WO1999031277A1 - Expression cloning and single cell detection of phenotype - Google Patents

Abstract

The present invention pertains to a new method of 'functional cloning' or 'expression cloning' based on single-cell detection of a conferred phenotype. More particularly, in a preferred embodiment the method involves co-transfecting an appropriate eukaryotic host cell, a test DNA sequence and an appropriate CDNA library. The test DNA sequence contains a test promoter coupled with a reporter. CDNA's coding for molecules that influence the expression of the reporter could be detected.

Description

EXPRESSION CLONING AND SINGLE CELL DETECTION OF PHENOTYPE

BACKGROUND

1. Field of the Invention

This invention pertains to a new method of "functional cloning" or "expression cloning" based on single-cell detection of a conferred phenotype. More particularly, in a preferred embodiment the method involves co-transfecting an appropriate eukaryotic host cell a test DNA sequence and an appropriate cDNA library, both of eukaryotic origin. The test DNA sequence contains a test promoter or portion thereof coupled with a reporter. The preferred reporter encodes the green fluorescent protein (GFP) or one of its variants. The cDNA library is preferably constructed in a high expression vector. If a cDNA present in the library codes for a molecule that influences the expression of the reporter, this will be seen as a change in fluorescence and can be detected in a single cell. The cell can thus be identified and the cDNA from it recovered in a number of ways.

The preferred method of identifying and selecting positive cells is fluorescence activated cell sorting (FACS). The cDNAs from an enriched population of positive cells can be purified and retransformed into bacterial cells. Positive cDNA clones can be tested by repeating the co-transfection experiment and analyzing the co-transfected cells by fluorescence microscopy. The procedure has been exemplified with a portion of the IL-8 promoter and it has been demonstrated that the pIL-8 construct is sensitive to cytokine activation in this co- transfection system.

2. Description of the Prior Art

"Chronic inflammatory diseases" are a wide spread health problem in the western world. This broad term covers a spectrum of conditions that include arthritis, atherosclerosis, various dermatological disorders such as psoriasis and other specific inflammatory diseases such as inflammatory bowel disease. It is a _. ?

broadly held view that the underlying driving mechanism of inflammatory diseases is cytokine over-expression. Cytokines produced transiently in normal responses to injury and infection can become persistently produced, leading to chronic inflamation. Such diseases tend to have a slow course, often spanning many years before symptoms become clinically significant. Consequently, the causes of the failure of the initial inflammatory responses are obscure, but likely derive from a complex interplay of environmental and genetic factors.

Current methods for treating such diseases employ surgical, immuno- suppressive (e.g., total lymphoid irradiation), and non-specific anti-inflammatory treatments (e.g., steroids). "Anti-cytokine" therapies based on inhibition of receptor/ligand binding for particular cytokines, while promising, have met with limited clinical success. Therefore, the development of a deeper understanding of the mechanisms of the gene regulation and action of pro-inflammatory cytokines continues to attract a great deal of interest in many academic and industrial institutions. Illucidation of the signal transduction pathways involved in cytokine activation of genes and identification of the components of these pathways and their model of action is therefore of importance.

Functional cloning methods have increasingly replaced conventional biochemical approaches to identifying genes of interest (Dower, S. ( 1993) in Neurobiology of Cytokines, vol. 16., E.B. DeSouza, ed. Academic Press, Inc.,

New York, p. 3-30). A particularly striking example is the application of the yeast two hybrid system for identifying partners to intracellular proteins in mammalian cells based on pairwise binding interactions which reconstitute transcription systems in yeast (Fields, S. and Song, 0. ( 1989) Nature 340:245-6). Based on the same principle (pairwise protein-protein binding), mammalian high expression cloning systems have proved particularly effective at identifying receptors for soluble factors and ligands for orphan receptors (Seed, B.

SUBSTTTUTE SHEET (RULE 26) ( 1995) Current Opinion In Biotechnology 6:567-573). This method usually relies on the use of autoradiography or immunocytochemistry to identify the presence of a clone in a complex pool of cDNAs that encodes the partner for a labeled probe molecule. Operationally, a library of cDNAs (typically 1-5 x 10⁶ independent transformants), prepared using random hexanucleotide primers (to minimize the representation of intrinsic 3' non coding regions), is cloned into a mammalian expression vector, which contains at least an E. coli origin, a selectable marker, and a high expression cassette (e.g., constaining a strong promoter such as CMV or adenovirus MLP; a polylinker and a splice/viral 3' noncoding/polyA+ tail). The library is transformed into E. coli, plated and pools of plasmids from 1,000 - 10,000 independent colonies are prepared.

Each pool is transfected into an indicator line, such as COS-7, and assayed for the phenotype of interest. Thus, when searching for a receptor, radio-labeled ligand is added, and cells binding high levels of label are detected by autoradiography. Typically, the presence of an appropriate cDNA in a pool of 10,000 will be indicated by 20-100 positive cells in a monolayer on a microscope slide of about 2 X 10⁵ COS-7 cells. The positive pool can then be broken down by replating an aliquot of bacteria held in reserve against the identification of that pool, and repeating the cycle. Three to four cycles plus a round of single colony picking, a process that takes about 1 month, will usually suffice to isolate a cDNA of interest.

