Reporter Gene Expression for Monitoring Gene Transfer Stephen Welsh∗ and Steve a Kay

617

Reporter gene expression for monitoring gene transfer Stephen Welsh∗ and Steve A Kay

The use of reporters such as green fluorescent protein (GFP) GFP has excitation peaks at 395 nm (largest peak) and and firefly luciferase permit highly sensitive and nondestructive 475 nm, and an emission peak at 509 nm with a small monitoring of gene transfer and expression. Modifications shoulder at 540 nm [5,6]. Reports from Prasher [6] describ- in GFP which increase intensity and thermostability, as well ing the cloning of Aequorea GFP, and Chalfie [7] showing as alter its spectral qualities, have facilitated the use of expression and fluorescence of GFP in a heterologous GFP in a variety of gene transfer methods. Improvements background (demonstrating that no exogenous substrates in imaging technologies and their increased application or cofactors were required to produce the active molecule), in biological research have allowed the expanded use of opened the door for use of GFP in a wide variety luciferase-based reporters in gene transformation, particularly of biological applications. GFP has been used in the in genetic screens and in monitoring temporal changes in measurement of gene expression, cell labeling, and in pro- gene expression. tein labeling localization studies [8••,9••,10,11•,12,13•,14]. GFP shows low toxicity, no interference with normal cellular activities, and is easy to assay (using fluorescence Addresses microscopy or fluorescence-activated cell sorting [FACS]). The Scripps Research Institute, Department of Cell Biology, Early modifications and uses of GFP in biological systems 10550 North Torrey Pines Road, La Jolla, CA 92037 USA • ∗e-mail: [email protected] have been reviewed elsewhere [5,6,15 ,16,17]; therefore, the focus of this section will be on the modification and Current Opinion in Biotechnology 1997, 8:617–622 use of GFP as a genetically encodable marker for use in http://biomednet.com/elecref/0958166900800617 monitoring gene transfer.  Current Biology Ltd ISSN 0958-1669 Modifications made in green fluorescent protein Abbreviations Modifications in GFP have been made using various CCD charge-coupled device FACS mutagenesis schemes. Mutants have been reported that fluorescence-activated cell sorting •• FRET fluorescence resonance energy transfer improve fluorescence intensity [18,19 ,20], thermostabil- GFP green fluorescent protein ity [21•], folding and formation of the chromophore [18], HER human epidermal growth factor receptor codon usage [22•,23], removal of cryptic intron sequences luc firefly luciferase [24••], and spectral qualities [19••]. The ability to combine RMGT retrovirus-mediated gene transfer these modifications in synthetic GFPs has led to many additive gains. The S65T mutant (amino acid single-letter code) is brighter and more resistant to photobleaching Introduction than wild-type GFP [19••]. Heim and co-workers [8••,19••] Gene transfer in its most useful sense must not only mutagenized GFP to generate a bright blue fluorescent include monitoring the successful transfer of desired genes protein which contains the T66H and T145F amino acid but also the establishment of the proper and predictable changes. Spectral variants (e.g. different colored GFPs) pattern of transgene expression. Detection and screening permit the simultaneous detection of expression from of such transformants can often pose serious challenges [1]. multiple reporters, tracking the transport and localization The use of reporters based on firefly luciferase (luc) and of more than one protein, and use of fluorescence green fluorescent protein (GFP), which allow transgene resonance energy transfer (FRET) to detect in vivo expression to be sensitively and noninvasively measured, protein–protein interactions [5]. Essentially one could is greatly facilitating gene transfer technology. Improve- monitor several cellular events at once in a noninvasive ments in the GFP molecule have increased its use in a manner in a living cell. The application of FRET in cells variety of gene transfer scenarios. The highly sensitive has recently been used to demonstrate the dimerization of nature of the luc assay, with the increasing availability the pituitary-specific transcription factor Pit-1, using GFP of detection and imaging