Reporter gene expression for monitoring gene transfer

617

Reporter gene expression for monitoring gene transfer Stephen Welsh* and Steve A Kay The use of reporters

such as green fluorescent

and firefly luciferase

permit

highly sensitive

of gene transfer

and expression.

monitoring

in GFP which

increase

as alter its spectral GFP in a variety in imaging

qualities,

have facilitated

research

luciferase-based

have allowed

Improvements application

the expanded

in gene transformation,

and in monitoring

as well

the use of

and their increased

reporters

screens

methods.

(GFP)

Modifications

and thermostability,

of gene transfer

technologies

in biological in genetic

intensity

protein

and nondestructive

temporal

use of particularly

changes

in

gene expression.

Addresses The Scripps Research institute, Department of Cell Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037 USA *e-mail: [email protected] Current

Opinion

in Biotechnology

1997, 8:617-622

http://biomednet.com/elecref/0958166900800617 0 Current

Biology Ltd ISSN

0956-l

GFP has excitation peaks at 395nm (largest peak) and 475 nm, and an emission peak at 509nm with a small shoulder at 540nm [5,6]. Reports from Prasher [6] describing the cloning of Aeguorea GFP, and Chalfie [7] showing expression and fluorescence of GFP in a heterologous background (demonstrating that no exogenous substrates or cofactors were required to produce the active molecule), opened the door for use of GFP in a wide variety of biological applications. GFP has been used in the measurement of gene expression, cell labeling, and in protein 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 microscopy or fluorescence-activated cell sorting [FACS]). Early modifications and uses of GFP in biological systems have been reviewed elsewhere [5,6,15*, 16,171; therefore, the focus of this section will be on the modification and use of GFP as a genetically encodable marker for use in monitoring gene transfer.

669

Modifications Abbreviations CCD

charge-coupled

FACS FRET GFP

fluorescence-activated cell sorting fluorescence resonance energy transfer

device

IUC

green fluorescent protein human epidermal growth factor receptor firefly luciferase

RMGT

retrovirus-mediated

HER

gene transfer

Introduction Gene transfer in its most useful sense must not only include monitoring the successful transfer of desired genes but also the establishment of the proper and predictable pattern of transgene expression. Detection and screening of such transformants can often pose serious challenges [ 11. The use of reporters based on firefly luciferase (luc) and green fluorescent protein (GFP), which allow transgene expression to be sensitively and noninvasively measured, is greatly facilitating gene transfer technology. Improvements in the GFP molecule have increased its use in a variety of gene transfer scenarios. The highly sensitive nature of the luc assay, with the increasing availability of detection and imaging technology, has made luciferase the reporter of choice in many transformation strategies. These versatile reporters complement other well validated reporter systems such as B-galactosidase, secreted alkaline phosphatase, chloramphenicol acetyltransferase, and B-glucuronidase [Z-l], by allowing accurate, continuous monitoring of gene expression in living tissues.

Green fluorescent

protein

The GFP of Aequona, a 238 amino acid polypeptide, is highly fluorescent and stable in many assay conditions [S].

made

in green

fluorescent

protein

Modifications in GFP have been made using various mutagenesis schemes. Mutants have been reported that improve fluorescence intensity [18,19**,20], thermostability [Zl’], folding and formation of the chromophore [18], codon usage [22*,23], removal of cryptic intron sequences [24**], and spectral qualities [19”]. The ability to combine 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 than wild-type GFP [19”]. Heim and co-workers [8**,19**] mutagenized GFP to generate a bright blue fluorescent protein which contains the T66H and T145F amino acid changes. Spectral variants (e.g. different colored GFPs) permit the simultaneous detection of expression from multiple reporters, tracking the transport and localization of more than one protein, and use of fluorescence resonance energy transfer (FRET) to detect in vivo protein-protein interactions [S]. Essentially one could monitor several cellular events at once in a noninvasive manner in a living cell. The application of FRET in cells has recently been used to demonstrate the dimerization of the pituitary-specific transcription factor Pit-l, using GFP and BFP fusions [ZS”]. Retrovirus-mediated

gene

transfer

GFP is rapidly gaining use in retrovirus-mediated gene transfer (RMGT) into mammalian cells, including tumor cell lines [26’,27]. Using a humanized (for codon usage) red-shifted mutant (S65T) version of GFP and FACS, Levy et a/. [26’] showed efficient RMGT in mammalian cells. Both A375 and PA317 cells expressing the vector were rapidly and easily identified, and showed no deleterious effects. The visualization of gene expression

