. RNA was transfected by electroporation as previously described (16). Briefly, BHK-21 cells were trypsinized, washed once in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and twice in icecold phosphate-buffered saline (PBS), pH 7.4, and then resuspended in PBS to 1 × 107 cells/mL. For electroporation, 5 µg of each RNA, 2 µL of RNasin (2.5 mM final concentration) were mixed

with 500 µL of cell suspension and placed in 0.2-cm cuvettes. Cells were then immediately pulsed at room temperature in a Gene Pulser apparatus (Bio-Rad, Hercules, CA, USA) with two consecutive pulses at 1.5 kV, 25 mF, with resistance set to infinity (time constant 0.7–1.0 ms). Cells were rested for 10 min at room temperature and were then diluted 1:20 in DMEM with 10% FBS and transferred to a 75-cm2 flask (Falcon; Becton Dickinson, Franklin Lakes, NJ, USA). Following incubation at 37°C for 24–48 h, TE/ 3′2J/GFP virus was harvested in tissue culture fluid from infected cells. Supernatant was removed, centrifuged (800× g, 5 min) and filtered through a 0.45µm cellulose acetate filter. The production of GFP by TE/3′2J/GFP virus was confirmed by infecting BHK- 21 cells grown to confluence on glass coverslips (8), and examination for fluores-

Figure 2. GFP expressed in stages of Ae. aegypti mosquitoes. (A) Comparison of control uninfected (upper) and infected (lower) larvae. (B) Ventral nerve chord and thoracic ganglia in larva. (C) Nerve fibres in larva. (D) Eye of pupa. (E) Comparison of control uninfected adult (center) with infected male (left) and female (right). (F) Common oviduct from dissected adult. Vol. 21, No. 4 (1996)

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Short Technical Reports cence using a BH-2 epifluorescence microscope (Olympus, Lake Success, NY, USA) with fluorescein isothiocyanate (FITC) filters (data not shown). Infection of Mosquitoes For arthropod studies, Aedes aegypti mosquitoes (a white-eyed strain) were inoculated (28) with 0.5–1.0 µL of TE/3′2J/GFP virus (tissue culture infectious dose 50% endpoint [TCID50] titer 7 log10/mL) either as larvae or adults. Viral persistence and replication in vivo was further demonstrated by titration of triturated insects on mammalian cell culture. Within 3 days postinoculation, TCID50 titers increased to 5.5 log10/mosquito. Observation of GFP Inoculated larvae, and adults emerging from these, were examined at various time points post-inoculation using an IMT-2 microscope (Olympus) with FITC filters for transmitted and epifluorescence. By attaching a VPC-920 1/2″ (1.27 cm) color CCD camera (5 Lux sensitivity), Panasonic AG-1960 video recorder and Sony PVM-1343MD monitor (S & M Microscopes, Colorado Springs, CO, USA), tissues in which GFP was expressed could be recorded. This relatively inexpensive system allowed nerves to be traced in living, moving larvae and to be recorded in real-time (data not shown). RESULTS AND DISCUSSION Within 48 h post-inoculation, fluorescence was observed in tissues of live larvae (Figure 2). Nervous tissues (the ventral nerve chord, brain and fine nerve fibers) were clearly visible, with dissemination into fat body tissues occurring with time. The virus survived metamorphosis; fluorescence was apparent in the head of pupae and was seen through the eyes and in other tissues, including those of the nervous and occasionally reproductive systems of the emerged adults (Figure 2). Since widespread fluorescence can hinder visualization of fine detail, we inoculated a noninfectious SIN replicon (14) expressing GFP. GFP was visible in the tissues (principally the nerve chord) of inoculated larvae but did not 662 BioTechniques