technology, has made luciferase and BFP fusions [25••]. the reporter of choice in many transformation strategies. These versatile reporters complement other well validated Retrovirus-mediated gene transfer β reporter systems such as -galactosidase, secreted alka- GFP is rapidly gaining use in retrovirus-mediated gene line phosphatase, chloramphenicol acetyltransferase, and transfer (RMGT) into mammalian cells, including tumor β -glucuronidase [2–4], by allowing accurate, continuous cell lines [26•,27]. Using a humanized (for codon usage) monitoring of gene expression in living tissues. red-shifted mutant (S65T) version of GFP and FACS, Levy et al. [26•] showed efficient RMGT in mammalian Green fluorescent protein cells. Both A375 and PA317 cells expressing the vector The GFP of Aequorea, a 238 amino acid polypeptide, is were rapidly and easily identified, and showed no highly fluorescent and stable in many assay conditions [5]. deleterious effects. The visualization of gene expression 618 Expression systems

in living tissues could become a powerful methodology Subramanian and Srienc [36•] performed quantitative in the evaluation of gene transfer in clinical trials. analysis of transient gene expression in single mammalian Zolotukhin et al. [22•] employed a humanized S65T cells to determine the ability of GFP to act as a GFP (92 base substitutions in 88 codons) combined with quantitative reporter. They found that green fluorescence a series of adeno-associated virus vectors, and showed is a quantitative measure of GFP in single cells. GFP successful retroviral transduction and expression of GFP has also been used in dicistronic expression cassettes in human 293 cells and neurosensory cells of the guinea (where GFP and the gene of interest are under the pig eye. Single integrated copies of virus-driven GFP control of the same promoter) to screen and select for cells were detectable. Fluorescence intensity levels for the expressing inducible gene products. Mosser et al. [37•] humanized GFP were 45-fold that of wild-type GFP as used GFP reporters to select tetracycline-regulated cells assayed by FACS. Bierhuizen et al. [28] used several from a mixed population of cells. A dicistronic cassette was variants of GFP to show their applicability as selectable used which incorporated a viral internal ribosome entry or screenable markers in RMGT and their expression site plasmid, encoding both GFP and the gene of interest. in primary hematopoietic cells. The positive phenotype Cells expressing GFP expressed the cotransfected gene selected by FACS provided greater than 90% pure and and minimized screening procedures for gene transfer and viable populations of transduced hematopoietic lines, with expression. the phenotype being stable for at least one month. Muldoon et al. [29] using murine replication-defective Green fluorescent protein in plants retroviral vectors and humanized GFP-S65T mutant [22•] GFP has been used extensively in plant systems, in showed that almost 100% of the selected cells were localization studies and as a screenable marker for gene GFP-positive. Detection of HIV-1 infection in HeLa cells transfer [9••,38••,39,40]. Some plant species (e.g. Arabidop- using the S65T mutant has also been reported by Dorsky sis) show little or no expression of GFP fluorescence as et al. [30]. a result of aberrant splicing of the GFP message [24••]. Modification of GFP to remove a cryptic intron site (a Baculovirus and yeast sequence recognised as an intron in Arabidopsis, leading to Baculovirus expression systems have gained wide popular- aberrant mRNA) resulted in successful detection of GFP ity for expressing genes from a variety of eukaryotes [31]. fluorescence [15••,24••]. Improvements in GFP to enhance Wilson et al. [32] describe a system utilizing novel its use in plants, including fluorescent signal and codon GFP baculovirus expression vectors that allow rapid usage, have been made by several groups [20,41]. Chiu identification of recombinant baculoviruses. Constructs et al. [42•], using the S65T mutant, showed 20-fold higher permit the gene of interest to be cloned in-frame with expression in maize leaf cells, and detected GFP driven the GFP open reading frame, and plaques expressing by weak promoters in a broad range of plant hosts. Sheen the fusion protein can be readily identified by exposure