616

Expression systems

in living

tissues

could

become

a powerful

methodology

in the evaluation of gene transfer in clinical Zolotukhin et al. [W] employed a humanized GFP

(92 base

substitutions

in 88 codons)

combined

trials. S65T with

Subramanian and analysis of transient cells to determine quantitative

Srienc [36*] performed quantitative gene expression in single mammalian the ability of GFP to act as a

reporter.

They

found

that green

fluorescence

a series of adeno-associated virus vectors, and showed successful retroviral transduction and expression of GFP in human 293 cells and neurosensory cells of the guinea pig eye. Single integrated copies of virus-driven GFP

is a quantitative measure of GFP in single cells. GFP has also been used in dicistronic expression cassettes (where GFP and the gene of interest are under the control of the same promoter) to screen and select for cells

were detectable. humanized GFP

expressing inducible gene products. Mosser et a/. [37-l used GFP reporters to select tetracycline-regulated cells from a mixed population of cells. A dicistronic cassette was used which incorporated a viral internal ribosome entry site plasmid, encoding both GFP and the gene of interest. Cells expressing GFP expressed the cotransfected gene and minimized screening procedures for gene transfer and expression.

Fluorescence were 45fold

intensity levels that of wild-type

for the GFP as

assayed by FACS. Bierhuizen et al. [ZS] used several variants of GFP to show their applicability as selectable or screenable markers in RMGT and their expression in primary hematopoietic cells. The positive phenotype selected by FACS provided greater than 90% pure and viable populations of transduced hematopoietic lines, with the phenotype being stable for at least one month. Muldoon et al. [29] using murine replication-defective retroviral vectors and humanized GFP-S65T mutant [Z.?] showed that almost 100% of the selected cells were GFP-positive. Detection of HIV-l infection in HeLa cells using the S65T mutant has also been reported by Dorsky et al. [30]. Baculovirus

and yeast

Baculovirus expression systems have gained wide popularity for expressing genes from a variety of eukaryotes [31]. Wilson et al. [32] describe a system utilizing novel GFP baculovirus expression vectors that allow rapid identification of recombinant baculoviruses. Constructs permit the gene of interest to be cloned in-frame with the GFP open reading frame, and plaques expressing the fusion protein can be readily identified by exposure to UV light. FACS analysis of yeast cells transformed with a yeast-enhanced (for codon usage) GFP mutant (yEGFP3) allows easy quantification of positive cells. The yEGFP3 fluorescence intensity is 7.5fold higher than that of wild-type GFP and can be detected at the single cell level

[33].

Mammalian

systems

Takada et a/. [34”] employed the the cytomegalovirus immediate-early

S65T GFP with enhancer and the

elongation factor 1 promoter to drive expression of S65T GFP in pre-implantation murine and bovine embryos. Using confocal laser scanning microscopy it was possible to detect S65T GFP positive cells at the morula and hatched blastocyst stages. Eight fetuses and four live-born mice were obtained from 55 S65T GFP-positive blastocysts. PCR analysis demonstrated that 11 of the 12 mice were transgenic. This method could increase the production of transgenic mice, combined with flow cytometry selection of GFP positive cells. Chiocchetti et a/. [35] used GFP, under the control of the human hemopexin and mouse Bt integrin promoter, as a marker for gene expression in transgenic mice. GFP proved to be a sensitive marker for detecting expression in targeted tissues at the single cell level.