This method is disadvantaged, however, in that single positive cells cannot be isolated and identified, requiring the sequential dilution and screening of pools of clones in order to narrow the search until a single positive clone is discovered. Furthermore, the currently used methds do not allow for the detection and isolation of single mammalian cells containing a test cDNA of interest. What is needed in the art, is a selection system that can be generalized to rapidly detect a phenotype conferred on mammalian cells at the single cell level. The invention described herein is such a cloning system, allowing single cell detection and isolation of positive cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Structure of the pIL-8 Construct Figure 2 Fluorescent Micrographs Converted to Histogram Format Figure 3 Effect of IL-1RI Transfection and IL-1 Stimulation on the Activity of Reporter Constructs in Cos-7 Cells

SUMMARY OF THE INVENTION

Briefly, the invention is a method of isolating a nucleotide sequence that increases (or otherwise modulates) expression from a test promoter. The method involves transfecting a mammalian host cell with a first nucleotide sequence to produce a transfected cell, wherein the first nucleotide sequence has at least a test promoter coupled with a reporter. Although any appropriate cell/promoter/reporter combination can be used, preferably, the mammalian host cell is a COS or Hela cell, the test promoter is a cytokine promoter, such as the IL- 8 promoter or portion thereof, and the reporter encodes a fluorescent protein, such as green fluorescent protein (GFP).

Next, the transfected cell is co-transfected with a second nucleotide sequence to produce a co-transfected cell, wherein the second nucleotide sequence has at least a cDNA coupled to a high expression promoter. The expression of the reporter in the co-transfected cell is measured and compared with a control cell transfected only with the first nucleotide sequence.

In an alternate embodiment of the invention, one or more agents, such as, for example, a cytokine or other hormone, may be provided to the cell to

SUBSTITUTE SHEET (RULE 25) determine if the reporter expression is further increased in response to the agent(s). The agent may be anything that regulated the expression of a gene and may include also chemical agents, inhibitory agents such as antisense molecules, peptides, metal, depending upon the signaling pathway that is being illucidated. Alternatively, an additional DNA sequence may be transfected into the cells to further enhance the synthesis of the reporter protein. For example, a cDNA encoding a receptor for the agent that is being provided to the cell as described above may be desired. In this way the cell may produce additional receptors with which the agent may interact, thereby increasing the level of signaling molecules within the cell. For example, the preferred embodiment of the invention may be altered to included the co-transfection of a cDNA encoding the IL-1 receptor (IL-1R). The resultant cells containing the IL-8-promoter-GFP cDNA and the IL-1R cDNA, thereby expressing increased levels of IL-1 R. These cells will thereby be more responsive to IL-1 treatment. Alternatively, other signaling molecules may be introduced, including, traf-6, myd-88, FADD, NIK, IRAKI, IRAK2, and REIA.

Although in the experiments devised herein, we have selected as positive those cDNA clones that increase the expression of a test promoter, it is of course equally possible to select for clones that decrease the expression of a highly active test promoter.

In yet another embodiment of the invention, the method allows for the enrichment of a cDNA library for a cDNA that modulates the expression of a test promoter. The method involves transfecting mammalian host cells with a first nucleotide sequence (as described above) to create transfected cells and then co- transfecting the transfected cells with a library of cDNA molecules to create a co- transfection library. Preferably, each cDNA molecule is coupled to a high expression promoter. Next, the co-transfection library is sorted by a fluorescence

SUBSTITUTE SHEET (RULE 25) activated cell sorter to create an enriched co-transfection cell library, wherein the co-transfection cells that produce a changed level of fluorescence, as compared with transfection control cells, are positively selected for inclusion in the enriched co-transfection cell library. Finally, the cDNA molecules from the enriched co- transfection library are purified, re-transformed into bacterial cells, amplified and repurified to create an enriched library of cDNA molecules. The method can be repeated until a sufficient level of enrichment for cDNA molecules that modulate the expression of the test promoter is achieved and each cDNA molecule can be isolated.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations:

BSA: bovine serum albumin CMV: cytomegalovirus

FACS: fluorescence activated cell sorter GFP: green fluorescent protein

IL-1 interleukin- 1

IL-8: interleukin-8 IL-1R: interleukin- 1 receptor

IL-1RP. interleukin- 1 receptor type I

PBS: phosphate buffered saline

TNF: tissue necrosis factor

Definitions:

SUBS 111 U I h SHEET (RULE 25) As used herein, the term "GFP" refers to green fluorescent protein or any mutant or variant thereof.

As used herein, the term, "gene regulatory sequences" refers to DNA or RNA sequences that act in cis to modulate gene transcription or a post- transcriptional mechanism that regulates protein synthesis. Such sequences can include, but are not limited to promoter sequences, enhancer sequences, other regions of DNA required for gene expression, and regions of RNA molecules that regulate, for example, mRNA turnover, translation efficieny, or polyadenylation.

As used herein, the term "reporter" or "reporter protein" refers to a nucleotide sequence encoding a detectable product (e.g., a protein). The reporter protein may be fluorescently, radioactively, biologically and/or enzymatically detectable. For example, the reporter protein may be detected by fluorescence activated cell sorting or fluorescence microscopy. Preferred reporter proteins are green fluorescent protein (GFP), EGFP™, d2-EGFP, S6ST, T203 Y and GFPmut or other GFP variant. Alternative fluorescent molecules may be used which differ from GFP in terms of its excitation wavelength (e.g., is a different color such as blue (BFP), yellow, orange or red) or its fluorescent intensity. Any reporter protein that is which is conducive to the use of the invention with live cells is preferred. As used herein, the term "test promoter" or "test promoter sequence" refers to a promoter, or portion thereof, for which it is desired to identify and clone sequences that influence its expression. In this application, the IL-8 test promoter is exemplified, but any promoter which is a component of a signal transduction pathway make be analysed by the method of the invention. As used herein, the term "test nucleotide sequence" or "test nucleic acid" refers to a nucleic acid which encodes a factor or polypeptide capable of interacting with a test promoter or other gene regulatory sequence. Preferred

SUBSTITUTE SHEET (RULE 25) nucleotide sequences encode a transactivating factor, such as, for example, a transcription factor or enhancer molecule.

As used herein, the term "transfection" means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.