and colleagues [43] have shown that GFP can be used to UV light. FACS analysis of yeast cells transformed as an efficient marker in flow cytometry sorting of plant with a yeast-enhanced (for codon usage) GFP mutant protoplasts for selection of positive clones. (yEGFP3) allows easy quantification of positive cells. The yEGFP3 fluorescence intensity is 75-fold higher than that By combining several modifications one can produce of wild-type GFP and can be detected at the single cell highly optimized versions of GFP that lead to cumulative level [33]. gains in GFP expression and utility. Haseloff et al. [24••] combined several changes in GFP that improve its func- Mammalian systems tion. Modification of codon usage ensures proper mRNA Takada et al. [34••] employed the S65T GFP with processing and removal of the cryptic intron. Amino the cytomegalovirus immediate-early enhancer and the acid substitutions for improved thermostability, folding, elongation factor 1 promoter to drive expression of S65T enhanced fluorescence, and altered spectral properties GFP in pre-implantation murine and bovine embryos. are useful for work in plants and in targeting to the Using confocal laser scanning microscopy it was possible to endoplasmic reticulum. The latter aspect was important detect S65T GFP positive cells at the morula and hatched in the recovery of transgenic plants from the most highly blastocyst stages. Eight fetuses and four live-born mice fluorescent cells. This recovery had been problematic were obtained from 55 S65T GFP-positive blastocysts. previously, most likely due to GFP toxicity (not normally PCR analysis demonstrated that 11 of the 12 mice were a problem at lower levels of expression). The use of GFP transgenic. This method could increase the production of has the potential to improve monitoring of both gene transgenic mice, combined with flow cytometry selection transfer and expression in plants, especially with the ease of GFP positive cells. Chiocchetti et al. [35] used GFP, (no addition of substrate, easy assay) and noninvasive under the control of the human hemopexin and mouse nature of the assay. β1 integrin promoter, as a marker for gene expression in transgenic mice. GFP proved to be a sensitive marker for Luciferase detecting expression in targeted tissues at the single cell Firefly luciferase offers features that make it useful as level. a marker for in vivo gene transfer. Luc, a monomer, Reporter gene expression for monitoring gene transfer Welsh and Kay 619

shows little toxicity or other deleterious effects on normal detection of hormonal regulation of gene expression from cellular metabolism, is conveniently extracted from plant relatively weak promoters in single cells. Frawley and and animal tissue, and is easily assayed by using a co-workers [52], using photon counting imaging of a luminometer or scintillation counter. The sensitivity [2], pituitary prolactin promoter–luc fusion in lactotrope cells, wide linear range (eight orders of magnitude), extremely showed a large degree of heterogeneity in the basal low background, and relative ease of the luciferase levels of pituitary prolactin gene expression in single assay has made it attractive to many researchers [2,4]. cells. This heterogeneity could provide a molecular basis The rapid turnover of luc activity has been useful in for the observed dynamic nature of lactotrope function. studying temporal gene expression [44••]. The ability to Kost et al. [53] analyzed luciferase activity in single identify transformants with desired expression patterns in tobacco protoplasts and small microcalli using a cooled, a sensitive and nondestructive assay makes luciferase a slow-scan CCD camera [53]. Transgenic tobacco lines powerful tool for monitoring in vivo gene transfer. These were eventually established from these single luminescent properties have also been used to develop novel genetic clones. screens and assays for measuring quantitative and temporal changes in gene expression. Luc has been used to evaluate gene transfer in a variety of recent transformation schemes [2,54]. Foster and Kern [55] tested the human epidermal growth factor Millar et al. [45,46] used an intensified video imaging receptor (HER)2 as a target for selective transfer to specific system for photon counting, and the luc gene fused to a cells. The authors noncovalently