Green

fluorescent

protein

in plants

GFP has been used extensively in plant systems, in localization studies and as a screenable marker for gene transfer [9**,38”,39,40]. Some plant species (e.g. Arabidopsis) show little or no expression of GFP fluorescence as a result of aberrant splicing of the GFP message [24**]. Modification of GFP to remove a cryptic intron site (a sequence recognised as an intron in Arabidopsis, leading to aberrant mRNA) resulted in successful detection of GFP fluorescence [ 15”,24”]. Improvements in GFP to enhance its use in plants, including fluorescent signal and codon usage, have been made by several groups [20,41]. Chiu et al. [42-l, using the S65T mutant, showed ZO-fold higher expression in maize leaf cells, and detected GFP driven by weak promoters in a broad range of plant hosts. Sheen and colleagues [43] have shown that GFP can be used as an efficient marker in flow cytometry sorting of plant protoplasts for selection of positive clones. By combining several modifications one can produce highly optimized versions of GFP that lead to cumulative gains in GFP expression and utility. Haseloff et al. [24**] combined several changes in GFP that improve its function. Modification processing and

of codon usage removal of the

ensures cryptic

proper intron.

mRNA Amino

acid substitutions for improved thermostability, folding, enhanced fluorescence, and altered spectral properties are useful for work in plants and in targeting to the endoplasmic reticulum. The latter aspect was important in the recovery of transgenic plants from the most highly fluorescent cells. This recovery had been problematic previously, most likely due to GFP toxicity (not normally a problem at lower levels of expression). The use of GFP has the potential to improve monitoring of both gene transfer and expression in plants, especially with the ease (no addition of substrate, easy assay) and noninvasive nature of the assay.

Luciferase Firefly luciferase offers features that a marker for in viva gene transfer.

make Luc,

it useful as a monomer,

Reporter gene expression for monitoring gene transfer Welsh and Kay

shows little toxicity or other deleterious effects on normal cellular metabolism, is conveniently extracted from plant and animal tissue, and is easily assayed by using a luminometer or scintillation counter. The sensitivity [Z], wide linear range (eight orders of magnitude), extremely and relative ease of the luciferase low background, assay has made it attractive to many researchers [2,4]. The rapid turnover of luc activity has been useful in studying temporal gene expression [44”]. The ability to identify transformants with desired expression patterns in a sensitive and nondestructive assay makes luciferase a powerful tool for monitoring in viva gene transfer. These properties have also been used to develop novel genetic screens and assays for measuring quantitative and temporal changes in gene expression.

Millar et a/. [45,46] used an intensified video imaging system for photon counting, and the luc gene fused to a portion of the chlorophyll A/B binding protein promoter (which has been shown to contain the cis elements necessary and sufficient for conferring both phytochrome and circadian clock control of gene expression on a heterologous reporter gene construct) to identify and isolate circadian clock mutants in Arabidopsis. Michelet and Chua [47”] have used luciferase to identify putative signal transduction components used by phytochrome A in Arabidopsis. The system utilized a chalcone synthase promoter fused to the luc gene and an intensified charge-coupled device (CCD) cooled slow-scan CCD imaging system. They were able to efficiently screen 170,000 seedlings for desirable mutant phenotypes. Inducible transcription systems compatible with plant systems are currently being developed [48*], such as the glucocorticoid-mediated induction system developed by Aoyama and Chua [49**]. Luc activity was used to evaluate the system’s ability to respond to a dose range of inducer, although luciferase’s half-life of three hours is too long to make it useful in kinetic studies of extremely rapid induction.