In one aspect, the instant invention relates to a novel cloning method for identifying transactivating factors that participate in gene regulation. In one embodiment, the invention relates to the identification of transcription factors or other transactivating regulatory factor required for the transcription of a gene. In an alternative embodiment, the invention relates to the identification of c/s-acting regions of genes that interact with the transactivating factors of the invention. In another embodiment, the invention relates to the determination of interactions between a test transactivating factor and the cw-acting region of a gene. In yet another embodiment, the invention relates to the above described embodiments which additionally involve the step of presenting to the transfected cells of the invention an agent which may modulate (e.g., enhance or inhibit) the ability of the transactivating factor to activate the cis-acύng region of the gene.

In a preferred embodiment, the instant invention relates to a cell signaling pathway dissection procedure, wherein the components of the pathway, and their role in signal transduction, are determined using the present method. For example, cellular signaling components that lie on the pathway that functionally connects a hormone receptor to a gene promoter can be identified and characterized. The method of the invention can be used to clone any nucleotide sequence whose product, or which itself, modulates the expression of a test promoter. It is also anticipated that other regulatory sequences, such as, for example, those present in the 5' untranslated region (UTR) or 3' UTR of a test

SUBSI I I U I b SHEET (RULE 26) mRNA may be analyzed in accordance with the present method. In this embodiment, the method will identify transactivating factors that interact with mRNA molecules to regulate protein synthesis post-transcriptionally. Such regulatory factors include, for example, those that regulate mRNA stability, mRNA polyadenylation or translation efficiency. Adaptation of the instant invention to achieve the study of post-transcriptional gene regulatory pathways would thus require appropriate reporter vectors that contain 5' UTR and 3'UTR sequences of interest.

In a preferred embodiment, the pathways functionally linking IL- 1 and TNF receptors to the promoter for the IL-8 gene may be identified, as an example of the broadly applicable novel cloning and pathway dissection procedure of the invention. IL-8 is a down-stream mediator of IL-1 and TNF action, playing a major role in activating the binding of neutrophils to, and their migration across, the endothelium of inflamed tissue (Huber, A., et al. (1991) Science 254:99-102). As in other IL-1 and TNF responsive genes, the IL-8 promoter contains both an AP- 1 and an NF-Kβ consensus site, and both have been shown to be critical for function (Sims, J., et al. (1994) in Signal Transduction Through Growth Factor Receptors., vol. 3. JIA Press, Greenwich, Conn., USA, London, U.K., p. 197- 222). We have previously shown that a small (200 bp) fragment from this promoter confers a high degree of IL-1 inducibility on reporter constructs incorporating it, using transient expression in COS-7 cells (Mitcham, J., et al. (1996) J. Biol. Chem. 271 :5777 '-5783). The instant invention was used to functionally clone cDNAs coding for signaling components that lie on the pathways connecting the IL-1 and TNF receptors to the IL-8 promoter. However, the method is of general applicability and can be used to clone any sequence that modulates the expression of a test promoter provided the host cell line is appropriately chosen and contains all of the required ancillary components.

SUBSTITUTE SHEET (RULE 25) In a preferred embodiment, the reporter protein is a fluorescing protein that can be expressed in a eukaryotic cell and not cause damage to the cell. In a particularly preferred embodiment, green fluorescent protein (GFP) from Aequorea victoria (Prasher, D.C. ( 1992) Gene 111:229-233). GFP is strongly fluorescent in GFP transfected E. coli or C. elegans cells and its fluorescence does not appear to depend upon exogenous substtrates or coenzymes. (Chalfie, N. et al. (1994) Science 263:802-805). GFP has also been used to construct chimeric proteins that fluoresce in Drosophila melanogaster (Wang and Hazelrigg (1994) Nature 369:400-403) and in human embryonic kidney cells (Marshall, et al. (1995), Neuron 14:21 1-215). More recently, GFP has been used to study in vivo gene expression and protein localization in eukaryotic cells (Cubitt, A.B. et al. (1995), Trends Biochem. Sci. 20:448-455).

GFP has been employed in a bacterial expression cloning system that differs from that described here (Valdivia, R.H. and Falkow, S. (1996), Mol. Microbiol. 22(2):367-78). Valdivia and Falkow applied a novel enrichment strategy, termed differential fluorescence induction (DFI), to screen a Salmonella typhimurium library for promoters that are upregulated at pH 4.5. DFI utilizes GFP and a fluorescence activated cell sorter (FACS) to perform genetic selection. In the presence of an inducing stimulus, such as low pH, a FACS is used to sort highly fluorescent bacterial clones bearing random promoters fused to a mutant GFP protein (GFPmut). This population is then amplified at neutral pH and the least fluorescent population is sorted to eliminate constitutive promoters. Sequential sorts for fluorescent and non-fluorescent bacteria in the presence or absence of inducing conditions enriches for promoter fusions that are regulated by the inducing stimulus.

The technique described by Valdivia and Falkow differs from the technique described herein in several respects. First, their system allows for the