linked a luc expression portion of the chlorophyll A/B binding protein promoter vector to a humanized HER2 antibody covalently modified (which has been shown to contain the cis elements with poly-L-lysine bridges. These constructs targeted necessary and sufficient for conferring both phytochrome gene delivery specifically to HER2 expressing cells. and circadian clock control of gene expression on a heterologous reporter gene construct) to identify and isolate circadian clock mutants in Arabidopsis. Michelet and Golman et al. [56] employed an adenovirus redirected to Chua [47••] have used luciferase to identify putative signal bind to fibroblast growth factor receptors expressed on transduction components used by phytochrome A in Ara- Kaposi’s sarcoma cells. Specific quantitative enhancement bidopsis. The system utilized a chalcone synthase promoter of luc in these cells demonstrated that this method could fused to the luc gene and an intensified charge-coupled be used in somatic gene therapy. Luc activity has also been device (CCD) cooled slow-scan CCD imaging system. used to evaluate cationic liposomes and peptides for use in They were able to efficiently screen 170,000 seedlings gene transfer protocols [57,58]. Liu et al. [57] redesigned for desirable mutant phenotypes. Inducible transcription cationic liposomes by using cholesterol as the neutral systems compatible with plant systems are currently being lipid and preparing them as multilamellar vesicles. These developed [48•], such as the glucocorticoid-mediated modified liposomes showed great improvement in cationic induction system developed by Aoyama and Chua [49••]. DNA delivery. Luc reporters have also been used to Luc activity was used to evaluate the system’s ability to evaluate several new peptide-mediated delivery systems. respond to a dose range of inducer, although luciferase’s Wadha et al. [59] tested cationic peptides containing a half-life of three hours is too long to make it useful in single cysteine, tryptophan and lysine repeat to define kinetic studies of extremely rapid induction. the minimal length necessary to act as mediators in in vitro gene transfer to mammalian cells (HepG2 and COS7). These peptides were modified to include lysine Imaging of luc activity has been demonstrated at the chains of varying lengths and then tested for their ability single cell level. White et al. [50], using viral promoters to condense plasmid DNA and to act as gene transfer fused to luciferase and a highly sensitive photon-counting vehicles (versus polylysine 19 and cationic peptides). Luc camera system, were able to measure real-time viral reporter constructs were used to evaluate the success of regulation of gene expression in single HeLa cells. They the gene transfer. Tryptophan-containing cationic peptides used three stable, transformed lines to show that in were found to be effective mediators of gene transfer, due, basal culture conditions there was a high degree of in part, to their low toxicity and ability to form small DNA heterogeneity in the viral promoter activity. This indicates condensates. a high degree of polymorphism in cultured cells at the level of gene transcription, even within cells from the same stably transformed line. The consequences are that Conclusions and future directions subtle changes in cells and/or their environment can have The diverse reports reviewed here demonstrate the dramatic effects at the level of gene transcription and versatility and power of luc- and GFP-based reporters that cell populations may have a much greater range in monitoring gene transfer events. Their ability to of responses than previously thought. Rutter et al. [51] conveniently and noninvasively monitor gene expression, examined the dynamics of insulin-stimulated activator pro- combined with their sensitivity, has made these reporters tein 1 dependent transcription in single living cells using increasingly useful for a wide variety of applications in microinjected luc reporters. Imaging of luc activity allowed biology. 620 Expression systems