Imaging of luc activity has been demonstrated at the single cell level. White et al. [SO], using viral promoters fused to luciferase and a highly sensitive photon-counting camera system, were able to measure real-time viral regulation of gene expression in single HeLa cells. They used three stable, transformed lines to show that in basal culture conditions there was a high degree of heterogeneity in the viral promoter activity. This indicates 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 subtle changes in cells and/or their environment can have dramatic effects at the level of gene transcription and that cell populations may have a much greater range of responses than previously thought. Rutter et al. [Sl] examined the dynamics of insulin-stimulated activator protein 1 dependent transcription in single living cells using microinjected luc reporters. Imaging of luc activity allowed

619

detection of hormonal regulation of gene expression from relatively weak promoters in single cells. Frawley and co-workers [SZ], using photon counting imaging of a pituitary prolactin promoter-luc fusion in lactotrope cells, showed a large degree of heterogeneity in the basal levels of pituitary prolactin gene expression in single cells. This heterogeneity could provide a molecular basis for the observed dynamic nature of lactotrope function. Kost et a/. [53] analyzed luciferase activity in single tobacco protoplasts and small microcalli using a cooled, slow-scan CCD camera [53]. Transgenic tobacco lines were eventually established from these single luminescent clones. Luc has been used to evaluate gene transfer in a variety of recent transformation schemes [2,54]. Foster and Kern [SS] tested the human epidermal growth factor receptor (HER)2 as a target for selective transfer to specific cells. The authors noncovalently linked a luc expression vector to a humanized HER2 antibody covalently modified with poly-L-lysine bridges. These constructs targeted gene delivery specifically to HER2 expressing cells. Golman et a/. [56] employed an adenovirus redirected to bind to fibroblast growth factor receptors expressed on Kaposi’s sarcoma cells. Specific quantitative enhancement of luc in these cells demonstrated that this method could be used in somatic gene therapy. Luc activity has also been used to evaluate cationic liposomes and peptides for use in gene transfer protocols [57,58]. Liu et a/. [57] redesigned cationic liposomes by using cholesterol as the neutral lipid and preparing them as multilamellar vesicles. These modified liposomes showed great improvement in cationic DNA delivery. Luc reporters have also been used to evaluate several new peptide-mediated delivery systems. Wadha et a/. [59] tested cationic peptides containing a single cysteine, tryptophan and lysine repeat to define 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 chains of varying lengths and then tested for their ability to condense plasmid DNA and to act as gene transfer vehicles (versus polylysine 19 and cationic peptides). Luc reporter constructs were used to evaluate the success of the gene transfer. Tiyptophan-containing cationic peptides were found to be effective mediators of gene transfer, due, in part, to their low toxicity and ability to form small DNA condensates.

Conclusions

and future directions

The diverse reports reviewed here demonstrate the versatility and power of luc- and GFP-based reporters in monitoring gene transfer events. Their ability to conveniently and noninvasively monitor gene expression, combined with their sensitivity, has made these reporters increasingly useful for a wide variety of applications in biology.

620

Expression

The

crystal

systems

structure

of GFP

has

by two research groups [60**,61”], direct future mutagenesis of GFP versions,

including

new

color

recently

been

solved

7.

and this should help to produce improved

variants.

Mutants

with

a

shorter half-life could facilitate GFP’s use in monitoring gene expression in real time. The solved structure, while locating sites for more directed mutagenesis towards improving GFP, also identifies certain features that may be off limits to modification (e.g., changes that disrupt the eleven P-strands surrounding the central a-helix). The use of combinatorial peptide technology may allow for the development of novel fluorescent proteins. The ability of luc to noninvasively measure real-time changes in gene expression will ensure its continued use in genetic screens and assays in which the timing, as well the magnitude of gene expression, are being examined. Different colors of luc will permit synchronous measurement of multiple transcriptional events and will facilitate normalization of transformation efficiencies. GFP and luc both have the potential to be used in enhancer trap assays (attachment of a reporter such as GFP or luc to a weak promoter so that expression of the reporter is driven by enhancer elements utilized by nearby genes) to identify important genes and regulatory elements involved in gene expression. Imaging of luc activity at the single cell level also opens the possibility of designing experiments in which reaction components can be precisely added and responses directly measured. The potential to quantitatively and noninvasively measure multiple cellular events (including multiple transcriptional events, intracellular CaZ+ regulation, and protein-protein interactions) in real time in response to changing conditions will greatly advance our understanding of the dynamic nature of the cell [62**].