disseminate from those cells initially infected (data not shown). These results are consistent with previous observations (14). We have previously described the neurotropism of dsSIN viruses (27), which may be due to the E1 and E2 genes derivation from a neurovirulent strain of SIN (17). The ability to observe viral gene expression without specimen processing represents a significant contribution to the study of viral infections in arthropod vectors. Since SIN viruses may replicate in a variety of insect species (15,30), and since the virus is able to survive metamorphosis, TE/3′2J/GFP may be suitable to study arthropod development. TE/3′2J/GFP replicates and expresses GFP in tissues of Culex pipiens, Anopheles gambiae (data not shown) and in D. melanogaster (A. Raymes-Keller, unpublished). Since SIN also replicates in vertebrates, TE/3′2J/GFP may also be useful for studies in higher organisms. With the development of improved GFP (6,11), the usefulness of GFP in a replicating expression system can be extended. As our ability develops to genetically engineer organisms such as Aedes densonucleosis virus (4), Trypanosoma cruzi (10), Rhodococcus rhodnii (2), Rickettsia-like organisms (3) and other pathogens of or transmitted by arthropods, GFP can facilitate studies of these organisms and of their insect hosts in a manner never before possible. A common problem associated with the generation of transgenic mosquitoes (19–21) is the differentiation of transformed and wild-type individuals. Genes encoding antibiotic resistence have been used exclusively as reporter genes. Variation in expression levels of the introduced gene and in the intraspecific and interspecific tolerances of wild-type mosquitoes to toxins means that the screening method may be relatively inefficient. Screening larvae requires the use of aqueous toxins and results in the generation of considerable volumes of waste. Our data demonstrates that GFP may be a useful marker for rapid and efficient screening of transgenic mosquitoes (4), and we are currently working to optimize the expression of GFP in systems such as the Aedes densonucleosis virus (4) and retroviruses (18) that have the potential

for DNA-based expression in mosquito cells and mosquitoes. REFERENCES 1.Amsterdam, A., S. Lin and N. Hopkins. 1995. Transient and transgenic expression of green fluorescent protein (GFP) in living zebrafish embryos. CLONTECHniques X:30. 2.Beard, C.B., P.W. Mason, S. Aksoy, R.B. Tesh and F.F. Richards. 1992. Transformation of an insect symbiont and expression of a foreign gene in the Chagas’ disease vector Rhodnius prolixus. Am. J. Trop. Med. Hyg. 46:195-200. 3.Beard, C.B., S.L. O’Neill, P. Mason, L. Mandelco, C.R. Woese, R.B. Tesh, F.F. Richards and S. Aksoy. 1993. Genetic transformation and phylogeny of symbionts from tsetse. Insect Mol. Biol. 1:123-131. 4.Carlson, J., K.E. Olson, S. Higgs and B.J. Beaty. 1995. Molecular genetic manipulation of mosquito vectors. Annu. Rev. Entomol. 40:359-388. 5.Chalfie, M., Y. Ti, G. Euskirchen, W.W. Ward and D.C. Prasher. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802-805. 6.Delagrave, S., R.E. Hawtin, C.M. Silva, M.M. Yang and D.C. Youvan. 1995. Redshifted excitation mutants of the green fluorescent protein. Bio/Technology 13:151-154. 7.Geigenmuller-Gnirke, U., B. Weiss, R. Wright and S. Schlesinger. 1991. Complementation between Sindbis viral RNAs produces infectious particles with bipartite genome. Proc. Natl. Acad. Sci. USA 88:32533257. 8.Gould, E.A., A. Buckley and N. Cammack. 1985. Use of a biotin-streptavidin interaction to improve flavivirus detection by immunofluorescence and ELISA tests. J. Virol. Methods 11:41-48. 9.Hahn, C.S., Y.S. Hahn, T.J. Braciale and C.M. Rice. 1992. Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. Proc. Natl. Acad. Sci. USA 89:2679-2683. 10.Hariharan, S., J. Ajioka and J. Swindle. 1993. Stable transformation of Trypanosoma cruzi: inactivation of the PUB12.5 polyubiquitin gene by targeted gene disruption. Mol. Biochem. Parasitol. 57:15-30. 11.Helm, R., A.B. Cubbit and R.Y. Tsien. 1995. Improved green fluorescent protein. Nature 373:663-664. 12.Higgs, S., A.M. Powers and K.E. Olson. 1993. Alphavirus expression systems: applications to mosquito vector studies. Parasitology Today 9:444-452. 13.Higgs, S., K.E. Olson, L. Klimowski, A.M. Powers, J.O. Carlson, R.D. Possee and B.J. Beaty. 1995. Mosquito sensitivity to a scorpion neurotoxin expressed using an infectious Sindbis virus vector. Insect Mol. Biol. 4:97103. 14.Kamrud, K.I., A.M. Powers, S. Higgs, K.E. Olson, C.D. Blair, J.O. Carlson and B.J. Beaty. 1995. The expression of chloramphenicol acetyltransferase in mosquitoes and mosquito cells using a packaged Sindbis replicon Vol. 21, No. 4 (1996)