SUBS I I I U IE SHEET (RULE 25) identification of bacterial promoters, not the analysis of eukaryotic regulatory elements as in the instant invention. The system of Valdivia and Falkow does not relate to the methods of the instant invention which begins with an isolated promoter and identify factors that interact with it. The Valdivia and Falkow method also does not require co-transfection of two nucleotide sequences into eukaryotic host cells in order to determine the interactions of the product of one nucleotide sequence with a region of the other nucletode sequence. Additionally, Valdivia and Falkow's system can isolate only those promoters that respond to simple environmental conditions such as low pH, high temperature, absence of essential nutrients, etc. The invention herein, in contrast, is more complex, allowing for the study of eukaryotic pathways and the selection of any sequence that modulates a eukaryotic test promoter. Thus, many different types of gene regulatory factors could be isolated and studied by the invention described herein. The instant invention relates to a single cell transcription reporter assay based on use of the Aequoria victorea green fluorescent protein (GFP) under the control of the human IL-8 promoter (pIL-8) to demonstrate the functional cloning of cDNAs important in the expression of the IL-8 test promoter. There is ample evidence from the recent literature that introduction of previously cloned cDNAs encoding components of cascades, such as MAP kinase cascades, can be read out as increased activity in downstream steps in the signaling pathways. Thus, there is reason to believe that this principle can be applied prospectively to identify cDNAs encoding novel signaling molecules. We have used a fragment of the IL- 8 promoter generated from human genomic DNA by PCR (Mitcham, J., et al. (1996) J. Biol. Chem. 271 :5777-5783) and a commercially available CLONTECH™ vector to construct a test promoter/reporter construct. The construct is shown in Figure 1 and is called pIL-8 herein. GFP and its variants are the preferred detection molecules of the invention. GFP has several advantages over other markers. First, it is relatively harmless to cells and no processing is required to visualize it. GFP is unique among fluorescent biomolecules in being composed solely of a polypeptide chain. The fluorescent form of the protein is generated by an auto-oxidation reaction after folding of the initial translation product. The only requirement for GFP to fluoresce is some amount of oxygen and time. Further, GFP photobleaches less quickly than other fluorescent molecules. In addition, because all the information for expression of GFP is contained in its gene sequence, the power of mutagenic techniques can be brought to bear on the generation and selection of GFPs with improved spectral, folding and stability properties. The EGFP in our construct is such a second generation protein, with improved autocatalytic conversion rate and spectral properties relative to wild type.

Detectable proteins useful in the practice of the invention include but are not limited to GFP, EGFP™ from CLONTECH™, d2-EGFP and other variants of GFP, S65T, T203Y, GFPmut, and other GFP mutants selected on the basis of altered spectral properties.

While other detectable labels could also be used (such as radio-labels, bio- labels such as biotin, histamine and enzymatic labels, fluorescent labels such as fluorescein, luciferase, and the like) the power of this system is that the reporter protein produced on stimulation of the test promoter enables direct selection of positive living cells. Thus, any reporter protein that is compatible with living cells and is conducive to enrichment and separation of positive cells may be used. Expression of GFP can be detected by techniques known to those skilled in the art, including, but not limited to, fluorescence microscopy, FACS, western blotting, immunodetection, and the like. Other labels are detected as appropriate for the type of label.

SUBSTITUTE SHEET (RULE 25) Any number of mammalian high-expression vectors may be employed in the present invention, including, but not limited to, pCDM8™, pCDNA, Amp™, and pCR-3™. We have tested these vectors in COS-7 cells for GFP expression using DEAE-dextran transfection. All give intense GFP fluorescence at approximately 40-50% transfection efficiency. However, the pCR-3™ vector is preferred because it provides slightly higher transfection frequencies and expression levels. An preferred vector should include a strong viral promoter, multiple cloning site, viral 3' non-coding region, E. coli selectable marker (such as Amp or SupF), mammalian selectable marker (such as G418 or GFP), viral origin of replications (such as OriP or SV40 Ori), and the large T or EBNA-1 sequences as appropriate.

Any number of cells may be used as the host cell, requiring only that the host cell contain all of the necessary ancillary components for signaling from a given nucleotide sequence to the test promoter. Thought not a requirement, in order to ensure that the system contains being studied compatible components, it is preferred that the cDNA library be made from cells that are compatible with the host cell. For example, we have shown that cDNA libraries made from CV-1 cells are compatible with COS cells (both cell types are derived from African green monkey cells), at least in their cytokine signal transduction machinery. The universal host/vector pairing is a COS cell and a vector with the SV40 origin of replication. Alternatively vectors with the OriP origin of replication can be used with CV- 1 cells stably expressing EBNA-1. Vectors are available from INVITROGEN™ to transfect CV-1 cells with EBNA- 1. Host cells include, but are not limited to, COS cells, human epithelial cells such as 293 or 293T, and Hela cells, but any cell type capable of transfection can be used. Specialized cells types, for example, that are IL-1 deficient, are also contemplated. Protocols for producing preparing and transfection DNA into host cells can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989) and other standard laboratory manuals.

Example 1 : Pilot Study: Detection of GFP in Single Cells in Response to IL-R Modulators

We describe herein a method for co-trans fecting an IL-8 test promoter-GFP reporter construct with a high expression cDNA library into COS-7 cells, and examination of pools of cDNA for the capacity to confer on single cells the following characteristics:

1) Expression of GFP in the absence of an external stimulus, and/or

2) Enhanced expression of GFP in response to low doses (~lpM) of IL-1. COS-7 monolayers in 4 well Lab-Tek chamber slides were transfected

(DEAE dextran/lμg each plasmid/well) (McMahon, C, et al. (1991) EMBO J. 10:2821-2832) with constructs containing no promoter (pO), the IL-8 promoter (pIL-8), the CMV promoter (pCMV) or co-transfected with pIL-8 and the human IL-1 receptor in pCDNA/I (pIL-lRI). Transfected cells were cultured for 48 hours to allow high expression of IL-IRI and then treated with lOnM IL-1 for 4 hours. Confocal Fluorescence Micrographs were taken (not shown) at Excitation 488 nm, 20 mW, 700 volts PMT*, 10X air objective. *Exceρt for pCMV 500 volts: these data were then scaled by a 16X multiplication since calibration data show that 50 volts = 2X increase in observed intensity. Images were analyzed in NIH IMAGE® and the numeric output converted to histograms with EXCEL®. Figure 2 shows the results of image analysis of confocal micrographs, both at 4 and 12 hours post IL-1 addition. Note that the x-axis in each of the three panels is different. In the absence of a functional promoter, GFP expression was

SUBSTITUTE SHEET (RULE 25) negligible and was not enhanced by IL-1 treatment. Expression driven by the CMV promoter gave approximately a 200-fold increase in GFP expression over background, and was unaffected by IL-1. Expression from the IL-8 promoter gives a basal level of expression of GFP that is about 4-fold over the pO control. IL-1 treatment (4 hours) induced an increase in GFP expression in 30% of the cells by about 2.5-fold over that seen without IL-1 , despite the fact that the basal transcription was ongoing for 24 hours by the time of IL-1 addition.