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science 1994, 263:802-805.

8. ..

Rizzuto R, Brini M, De Giorgi F, Rossi R, Heim R, Tsien RY, Pozzan T: Double labeling of subcellular structures with organelle-targeted GFP mutants in Go. Curr Biol 1996, 6:183108. An example of the successful use of green fluorescent protein and blue fluorescent protein in a double labeling study. It also demonstrates targeting and detection of green fluorescent protein in organelle compartments. 9. ..

Kohler RH, Cao J, Zipfel WR, Webb WW, Hanson M: Exchange of protein molecules through connections between higher plant plastids. Science 1997, 276:2039-2042. This study reveals a level of direct communication between plastids not previously seen. Tubules facilitating exchange of protein between individual plastids are shown. This will almost certainly be shown to have an impact on gene expression and function in plastids. 10.

Goetz-Zernicka M, Pines J, Hunter McLean S, Dixon JP, Siemering KR, Haseloff J, Evans MJ: Following cell fate in the living mouse embryo. Development 1997, 124:1133-l 137.

11. .

Yokoe H, Meyer T: Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement Nat Biotechnoll996, 14:1252-l 256. This study uses the local fluorescent enhancement technique to measure 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. 12.

Yano M, Kanazawa M, Terada K, Namchai C, Yamaizumi M, Hanson B, Hoogenraad N, Mori M: Visualization of mitochondrial protein import in cultured mammalian cells with green fluorescent protein and effects of over expression of the human import receptor TomSO. J Biol Chem 1997, 272:84598465.

13. .

Tarasova N, Stauber RH, Choi JK, Hudson EA, Czerwinski G, Miller JL, Pavlakis GN, Michejda CJ, Wank SA: Visualization of G-protein-coupled receptor trafficking with the aid of the green fluorescent protein. J Biol Chem 1997, 272:14817-l 4824. The authors use a chimeric protein consisting of cholecystokinin receptor and green fluorescent protein to monitor receptor dynamics. The study shows that green fluorescent protein can be used to report on temporal changes in receptor physiology. 14.

Straight AF, Belmont AS, Robinett CC, Murray AW: GFP tagging of budding veast chromosomes reveals that orotein-Drotein interactions can mediate sister chromatid cohesion. Curr Biol 1996, 6:1599-l 608.

15. .

Acknowledgements \\‘e\vish to thank ‘l‘heresa \\‘clsh, and editorial

Jwl Krcps and Jeff I’lautz for rhclr rcadlng

review

Plautz JD, Day RN, Dailey G, Welsh SB, Hall JC, Halpain SL, Kay SA: Green fluorescent protein and its derivatives as versatile markers for gene expression in living Drosophila, plant and mammalian cells. Gene 1996, 173:83-87. This is a good applications paper, showing green fluorescent protein as a marker in several biological systems. 16.

Gerdes H, Kaether C: Green fluorescent cell biology. FEBS Leff 1996, 389:44-47.

1 7.

Heim R, Prasher DC, Tsien RY: Wavelength mutations posttranslational auto-oxidation of green fluorescent Proc Nat/ Acad Sci USA 1994, 91 :12501-l 2504.

of special interest of outstanding interest

18.

Crameri A, Whitehorn EA, Tate E, Stemmer WPC: Improved green fluorescent protein by molecular evolution using DNA shuffling. Biofechnology 1996, 14:315-319.

1.

Birch RG: Plant transformation: problems and strategies for practical application. Annu Rev Plant Physiol Plant MO/ Biol 1997, 48:297-326.

19. ..

2.

Alam J, Cook JL: Reporter genes: applications mammalian gene transcription. Anal Biochem 254.

3.

Martin CS, Wight PA, Dobretsova A, Bronstein I: Dual luminescent-based reporter gene assay for luciferase P-galactosidase. Biotechniques 1996, 21:520-524.