The crystal structure of GFP has recently been solved 7. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green by two research groups [60••,61••], and this should help fluorescent protein as a marker for gene expression. Science 1994, 263:802-805. direct future mutagenesis of GFP to produce improved 8. Rizzuto R, Brini M, De Giorgi F, Rossi R, Heim R, Tsien RY, versions, including new color variants. Mutants with a •• Pozzan T: Double labeling of subcellular structures with shorter half-life could facilitate GFP’s use in monitoring organelle-targeted GFP mutants in vivo. Curr Biol 1996, 6:183- 188. gene expression in real time. The solved structure, while An example of the successful use of green fluorescent protein and blue locating sites for more directed mutagenesis towards fluorescent protein in a double labeling study. It also demonstrates targeting and detection of green fluorescent protein in organelle compartments. improving GFP, also identifies certain features that may 9. Kohler RH, Cao J, Zipfel WR, Webb WW, Hanson M: Exchange be off limits to modification (e.g., changes that disrupt •• of protein molecules through connections between higher the eleven β-strands surrounding the central α-helix). The plant plastids. Science 1997, 276:2039-2042. This study reveals a level of direct communication between plastids not use of combinatorial peptide technology may allow for the previously seen. Tubules facilitating exchange of protein between individual development of novel fluorescent proteins. The ability of plastids are shown. This will almost certainly be shown to have an impact luc to noninvasively measure real-time changes in gene on gene expression and function in plastids. 10. Goetz-Zernicka M, Pines J, Hunter McLean S, Dixon JP, expression will ensure its continued use in genetic screens Siemering KR, Haseloff J, Evans MJ: Following cell fate in the and assays in which the timing, as well the magnitude living mouse embryo. Development 1997, 124:1133-1137. of gene expression, are being examined. Different colors 11. Yokoe H, Meyer T: Spatial dynamics of GFP-tagged proteins of luc will permit synchronous measurement of multiple • investigated by local fluorescence enhancement. Nat Biotechnol 1996, 14:1252-1256. transcriptional events and will facilitate normalization of This study uses the local fluorescent enhancement technique to measure the transformation efficiencies. GFP and luc both have the dissociation rate of green fluorescent protein tagged K-Ras, demonstrating green fluorescent protein’s ability to act as a dynamic reporter of cell events. potential to be used in enhancer trap assays (attachment of 12. Yano M, Kanazawa M, Terada K, Namchai C, Yamaizumi M, a reporter such as GFP or luc to a weak promoter so that Hanson B, Hoogenraad N, Mori M: Visualization of mitochondrial expression of the reporter is driven by enhancer elements protein import in cultured mammalian cells with green fluorescent protein and effects of over expression of the utilized by nearby genes) to identify important genes human import receptor Tom20. J Biol Chem 1997, 272:8459- and regulatory elements involved in gene expression. 8465. Imaging of luc activity at the single cell level also 13. Tarasova N, Stauber RH, Choi JK, Hudson EA, Czerwinski G, • Miller JL, Pavlakis GN, Michejda CJ, Wank SA: Visualization of opens the possibility of designing experiments in which G-protein-coupled receptor trafficking with the aid of the green reaction components can be precisely added and responses fluorescent protein. J Biol Chem 1997, 272:14817-14824. directly measured. The potential to quantitatively and The authors use a chimeric protein consisting of cholecystokinin receptor and green fluorescent protein to monitor receptor dynamics. The study noninvasively measure multiple cellular events (including shows that green fluorescent protein can be used to report on temporal multiple transcriptional events, intracellular Ca2+ regu- changes in receptor physiology. lation, and protein–protein interactions) in real time in 14. Straight AF, Belmont AS, Robinett CC, Murray AW: GFP tagging of budding yeast chromosomes reveals that protein–protein response to changing conditions will greatly advance our interactions can mediate sister chromatid cohesion. Curr Biol understanding of the dynamic nature of the cell [62••]. 1996, 6:1599-1608. 15. Plautz JD, Day RN, Dailey G, Welsh SB, Hall JC, Halpain SL, • Kay SA: Green fluorescent protein and its derivatives as Acknowledgements versatile markers for gene expression in living Drosophila, We wish to thank Theresa Welsh, Joel Kreps and Jeff Plautz for their reading plant and mammalian cells. Gene 1996, 173:83-87. and editorial review. This is a good applications paper, showing green fluorescent protein as a marker in several biological systems. 