system. Exp. Parasitol. 81:395-403. 15.Karabatsos, N. (Ed.). 1985. International Catalogue of Arboviruses, 3rd ed. Am. Soc. Trop. Med. Hyg. San Antonio, TX. 16.Liljestrom, P. and H. Garoff. 1991. A new generation of animal expression vectors based on the Semliki forest virus replicon. Bio/Technology 9:1356-1361. 17.Lustig, S., A.C. Jackson, C.S. Hahn, D.E. Griffin, E.G. Strauss and J.H. Strauss. 1988. Molecular basis of Sindbis virus neurovirulence in mice. J. Virol. 62:2329-2336. 18.Matsubara, T., R.W. Beeman, N. Besansky, O. Mukabayire, S. Higgs, A.A. James and J.C. Burns. 1996. Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae. Proc. Natl. Acad. Sci. USA 93:6181-6185. 19.Mc Grane, V., J.O. Carlson, B.R. Miller and B.J. Beaty. 1988. Microinjection of DNA into Aedes triseriatus ova and detection of integration. Am. J. Trop. Med. Hyg. 39:501-510. 20.Miller, L.H., R.K. Sakai, P. Romans, R.W. Gwadz, P. Kantoff and H.G. Coon. 1987. Stable integration and expression of a bacterial gene in the mosquito Anopheles gambiae. Science 237:779-781. 21.Morris, A.C., P. Eggleston and J.M. Crampton. 1989. Genetic transformation of mosquito Aedes aegypti by microinjection of DNA. Med. Vet. Entomol. 3:1-7. 22.Okabe, M. and I. Masahito. 1996. Generation of GFP transgenic mice. CLONTECHniques XI:21. 23.Olson, K.E., S. Higgs, P.A. Gaines, A.M. Powers, B.S. Davis, K.I. Kamrud, J.O. Carlson, C.D. Blair and B.J. Beaty. 1996. Genetically engineered resistance in mosquitoes to dengue virus transmission. Science 272:884-886. 24.Olson, K., S. Higgs, C.S. Hahn, C.M. Rice, J.O. Carlson and B.J. Beaty. 1994. Expression of chloramphenicol acetyltransferase in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes using double subgenomic recombinant Sindbis virus vectors. Insect Biochem. Mol. Biol. 24:39-48. 25.Powers, A.M., K.I. Kamrud, K.E. Olson, S. Higgs, J.O. Carlson and B.J. Beaty. 1996. Molecularly engineered resistance to California serogroup virus replication in mosquito cells and mosquitoes. Proc. Natl. Acad. Sci. USA 93:4187-4191. 26.Prasher, D.C., V.K. Eckenrode, W.W. Ward, F.G. Prendergast and M.J. Cormier. 1992. Primary structure of the Aequorea victoria green fluorescent protein. Gene 111:229- 233. 27.Rayms-Keller, A., A.M. Powers, S. Higgs, K.E. Olson, K.I. Kamrud, J.O. Carlson and B.J. Beaty. 1995. Replication and expression of a recombinant Sindbis virus in mosquitoes. Insect Mol. Biol. 4:245-251. 28.Rosen, L. and D. Gubler. 1974. The use of mosquitoes to detect and propagate dengue viruses. Am. J. Trop. Med. Hyg. 23:11531160. 29.Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 30.Schiefer, B.A. and J.R. Smith. 1974. Comparative susceptibility of eight mosquito Vol. 21, No. 4 (1996)

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Short Technical Reports species to Sindbis virus. Am. J. Trop. Med. Hyg. 23:131-134. 31.Wang, S. and T. Hazelrigg. 1994. Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 369:400-403.