COS cells express about 500 IL-1 receptors per cell (Sims, J., et al. (1988) Science 241 :585-589). However, the data clearly show that these cells are capable of responding to IL-1 even with this low level of receptor expression and therefore contain all the components necessary to transcribe off pIL-8, albeit at low efficiency. These observations are consistent with our findings that a pIL-8- IL2Rα construct, containing the same promoter fragment, was IL-1 responsive in COS-7 cells (Mitcham, J., et al. (1996) J. Biol. Chem. 271 :5777-5783). In order to test the principle that introducing a DNA for an IL-lRI-pIL-8 pathway element into this system will enhance the IL-1 responsiveness of these cells, we co-transfected the IL-1 receptor type I (IL-IRI) into the pIL-8 containing cells. The use of the receptor in this control experiment is rational, since the system should be most sensitive to rate limiting steps in the pathway such as recptor expression. It is logical that the receptor would be a primary control or rate limiting step, being "switched" from the "off to the "on" state when some effector molecule binds.

The data in Figure 3 show that when IL- 1 receptors are over-expressed, there is a considerable increase in the activity of pIL-8 even in the absence of IL- 1. We have observed similar results in previous experiments using the pIL-8- IL2Rα reporter and controls (not shown) have shown that this response cannot be blocked with high concentrations of anti-IL-lR monoclonal antibodies or IL-1

SUBSTITUTE SHEET (RULE 25) receptor antagonist (Id.). The increase in pIL-8 expression was, therefore, not due to endogenous IL- 1 production but to spontaneous IL- 1 R activation when the receptor was expressed at levels of about 10⁵ copies/cell. Hence, we could easily detect those cells expressing the IL-1R cDNA. Stimulation with IL-1 for 4 hours produced a further increase in GFP expression in the majority of transfected cells. The data showed that at saturating DNA levels (about 1 μg/100,000 cells), about 30-40 cells/field (200 cells total/field- 1 7mm²) was detected as strongly positive in either the plus or minus IL-1 added to pIL-8/pIL-lRI co-transfected cultures. Therefore, in each 2cm² well there would be about 5,000 positive cells suggesting that a pool size of 500- 1000 is feasible for screening (5-10 positive cells/well). Based on these numbers, there is every reason to suppose that this procedure can form the basis of a practical expression screen based on pool screening techniques.

Example 2. Single Cell Detection: Hirt DNA Purification and Transformations

The power of this functional cloning system is that the individual cells can be selected and the cDNA clones therein can be purified immediately on detection. For example, using a cloning ring coupled with the single cell DNA purification techniques of Hirt, the DNA from a positive cell can be purified, and retransformed into bacteria for sequencing and further analysis.

The Hirt preparation of episomal DNA is generally as follows: to 1-3 X 10⁶ transfected cells on a plate add 1ml of buffer A (10 mM Tris, pH 7.5, 10 mM EDTA, 0. 6% SDS) and incubate 3-5 minutes at room temperature. Then add 0.25 ml of 5 M NaCl. Gently scrape the plate to avoid shearing the genomic DNA and transfer the entire solution to a micro fuge tube. Leave at 40 °C for 4 to 12 hours. Spin at 15,000 rpm for 15 minutes. If a small amount of DNA is expected,

SUBSTTTUTE SHEET (RULE 26) add carrier DNA (1-10 μg salmon sperm DNA). Phenol extract, ethanol precipitate and use the purified DNA to transform E. coli cells. This procedure can be scaled down to use on single cells isolated with a cloning ring. However, the amount of DNA recovered is very small. Therefore, an alternative approach (described below) uses a FACS to sort and collect positive cells from which DNA is purified and transformed into bacteria for amplification and further screening.

Example 3. Single Cell Detection: PCR Amplification

Because single cell DNA preparations and transformations are technically difficult, it may be desired to first amplify the cDNA insert of interest. This can be done by using oligonucleotides that are unique to the plasmid just 5' of the cDNA insert. The other primer can be selected to complement the 5' end reporter sequence or the junction sequence. Either single positive cells can be amplified in this way, or pools of FACS selected positive cells can be amplified. The PCR amplified DNA can then be re-ligated into a suitable vector and transformed into bacterial cells for ease of manipulation.

PCR amplification is very well known in the art and the details need not be described herein. However, for reference the reader is referred to the following: for general advice about PCR see: httpJ/www.fermentus.1 t/fermentus/catalog/PCR/DNA_amp/Protocol.htm; a variety of specific protocols can be found at http://research.nwfsc.noaa.gov/protocols.html; and a protocol for the amplication of a single mammalian cell is provided by Holding, C. and Monk, M. (1990) Genet. Res. 55(2): 120.

PCR amplification is a powerful technique, well able to amplify the DNA from single cells, as described above. However, unless the dNTP pools are well

SUBSTITUTE SHEET (RULE 25) balanced and the magnesium and polymerase levels optimized, the polymerase can tend toward error-prone replication. An error early in the amplification process could profoundly affect the functionality of a given cDNA. If this problem is suspected for a particular clone, it can be corrected by using the PCR amplified DNA or PCR generated clone to isolate the original cDNA from the starting library. However, careful selection of PCR conditions should minimize this problem.