References

and recommended

reading

Papers of particular interest, published within the annual period of review, have been highlighted as: . l

4.

*

to the study of 1990, 188:245-

applications

in

and protein.

Heim R, Tsien RY: Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr B/o/ 1996, 6:178-l 82. The authors in this landmark paper use mutagenesis to generate some very important and widely used mutant green fluorescent proteins. The authors are noted for their pioneering and thorough work in developing green fluorescent protein as a reporter. 20.

and

Bronstein I, Fortin J, Stanley PE, Stewart GSAB, Kricka LJ: Chemiluminescent and bioluminescent reporter 9ene assays. Anal Biochem 1994, 219:169-181.

protein:

Pang SZ, DeBoer DL, Wan Y, Ye G, Layton JG, Neher MK, Armstrong CL, Fry JE, Hinchee MA, Fromm ME: An improved green fluorescent protein gene as a vital marker in plants. Physiol Plant 1996, 112:893-900.

5.

Cubitt AB. Heim R, Adams SR. Boyd AE, Gross LA, Tsien RY: Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 1995, 20:448-455.

Siemering KR, Golbik R, Sever R, Haseloff J: Mutations that suppress the thermosensitivity of green fluorescent protein. Curr Biol 1996, 6:1653-l 663. This paper demonstrates modified codon usage to improve green fluorescent protein thermosensitivity, an important concern especially when using mammalian systems.

6.

Prasher DC: Using GFP to see the light 11:320-323.

22. .

Trends Gener 1995,

21. .

Zolotukhin S, Potter M, Hauswirth W, Guy J, Muzyczka N: A ‘humanized’ green fluorescent protein cDNA adapted for high-

Reporter

level expression in mammalian cells. I V&o/ 1996, 70:46464654. A good example of modifying green fluorescent protein for optimal performance in a specific system. 23.

Yang T, Cheng L, Kain SR: Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res 1996, 24:45924593.

Haseloff J, Siemering KR, Prasher DC, Hodge S: Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arebidopsis plants brightly. Proc Nat/ Acad Sci USA 1997, 94:2122-2127. An excellent paper that combines several important modifications to green fluorescent protein (GFP) for work in plant systems. Previous attempts to use GFP in Arabidopsis had been unsuccessful. This group continues to produce ground-breaking work with GFP.

gene

Levy JP, Muldoon RR, Zolotukhin S, Link CJ Jr: Retroviral transfer and expression of a humanized, red-shifted green fluorescent protein gene into human tumor cells. Biotechnology 1996, 14:61 O-61 4. A good example of green fluorescent protein as a reporter for monitoring viral gene transfer, including a good analysis of this process using a variety of approaches. 27.

Cheng L, Fu J, Tsukamoto A, Hawley R: Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nat Biotechnol 1996, 14:606-609.

26.

Bierhuizen MFA, Westerman Y, Visser TP, Wognum AW, Wagemaker G: Green fluorescent protein variants as markers of retroviral-mediated gene transfer in primary hematopoietic cells and cell lines. Biochem Biophys f?es Commun 1997, 234:371-375.

29.

Muldoon RR, Levy JP, Kain SR, Kitts PA, Link CJ: Tracking and quantitation of retroviral-mediated transfer using a completely humanized, red-shifted green fluorescent protein gene. Biotechnigues 1997, 22:162-l 64.

30.

Dorsky D, Wells M, Harrington R: Detection of HIV-1 infection with a green fluorescent protein reporter system. J Acquir immune Defic Syndr Hum Retrovirol 1997, 13:306-313.

31.

Wu C, Liu H, Crossen R, Gruenwald S, Singh S: Novel green fluorescent protein (GFP) baculovirus expression vectors. Gene 1997, 190:157-l 62.

32.

Wilson LE, Wilkinson N, Marlow SA, Possee RD, King LA: Identification of recombinant baculovirus using green fluorescent protein as a selectable marker. Biotechniques 22:674-662.

33.