Fluorometric Assay for DNA Polymerases and Reverse Transcriptase

This work was supported by Grant Nos. AI-37671, AI-28781 and AI-34014 from the NIH, by a grant from the John D. and Catherine T. MacArthur Foundation and by a CRC grant from Colorado State University. Anopheles gambiae mosquitoes were kindly supplied by Dr. F.H. Collins (Centers for Disease Control and Prevention, Atlanta, GA). Address correspondence to Stephen Higgs, The Arthropod-borne and Infectious Diseases Laboratory, Foothills Campus, Colorado State University, Fort Collins, CO, 80523-1682 USA. Internet: shiggs@ vines.colostate.edu

BioTechniques 21:664-672 (October 1996)

Received 15 March 1996; accepted 21 May 1996.

S. Higgs, D. Traul, B.S. Davis, K.I. Kamrud, C.L. Wilcox and B.J. Beaty Colorado State University Fort Collins, CO, USA

ABSTRACT We report a quick, easy and inexpensive fluorometric assay that measures the activity of replication enzymes using PicoGreen. The systems tested include replication of the natural template M13 Gori by E. coli DNA polymerase III holoenzyme and the replication of a synthetic homopolymer by human immunodeficiency virus reverse transcriptase. A direct comparison of the fluorometric assay with the conventional isotopic assay shows that the fluorometric assay accurately reflects the extent of replication. By performing the assay reactions directly in 96-well plates and using a fluorescence plate reader to determine the extent of reaction, the time required to measure replication activities is significantly shortened.

INTRODUCTION Conventional assays of polymerase activity involve measuring the extent of incorporation of radiolabeled nucleotides into acid-insoluble polynucleotides (5,6,8). Such procedures are cumbersome and time-consuming because of the need to separate incorporated and free nucleotides by a series of filtration and washing steps. Several spectroscopic assays have been introduced recently. These include a reverse transcriptase (RT) assay that uses 4′,6-diamidino-2-phenylindole, the fluorescence of which is enhanced by binding to poly(A)·poly(dT) during the course of the reaction (1). The fluorescence of this dye is unchanged in the presence of double-stranded DNA (dsDNA) and, therefore, cannot be used to assay conventional DNA-dependent DNA polymerases (1). To detect transient intermediates in the reactions catalyzed by E. coli DNA polymerase I and T4 DNA polymerase, Frey et al. (3) have used changes in the fluorescence of the base analog 2aminopurine upon incorporation into

DNA. In a different approach, Griep (4) has recently described a fluorometric assay for E. coli DNA polymerase III holoenzyme based on changes in the fluorescence of single-stranded (ss) DNA-binding protein (SSB) during DNA replication. However, this assay is limited to systems requiring SSB. The fluorescence of the recently introduced dye PicoGreen is enhanced specifically upon binding to dsDNA, and it is relatively nonfluorescent in the presence of ssDNA. Here, we report a quick, easy, sensitive and inexpensive fluorometric assay, utilizing PicoGreen, that can be used to monitor the synthesis of both dsDNA and DNA:RNA hybrids and is, therefore, applicable to a wide range of DNA polymerases and reverse transcriptases. MATERIALS AND METHODS E. coli DNA polymerase III holoenzyme (pol III holoenzyme), human immunodeficiency virus (HIV) RT, dnaG primase, SSB and M13 Gori DNA are commercially available from Enzyco (Denver, CO, USA). PicoGreen was obtained from Molecular Probes (Eugene, OR, USA). Synthetic polynucleotides were obtained from Pharmacia Biotech (Piscataway, NJ, USA). Fluorescence Instrumentation Fluorescence measurements were made using either an SLM Model 48 000 fluorometer (SLM Instruments, Urbana, IL, USA) or an SLT Fluorostar microplate fluorometer (SLT Labinstruments, Research Triangle Park, NC, USA). The SLM fluorometer was configured with excitation and emission wavelengths of 500 and 526 nm, respectively. The slit widths were varied between 4 and 16 nm. All measurements were performed at room temperature except where noted. The SLT microplate fluorometer was equipped with 485 and 538 nm interference filters on excitation and emission, respectively. Solutions Pol III holoenzyme primer-template solution: 60 mM HEPES, pH 7.5, 14 mM magnesium acetate, 2.8 mM ATP, GTP, CTP and UTP, 14% Vol. 21, No. 4 (1996)