16. Gerdes H, Kaether C: Green fluorescent protein: applications in References and recommended reading cell biology. FEBS Lett 1996, 389:44-47. 17. Heim R, Prasher DC, Tsien RY: Wavelength mutations and Papers of particular interest, published within the annual period of review, posttranslational auto-oxidation of green fluorescent protein. have been highlighted as: Proc Natl Acad Sci USA 1994, 91:12501-12504. • of special interest 18. Crameri A, Whitehorn EA, Tate E, Stemmer WPC: Improved •• of outstanding interest green fluorescent protein by molecular evolution using DNA shuffling. Biotechnology 1996, 14:315-319. 1. Birch RG: Plant transformation: problems and strategies for 19. Heim R, Tsien RY: Engineering green fluorescent protein for practical application. Annu Rev Plant Physiol Plant Mol Biol •• improved brightness, longer wavelengths and fluorescence 1997, 48:297-326. resonance energy transfer. Curr Biol 1996, 6:178-182. The authors in this landmark paper use mutagenesis to generate some very 2. Alam J, Cook JL: Reporter genes: applications to the study of important and widely used mutant green fluorescent proteins. The authors mammalian gene transcription. Anal Biochem 1990, 188:245- are noted for their pioneering and thorough work in developing green fluo- 254. rescent protein as a reporter. 3. Martin CS, Wight PA, Dobretsova A, Bronstein I: Dual 20. Pang SZ, DeBoer DL, Wan Y, Ye G, Layton JG, Neher MK, luminescent-based reporter gene assay for luciferase and Armstrong CL, Fry JE, Hinchee MA, Fromm ME: An improved β-galactosidase. Biotechniques 1996, 21:520-524. green fluorescent protein gene as a vital marker in plants. Physiol Plant 1996, 112:893-900. 4. Bronstein I, Fortin J, Stanley PE, Stewart GSAB, Kricka LJ: Chemiluminescent and bioluminescent reporter gene assays. 21. Siemering KR, Golbik R, Sever R, Haseloff J: Mutations that Anal Biochem 1994, 219:169-181. • suppress the thermosensitivity of green fluorescent protein. Curr Biol 1996, 6:1653-1663. 5. Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY: This paper demonstrates modified codon usage to improve green fluores- Understanding, improving and using green fluorescent cent protein thermosensitivity, an important concern especially when using proteins. Trends Biochem Sci 1995, 20:448-455. mammalian systems. 6. Prasher DC: Using GFP to see the light. Trends Genet 1995, 22. Zolotukhin S, Potter M, Hauswirth W, Guy J, Muzyczka N: A 11:320-323. • ‘humanized’ green fluorescent protein cDNA adapted for high- Reporter gene expression for monitoring gene transfer Welsh and Kay 621

level expression in mammalian cells. J Virol 1996, 70:4646- 38. Padgett HS, Epel BL, Kahn TW, Heinlein M, Watanabe Y, 4654. •• Beachy RN: Distribution of tobamovirus movement in infected A good example of modifying green fluorescent protein for optimal perfor- cells and implications for cell-to-cell spread of infection. Plant J mance in a specific system. 1996, 10:1079-1088. A classic paper demonstrating the use of green fluorescent protein in study- 23. Yang T, Cheng L, Kain SR: Optimized codon usage and chromophore mutations provide enhanced sensitivity with the ing viral infection dynamics in vivo. Essential reading for those involved in this area of research. green fluorescent protein. Nucleic Acids Res 1996, 24:4592- 4593. 39. Sheen J, Hwang SB, Niwa Y, Kobayashi H, Galbraith DW: Green- fluorescent protein as a new vital marker in plant cells. 24. Haseloff J, Siemering KR, Prasher DC, Hodge S: Removal of a Plant J •• cryptic intron and subcellular localization of green fluorescent 1995, 8:777-784. protein are required to mark transgenic Arabidopsis plants 40. Kohler RH, Zipfel WR, Webb WW, Hanson M: The green brightly. Proc Natl Acad Sci USA 1997, 94:2122-2127. fluorescent protein as a marker to visualize plant mitochondria An excellent paper that combines several important modifications to green in vivo. Plant J 1997, 11:613-621. fluorescent protein (GFP) for work in plant systems. Previous attempts to use GFP in Arabidopsis had been unsuccessful. This group continues to 41. Rouwendal GJA, Mendes O, Wolbert EJH, Douwe de Boer A: produce ground-breaking work with GFP. Enhance