1997,

Cormack BP, Bertram G, Egerton M, Gow NAR, Falkow S, Brown AJP: Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicens. Microbiology 1997, 143:303-311.

Takada T, lida K, Awaji T, ltoh K, Takahashi R, Shibui A, Yoshida K, Sugano S, Tsujimoto G: Selective production of transgenic mice using green fluorescent protein as a marker. Nat Biotechnol 1997, 15:456-461. Demonstration of green fluorescent protein in the production of transgenic mice, showing practical aspects of this application. Chiocchetti A, Tolosano E, Hirsch E, Silengo L, Altruda F: Green fluorescent protein as a reporter of gene expression in transgenic mice. Biochim Siophys Acta 1997, 1352:193-202.

Subramanian S, Srienc F: Quantitative analysis of transient gene expression in mammalian cells using the green fluorescent protein. J Biotechnol 1996, 49:137-l 51. This paper deals with often neglected aspects of quantification in applying green fluorescent protein as a reporter in transient assays. Mosser DD, Caron AW, Bourget L, Jolicoeur P, Massie B: Use of a dicistronic expression cassette encoding the green fluorescent protein for the screening and selection of cells expressing inducible gene products. Biotechniques 1997, 22:150-152. A potentially useful and time-saving tool for use in screening for transformed cell lines.

621

Sheen J, Hwang SB, Niwa Y, Kobayashi H, Galbraith DW: Greenfluorescent protein as a new vital marker in plant cells. P/ant J 1995, 8:777-784.

40.

Kohler RH, Zipfel WR, Webb WW, Hanson M: The green fluorescent protein as a marker to visualize plant mitochondria in viva. Plant J 1997, II:61 3-621.

41.

Rouwendal GJA, Mendes 0, Wolbert EJH, Douwe de Boer A: Enhance expression in tobacco of the gene encoding green fluorescent protein by modification of its codon usage. Plant MO/ Biol 1997, 33:969-999.

Chiu W-L, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J: Engineered GFP as a vital reporter in plants. Gun Biol 1996, 6:325-330. This study deals with many aspects of working with green fluorescent protein in plants.

42. .

43.

Galbraith DW, Lambert GM, Grebnok RJ, Sheen J: Flow cytometry analysis of transgene expression in higher plants: green fluorescent protein. Methods Cell Biol 1995, 50:3-l 4.

44. ..

Brandes C, Plautz JD, Stanewsky R, Jamison CF, Straume M, Wood KV, Kay SA, Hail JC: Novel features of Drosophila period transcription revealed by real-time luciferase reporting. Neuron 1996, 16:667-692. This paper clearly demonstrates use and aspects of luciferase as a realtime reporter of gene expression. It shows use of an automated system for assaying multiple samples over several days. 45.

Millar Al, Car& IA, Strayer CA, Chua N-H, Kay SA: Circadian clock mutants in Arabidopsis identified by luciferase imaging. Science 1995, 267:1161-l 163.

46.

Millar Al, Straume M, Chary J, Chua N-H, Kay SA: The regulation of circadian period by phototransduction pathways in Arabidopsis. Science 1995, 267:1163-l 166.

4% ..

Michelet B, Chua N: Improvement of Arabidopsis mutant screens based luciferase imaging in planta. Plant MO/ Biol Rep 1997, 14:321-329. An example of using luciferase as a dynamic reporter of gene expression. This paper, along with Millar et a/. 1995 [45,46], show use of luciferase in novel genetic screens. 46.

Gatz C: Chemical control of gene expression. Annu Rev Plant Physiol Plant MO/ Biol 1997, 46:69-l 06. ; comprehensive review of the state of inducible gene expression systems in plants. This is a landmark paper for the use of inducible reporter genes in plants. 49. ..

Aoyama T, Chua N-H: A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 1997, 11:605612. This break-through paper shows development and use of a chemically inducible system for studying transcription in plants. 50.

White MRH, Masuko M, Amet L, Elliott G, Braddock M, Kingsman AJ, Kingman SM: Real-time analysis of the transcriptional regulation of HIV and CMV promoters in single mammalian cells. J Cell Sci 1995, 108:441-455.