Example 4. Single Cell Detection: FACS Selection of Positive Cells

A simple and powerful approach for positive cell selection is to FACS enrich for a population of positive cells. The histograms in Figure 2 show that the fluorescence in positive cells is much stronger than background fluorescence. Because the GFP-expressing cells are not destroyed by the detection means employed, the living cells can be selected by FACS (Yamasaki, et al. (1988) Science, 241 :825-28). The cDNA constructs therein are then purified (or their inserts amplified and ligated into an appropriate vector) and retransformed into bacteria to create a pool of positive clones. Positive clones can be confirmed by the co-transfection procedure above, sequenced and further analyzed. Though the preferred method of the invention is to detect an increase in reporter gene expression, decreases may also be detected using the invention. Further, an altered reporter readout could be due to the change in color of the reporter protein, for example.

The FACS sorting procedure is generally as follows: COS cells are co- transfected with pIL-8 and library DNA at 50 μg DNA per 10⁷ cells. Co- transfected COS cells are lifted from the plate by treatment with trypsin and EDTA. The cells are resuspended at 10⁶ cells/ml in PBS/BSA (0.01%). Positive cells are separated by sorting on a FACSTAR plus (Ar 488 line excite, 530bp filter detect). At 10-50,000 cells per minute, it will require about 1 to 2 hours to sort the library. The brightest 10% cells are subject to HIRT DNA preparation and transformation into E. coli. Plasmids are prepared from the transformed E. coli and used to again co-transfect COS cells. This can be repeated three times, if necessary, thus repeatedly enriching for positive clones. Individual colonies can be tested by co-transfection with pIL-8 and pCMV and confocal microscopy. Double positive clones are eliminated and the pIL8 (+)/pCMV(-) clones are chosen for further study.

Example 5. Design of Pool Screens

In the event that the above techniques are not readily available, one may still employ pool screening techniques with the expression cloning method described herein. The data presented above show that it will be possible to identify from a pool of cDNAs, a single cDNA encoding a cellular component controlling the expression of GFP via the pIL-8 construct. In previous applications of mammalian expression cloning techniques, the search has been for a unique protein, for example a receptor subunit. However in the experiments proposed here multiple gene products can be expected to read out in the screen. This fact raises two issues that need to be addressed: i) Elimination of false positives:

To eliminate from further analysis those cDNAs that affect steps other than the rate of transcription and lead to GFP accumulation. These include plasmid replication, mRNA stability, rate of translation, and rate of GFP degradation. While many of the cDNAs that read out for these reasons may be of interest, within the scope of the proposed studies they will be taken as "false positives." These can be screened out by testing against the pCMV reporter and eliminated if

SUBSTITUTE SHEET (RULE 25) they enhance expression, because even though the CMV promoter contains several mammalian transcription factor binding sites in the 600 bp core fragment, it is constitutively active in COS-7 cells and is unaffected by IL-1 , for example.

ii) Optimization of Pool Size

It is important to optimize the pool versus library size in order to minimize the time taken to screen the entire library while maximizing the discriminatory power of the screen, bearing in mind that signal strength, as measured by positive cells per pool transfected, increases linearly with decreasing pool size. Some estimates of the overall "behavior" of the library may be made from general information about eukaryotic message levels and also from our previous experience. General estimates suggest that translationally active and proliferating eukaryotic cells express about 400,000 copies per cell of poly A+ MRNA. A "rare" mRNA is expressed at about 10-20 copies per cell, and therefore about one in 30,000 mRNAs would be found to encode any particular rare protein (-100,000) protein molecules / cell).

We can compare these estimates with the results from our cloning of IL-1- RII (McMahon, C, et al. (\99 \) EMBO J. 10:2821-2832). A library of 5 X 10⁶ cDNAs was prepared from polyA+RNA (from CB23 cells: expressing about 20,000 copies per cell of the protein) using random hexanucleotide primers and screened in pools of 4,000 cDNAs. One of the first 85 pools tested by CV-1- EBNA transfection and ¹²⁵IL-1 binding autoradiography was positive and the screen was then terminated. Thus, one functional transcript was detected in 9% of the library, suggesting that there were about 1 1 such transcripts in the entire library, or one in 350,000.

This would be consistent with the expected mRNA frequency if only 10% of the relevant message was converted to cDNAs that gave functional transcripts.

SUBSTITUTE SHEET (RULE 25) Since the coding region and the 3' UTR of the IL-IRI mRNA are of approximately equal length (about 1.2 Kb) this suggests that half the transcripts would have primed 5' to the stop codon and hence failed to give functional mRNAs because our vector lacked a universal terminator. Bearing in mind the semi quantitative nature of the above estimates, this leaves a factor of 5 which could be accounted for by premature termination during cDNA synthesis and destabilizing sequences in the 3' UTR.

These calculations suggest that, in a library made from polyA+RNA using random hexanucleotide primers, an estimated one in 300,000 cDNAs will express a functional protein product corresponding to a unique gene expressed at low levels. As an illustration, the results of a simulated screen of 10⁶ cDNAs, in pools of either 10,000 or 1,000, assuming 5 functional cDNAs per unique mRNA, as the number of unique cDNAs that read out in the screen rises from 1 to 10,000 is shown in Table I. The figure of 10 is likely to be a realistic minimum in our screens because there are already known to be at least this number of components controlling the three known transcription factors with binding sites in pIL-8. The true number of positives cannot be reasonably estimated, since i) we do not know how many unknown components of the relevant signal transduction pathways exist, and ii) while it is likely that "signal transduction systems are degenerate," we do not know how degenerate.