expression in tobacco of the gene encoding green fluorescent protein by modification of its codon usage. Plant 25. Periasamy A, Kay SA, Day RN: Fluorescence resonance Mol Biol 1997, 33:989-999. •• energy transfer (FRET) imaging of a single living cell using green fluorescence protein. Proc Int Soc Optical Eng 1997, 42. Chiu W-L, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J: 2983:58-66. • Engineered GFP as a vital reporter in plants. Curr Biol 1996, This paper shows in vivo demonstrations of protein–protein interactions with 6:325-330. fluorescence resonance energy transfer, a technique which promises to rev- This study deals with many aspects of working with green fluorescent protein olutionize the in vivo study of protein–protein interactions. in plants. 26. Levy JP, Muldoon RR, Zolotukhin S, Link CJ Jr: Retroviral transfer 43. Galbraith DW, Lambert GM, Grebnok RJ, Sheen J: Flow • and expression of a humanized, red-shifted green fluorescent cytometry analysis of transgene expression in higher plants: protein gene into human tumor cells. Biotechnology 1996, green fluorescent protein. Methods Cell Biol 1995, 50:3-14. 14 :610-614. 44. Brandes C, Plautz JD, Stanewsky R, Jamison CF, Straume M, A good example of green fluorescent protein as a reporter for monitoring •• Wood KV, Kay SA, Hall JC: Novel features of Drosophila period viral gene transfer, including a good analysis of this process using a variety transcription revealed by real-time luciferase reporting. Neuron of approaches. 1996, 16:687-692. 27. Cheng L, Fu J, Tsukamoto A, Hawley R: Use of green fluorescent This paper clearly demonstrates use and aspects of luciferase as a real- protein variants to monitor gene transfer and expression in time reporter of gene expression. It shows use of an automated system for mammalian cells. Nat Biotechnol 1996, 14:606-609. assaying multiple samples over several days. 28. Bierhuizen MFA, Westerman Y, Visser TP, Wognum AW, 45. Millar AJ, Carre´ IA, Strayer CA, Chua N-H, Kay SA: Circadian Wagemaker G: Green fluorescent protein variants as markers clock mutants in Arabidopsis identified by luciferase imaging. of retroviral-mediated gene transfer in primary hematopoietic Science 1995, 267:1161-1163. cells and cell lines. Biochem Biophys Res Commun 1997, The regulation 234 46. Millar AJ, Straume M, Chory J, Chua N-H, Kay SA: :371-375. of circadian period by phototransduction pathways in 29. Muldoon RR, Levy JP, Kain SR, Kitts PA, Link CJ: Tracking and Arabidopsis. Science 1995, 267:1163-1166. quantitation of retroviral-mediated transfer using a completely humanized, red-shifted green fluorescent protein gene. 47. Michelet B, Chua N: Improvement of Arabidopsis mutant •• screens based luciferase imaging . Biotechniques 1997, 22:162-164. in planta Plant Mol Biol Rep 1997, 14:321-329. 30. Dorsky D, Wells M, Harrington R: Detection of HIV-1 infection An example of using luciferase as a dynamic reporter of gene expression. with a green fluorescent protein reporter system. J Acquir This paper, along with Millar et al. 1995 [45,46], show use of luciferase in Immune Defic Syndr Hum Retrovirol 1997, 13:308-313. novel genetic screens. 31. Wu C, Liu H, Crossen R, Gruenwald S, Singh S: Novel green 48. Gatz C: Chemical control of gene expression. Annu Rev Plant fluorescent protein (GFP) baculovirus expression vectors. • Physiol Plant Mol Biol 1997, 48:89-108. Gene 1997, 190:157-162. A comprehensive review of the state of inducible gene expression systems 32. Wilson LE, Wilkinson N, Marlow SA, Possee RD, King LA: in plants. This is a landmark paper for the use of inducible reporter genes in Identification of recombinant baculovirus using green plants. fluorescent protein as a selectable marker. Biotechniques 1997, 49. Aoyama T, Chua N-H: A glucocorticoid-mediated transcriptional 22:674-682. •• induction system in transgenic plants. Plant J 1997, 11:605- 33. Cormack BP, Bertram G, Egerton M, Gow NAR, Falkow S, 612. Brown AJP: Yeast-enhanced green fluorescent protein This break-through paper shows development and use of a chemically in- (yEGFP): a reporter of gene expression in Candida albicans. ducible system for studying transcription in plants. Microbiology 1997, 143:303-311. 