51 I

Rutter GA, White MRH, Tavarb JM: Involvement of MAP kinase in insulin sianalina revealed bv non-invasive imaaina of luciferase g&e eipression in iingle living cells. &J; Biol 1995, 5:690-699.

52.

Castano JP, Kineman RD, Frawley LS: Dynamic monitoring and quantification of gene expression in single, living cells: a molecular basis for secretory cell heterogeneity. Molecular 1996, 10:599-605.

53.

Kost B, Schnorl M, Potrykus I, Neuhaus G: Non-destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow-scan CCD camera and an optimized assay. Plant J 1995, 6:155-l 66.

54.

Fominaya J, Wels W: Target cell-specific DNA transfer mediated by a chimeric multidomain protein. J Biol Chem 1996, 271 :10560-l 0566.

55.

Foster BJ, Kern JA: HERP-targeted Ther 1997, 6:719-727.

36. .

37. .

Welsh and Kay

39.

34. ..

35.

gene transfer

Padgett HS, Epel BL, Kahn TW, Heinlein M, Watanabe Y, Beachy RN: Distribution of tobamovirus movement in infected cells and implications for cell-to-cell spread of infection. PlantJ 1996, 10:1079-l 066. A classic paper demonstrating the use of green fluorescent protein in studying viral infection dynamics in viva. Essential reading for those involved in this area of research.

25. ..

26. .

for monitoring

36. ..

24. ..

Periasamy A, Kay SA, Day RN: Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescence protein. Proc Int Sot Optical fng 1997, 2963:56-66. This paper shows in viva demonstrations of protein-protein interactions with fluorescence resonance energy transfer, a technique which promises to revolutionize the in viva study of protein-protein interactions.

expression

gene transfer. Hum Gene

622

Expression

systems

56.

Golman CK, Rogers BE, Douglas JT, Sosnoswski BA, Ying W, Siegal GP, Baird A, Campain JA, Curie1 DT: Targeted gene delivery to kaposi’s sarcoma cells via the fibroblast growth factor receptor. Cancer Res 1997, 57:1447-l 451.

5%

Liu Y, Mounkes LC, Liggitt HD, Brown CS, Solodin I, Heath TD, Debbs RJ: Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery. Nat Biofechnol 1997, 15:167-l 73.

58.

Wyman TB, Nicol F, Zelphati 0, Scaria PV, Plank C, Szoka FC Jr: Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 1997, 36:3006-3017.

59.

Wadhwa MS, Collard WT, Adami RC, McKenzie DL, Rice KG: Peptide mediated gene delivery: influence of peptide structure on gene expression. Bioconjug Chem 1997, 8:81-88.

Yang F, Moss LG, Phillips GN: The molecular structure of green 60. .. fluorescent protein. Nat Biotechnol 1996, 14:1246-l 251. The determination of the crystal structure of green fluorescent protein will bring further improvements to an already versatile reporter. Good discus-

sion on aspects of structure-function interplay and potential improvement of green fluorescent protein for use as a reporter. Essential reading. 61. ..

Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ: Crystal structure of the Aequorea Victoria green fluorescent protein. Science 1996, 273:1392-l 395. This paper describes the crystal structure of green fluorescent protein (GFP), with an excellent discussion of possible improvements to GFP and of some of the limitations imposed on engineering GFP. Excellent discussion section deals with numerous aspects of GFP mutants and structure; as with Yang et a/. 1996 [60”1, essential reading. 62. ..

Romoser VA, Hinkle PM, Persechini A: Detection in living cells of Ca2+-dependent changes in the fluorescence emission of a indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. J Biol Chem 1997, 272:13270-l 3274. This authors’ demonstration the of application of green fluorescent protein as a sensitive fluorosensor molecule. Demonstrates potential use of green fluorescent protein in developing a new class of novel and sensitive fluorosensors for studying cell function.