Since the initial screen is likely a minor component of the work in the project, it would be prudent to test a small number of pools (20) at both pool sizes against pIL-8±-IL-l and pCMV±IL-1 (see above) to determine both the number of positive pools and the number of positive cells per pool versus pool size. These data will allow us to estimate how many cDNA clones are likely to be isolated and to choose an optimal pool size. . 97

Table I: Number of Positive Pools as a Function of Number of Positive cDNAs in Library. Entries show the predicted number of pools expressing a given number of positive cDNAs, calculated using the binomial theorem. The approximate number of positive cells/ 10⁵ can be estimated by multiplying the cDNAs per pool column by 30 for the 10,000 pool size and 300 for the 1 ,000 pool size. The cDNAs per library row represents unique cDNAs, each present at 5 copies in the library.

SUBSTITUTE SHEET (RULE 25) Example 6. Strategic Considerations for Future Experiments

Additional experiments can be performed using functional cloning and single cell detection system of the invention. These experiments include:

(1) Isolation of novel cDNA clones which encode elements of the cytokine signal transduction systems. cDNA clones that modulate the test IL-8 promoter can be isolated by the direct expression cloning methods described above. The cDNA library is prepared from CV-1 cells. This will help to avoid any species incompatibility problems between over expressed proteins and the background signaling machinery in COS cells.

(2) Mapping the functions of the products of all cDNAs isolated against the test promoter using a series of test promoter deletion constructs, containing all six pairwise and single binding site combinations. For example, the following promoter combinations can be tested and should define the sites through which, for any cloned sequence, IL-8 activity is modulated.

(3) Elucidating pathways by stimulating cells transfected with pIL-8± novel cDNAs with IL-1 and both, to map which elements that lie on pathways from TNFR, IL-1R and which are common.

(4) Further elucidating pathways by combinatorial transfections with cloned cDNAs and stimulating with TNF, IL-1 , and both, to determine quantitatively which cDNA regulates the most rate critical steps in the pathways and in which order the steps are organized by measuring the rates of GFP accumulation.

The methods we will use have been described in the previous sections. We are, as the pilot data illustrate, already performing GFP transfections and measurements on a routine basis. These experiments will allow us to clone cDNAs encoding novel signal transduction pathway elements, to determine which are most critical for setting the rate of pathway activity, to determine which pathways they control, and to gain some initial ideas of to what extent the pathways for IL-1 and TNF overlap. This information can all be generated within the experimental framework outlined above. In many instances the sequence of each cDNA clone will provide a clear cut guide as to its function, based on homology to known families of molecules. In addition, the frequency of positive clones in our libraries will allow us to estimate the number of different cellular components which can impact on the activity of this limited set of pathways. This will allow us to begin to address broader issues of redundancy and cross talk in the network of interacting signals that control patterns of gene expression in mammalian cells. Although we have demonstrated our functional cloning and single cell detection methodology with respect to the cytokine signal transduction pathway, in theory the system could be used to isolate any sequence that modulates the transcription of a test promoter or other regulatory gene sequence. Therefore, it would be possible to isolate those sequences important in the control of the P selectin, ELAM- 1 , and CD 1 la promoters, to name a few.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific polypeptides, nucleic acids, methods, assays and reagents described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

All articles and patents cited herein are incorporated by reference and repeated here for convenience only:

References

1. Fanslow, W., et al. 1990 Science 248:739-742.

₁ 1 I D5 c

2. MoWer, K., D. et al. 1993 J. Immunol. 151 : 1548-1561.

3. Sims, J., et al. 1988 Science 241 :585-589.

20 4. Smith, C, D. et al. 1990 Science 248:1019-1023.

5. Gruss, H. J., and Dower, S.K. 1995 Cyto/dnes and Molecular Therapy 1 :75-105.

6. Sims, J. E., and Dower, S.K. 1994 European Cytokine Network 5:539-546.

25

7. Rothe, M. , 1994 Cell 78:681-692.

8. Karin, M., and Smeal, T. 1992 Trends In Biochem. Sci. 17:418-422.

30 9. Sha. W.H. el al. 1995 Cell 80:321-330.

10. Huber, A., et al. 1991 Science 254:99-102.

1 1. Sims, J., et al. 1994 IL-1 SIGNAL TRANSDUCTION in Signal Transduction 35 Through Growth Factor Receptors., vol. 3. JAI Press, Greenwich, Conn., USA,

London, U,K., p. 197-222.

SUBSTITUTE SHEET (RULE 25) 12. Mitcham, J., et al. \996 JBiol. Chem. 271 :5777-5783.

13. Dower, S. 1993 P PHARMACOLOGICAL. BIOCHEMICAL AND MOLECULAR BIOLOGICAL STUDIES ON CYTOKINE RECEPTORS in Neiirobiology of Cytokines, vol. 16., E. B. De Souza, ed. Academic Press, Inc., New York, p. 3-30.

14. Fields, S. & Song, 0. 1989 Nature 340:245-6. 15. Seed, B. 1995 Current Opinion In Biotechnology 6:567-573.

16. Davis, S., et al. 1996 Cell 87:1 161-1 169.

17. Chalfie, M„ et al. 1994 Science 263:802-805.

18. McMahon, C., et α/. 1991 EMBO J. 10:2821-2832.

19. Valdivia, R.H., Falkow, S., 1996 Mol. Microbiol. 22(2):367-78. 20. Holding, C. & Monk, M. 1990 Genet. Res. 55(2): 120.