50. White MRH, Masuko M, Amet L, Elliott G, Braddock M, 34. Takada T, Iida K, Awaji T, Itoh K, Takahashi R, Shibui A, Yoshida K, Kingsman AJ, Kingman SM: Real-time analysis of the •• Sugano S, Tsujimoto G: Selective production of transgenic mice transcriptional regulation of HIV and CMV promoters in single using green fluorescent protein as a marker. Nat Biotechnol mammalian cells. J Cell Sci 1995, 108:441-455. 1997, 15:458-461. 51. Rutter GA, White MRH, Tavare´ JM: Involvement of MAP kinase Demonstration of green fluorescent protein in the production of transgenic in insulin signaling revealed by non-invasive imaging of mice, showing practical aspects of this application. luciferase gene expression in single living cells. Curr Biol 35. Chiocchetti A, Tolosano E, Hirsch E, Silengo L, Altruda F: Green 1995, 5:890-899. fluorescent protein as a reporter of gene expression in 52. Castano JP, Kineman RD, Frawley LS: Dynamic monitoring and transgenic mice. Biochim Biophys Acta 1997, 1352:193-202. quantification of gene expression in single, living cells: a 36. Subramanian S, Srienc F: Quantitative analysis of transient gene molecular basis for secretory cell heterogeneity. Molecular • expression in mammalian cells using the green fluorescent 1996, 10:599-605. protein. J Biotechnol 1996, 49:137-151. 53. Kost B, Schnorf M, Potrykus I, Neuhaus G: Non-destructive This paper deals with often neglected aspects of quantification in applying detection of firefly luciferase (LUC) activity in single plant green fluorescent protein as a reporter in transient assays. cells using a cooled, slow-scan CCD camera and an optimized 37. Mosser DD, Caron AW, Bourget L, Jolicoeur P, Massie B: Use assay. Plant J 1995, 8:155-166. • of a dicistronic expression cassette encoding the green 54. Fominaya J, Wels W: Target cell-specific DNA transfer mediated fluorescent protein for the screening and selection of cells by a chimeric multidomain protein. J Biol Chem 1996, expressing inducible gene products. Biotechniques 1997, 271:10560-10568. 22:150-152. A potentially useful and time-saving tool for use in screening for transformed 55. Foster BJ, Kern JA: HER2-targeted gene transfer. Hum Gene cell lines. Ther 1997, 8:719-727. 622 Expression systems

56. Golman CK, Rogers BE, Douglas JT, Sosnoswski BA, Ying W, sion on aspects of structure–function interplay and potential improvement Siegal GP, Baird A, Campain JA, Curiel DT: Targeted gene of green fluorescent protein for use as a reporter. Essential reading. delivery to kaposi’s sarcoma cells via the fibroblast growth factor receptor. Cancer Res 1997, 57:1447-1451. 61. Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ: •• Crystal structure of the Aequorea victoria green fluorescent 57. Liu Y, Mounkes LC, Liggitt HD, Brown CS, Solodin I, Heath TD, protein. Science 1996, 273:1392-1395. Debbs RJ: Factors influencing the efficiency of cationic This paper describes the crystal structure of green fluorescent protein (GFP), liposome-mediated intravenous gene delivery. Nat Biotechnol with an excellent discussion of possible improvements to GFP and of some 1997, 15:167-173. of the limitations imposed on engineering GFP. Excellent discussion section 58. Wyman TB, Nicol F, Zelphati O, Scaria PV, Plank C, Szoka FC Jr: deals with numerous aspects of GFP mutants and structure; as with Yang Design, synthesis, and characterization of a cationic peptide et al. 1996 [60••], essential reading. that binds to nucleic acids and permeabilizes bilayers. Biochemistry 1997, 36:3008-3017. 62. Romoser VA, Hinkle PM, Persechini A: Detection in living cells •• of Ca2+-dependent changes in the fluorescence emission of a 59. Wadhwa MS, Collard WT, Adami RC, McKenzie DL, Rice KG: indicator composed of two green fluorescent protein variants Peptide mediated gene delivery: influence of peptide structure linked by a calmodulin-binding sequence. J Biol Chem 1997, on gene expression. Bioconjug Chem 1997, 8:81-88. 272:13270-13274. 60. Yang F, Moss LG, Phillips GN: The molecular structure of green This authors’ demonstration the of application of green fluorescent protein •• fluorescent protein. Nat Biotechnol 1996, 14:1246-1251. as a sensitive fluorosensor molecule. Demonstrates potential use of green The determination of the crystal structure of green fluorescent protein will fluorescent protein in developing a new class of novel and sensitive fluo- bring further improvements to an already versatile reporter. Good discus- rosensors for studying cell function.