617 Reporter gene expression for monitoring gene transfer Stephen Welsh∗ and Steve A Kay The use of reporters such as green ¯uorescent protein (GFP) GFP has excitation peaks at 395 nm (largest peak) and and ®re¯y luciferase permit highly sensitive and nondestructive 475 nm, and an emission peak at 509 nm with a small monitoring of gene transfer and expression. Modi®cations 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 Chal®e [7] showing as alter its spectral qualities, have facilitated the use of expression and ¯uorescence 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 ¯uorescence Addresses microscopy or ¯uorescence-activated cell sorting [FACS]). The Scripps Research Institute, Department of Cell Biology, Early modi®cations 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 modi®cation 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 Modi®cations made in green ¯uorescent protein Abbreviations Modi®cations in GFP have been made using various CCD charge-coupled device FACS mutagenesis schemes. Mutants have been reported that ¯uorescence-activated cell sorting •• FRET ¯uorescence resonance energy transfer improve ¯uorescence intensity [18,19 ,20], thermostabil- GFP green ¯uorescent 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 ®re¯y luciferase [24••], and spectral qualities [19••]. The ability to combine RMGT retrovirus-mediated gene transfer these modi®cations 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 ¯uorescent 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 ®re¯y luciferase (luc) and of more than one protein, and use of ¯uorescence green ¯uorescent 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-speci®c 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 ef®cient RMGT in mammalian Green ¯uorescent protein cells. Both A375 and PA317 cells expressing the vector The GFP of Aequorea, a 238 amino acid polypeptide, is were rapidly and easily identi®ed, and showed no highly ¯uorescent 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 ¯uorescence 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 ¯uorescent 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 ¯uorescence as et al. [30]. a result of aberrant splicing of the GFP message [24••]. Modi®cation 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]. ¯uorescence [15••,24••]. Improvements in GFP to enhance Wilson et al. [32] describe a system utilizing novel its use in plants, including ¯uorescent signal and codon GFP baculovirus expression vectors that allow rapid usage, have been made by several groups [20,41]. Chiu identi®cation 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 identi®ed by exposure and colleagues [43] have shown that GFP can be used to UV light. FACS analysis of yeast cells transformed as an ef®cient marker in ¯ow cytometry sorting of plant with a yeast-enhanced (for codon usage) GFP mutant protoplasts for selection of positive clones. (yEGFP3) allows easy quanti®cation of positive cells. The yEGFP3 ¯uorescence intensity is 75-fold higher than that By combining several modi®cations 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. Modi®cation 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