21. Yamasaki, et al. (1988) Science, 241 :825-28

SUBSTITUTE SHEET (RULE 25)

Claims

CLAIMSWe claim:

1. A method of determining whether a the expression from a test promoter or

other gene regulatory sequence is modulated by a test nucleic acid or test

compound , comprising:

a. transfecting a host cell with a first nucleic acid comprising a first test

promoter and/or other gene regulatory sequence coupled with a nucleotide

sequence encoding a detectable reporter protein, to produce a transfected

cell;

b. providing to the transfected cell (i) a second nucleic acid comprising a

second promoter coupled to a test nucleotide sequence, to produce a co-

transfected cell or (ii) a test compound; and

c. detecting the expression of said reporter protein in said co-transfected

cell if the expression levels of said reporter protein is different from the

level found in a cell transfected only with said first nucleic acid sequence.

2. The method of claim 1 , wherein said host cell is a mammalian cell.

3. The method of claim 2, wherein said mammalian cell is a COS or a Hela

cell.

SUBSTITUTE SHEET (RULE 25)

4. The method of claim 1 , wherein said reporter protein is fluorescently,

radioactively, biologically and or enzymatically labeled.

5. The method of claim 1 , wherein said reporter protein is selected from the

group consisting of green fluorescent protein (GFP), EGFPΓäó, d2-EGFP,

S6ST, T203Y, BFP and GFPmut or any other GFP variant.

6. The method of claim 1 , wherein said reporter gene expression is detected

by fluorescence activated cell sorting or fluorescence microscopy.

7. The method of claim 1 , wherein said test promoter comprises at least a

portion of a cytokine promoter.

8. The method of claim 7, wherein said test promoter comprises at least a

portion of a promoter selected from the group consisting of an IL-8. a P-

selectin, an ELAM- 1 and a CD11 promoter.

9. The method of claim 7, wherein said cytokine promoter is modulated by

IL- 1 or TNF╬▒ other components that directly or indirectly induce the IL-8

promoter.

SUBSTTTUTE SHEET (RULE 26)

10. The method of claim 1 , wherein said test nucleotide sequence modulates

one or more of the rate of transcription, plasmid replication, mRNA

stability, rate of translation and rate of degradation of said detectable

reporter protein.

1 1. A method for determining whether the expression of a test promoter or

other gene regulatory sequence is modulated by a test nucleic acid in

response to one or more agents, comprising:

a. transfecting a host cell with a first nucleic acid comprising a first

test promoter and/or other gene regulatory sequence coupled with a

nucleotide sequence encoding a detectable reporter protein, to

produce a transfected cell;

b. transfecting said transfected cell with a second nucleic acid

comprising a second promoter coupled to a test nucleotide

sequence, to produce a co-transfected cell;

c. providing to said co-transfected cell one or more agents; and

d. detecting the expression of said reporter protein in said agent-

stimulated co-transfected cell if the expression levels of said

reporter protein is different from the level found in said co-

transfected cell that was not provided with said agent(s).

SUBSTITUTE SHEET (RULE 25)

12. The method of claim 1 1 , wherein said host cell is a mammalian cell.

13. The method of claim 12, wherein said mammalian cell is a COS or a Hela

cell.

14. The method of claim 11 , wherein said reporter protein is fluorescently,

radioactively, biologically and/or enzymatically labeled.

15. The method of claim 14, wherein said reporter protein is selected from the

group consisting of green fluorescent protein (GFP), EGFPΓäó, d2-

EGFPS6ST, T203Y, BFP and GFPmut or other GFP mutant.

16. The method of claim 14, wherein said reporter gene expression is detected

by fluorescence activated cell sorting or fluorescence microscopy.

17. The method of claim 1 1 , wherein said test promoter comprises at least a

portion of a cytokine promoter.

18. The method of claim 1 1 , wherein said test promoter comprises at least a

portion of a promoter selected from the group consisting of an IL-8, a P

selectin, an ELAM- 1 and a CD1 1 promoter.

SUBSTTTUTE SHEET (RULE 26)

19. The method of claim 1 1 , wherein said agent(s) participate in a cytokine

signal transduction pathway.

20. The method of claim 11 , wherein said agent is a cytokine selected from the

group consisting of IL- 1 and TNF╬▒.

21. The method of claim 1 1 , wherein said expression levels are increased in

response to said agent(s).

22. The method of claim 1 1 , wherein said expression levels are decreased in

response to said agent(s).

23. The method of claim 1 1, further comprising the step of selecting against

molecules that stimulate expression of said reporter protein when coupled

to a test promoter which is unresponsive to said agent(s), thereby

eliminating false positives if expression is enhanced.

24. A method of identifying and isolating from a live cell a nucleic acid

encoding a molecule that modulates the expression of a test promoter or

other gene regulatory sequence, comprising the steps of:

SUBSTITUTE SHEET (RULE 25) a. transfecting a host cell with a nucleic acid comprising a test

promoter and/or other gene regulatory sequence coupled with a

nucleotide sequence encoding a detectable reporter protein, to

produce transfected cells;

b. transfecting said transfected cells with a library of cDNA

molecules, to produce a co-transfection library;

c. detecting the expression of said reporter protein in said co-

transfection library;

d. sorting the co-transfection library to create an enriched co-

transfection library, wherein said co-transfected cells are selected

for inclusion of cells expressing said reporter protein at levels that

are different from those found in unsorted co-transfected cells;

e. purifying, transforming, amplifying and repurifying the cDNA

molecules from step (b) until a sufficient level of enrichment for

cDNA molecules that modulate the expression of said reporter is

obtained; and

f. isolating said cDNA molecules.

25. The method of claim 24. wherein said host cell is a mammalian host cell.

SUBSTTTUTE SHEET (RULE 26)

26. The method of claim 25, wherein said mammalian cell is a COS or a Hela

cell.

27. The method of claim 24, wherein said reporter protein is fluorescently,

radioactively, biologically and or enzyrnatically labeled.

28. The method of claim 27, wherein said reporter protein is selected from the

group consisting of green fluorescent protein (GFP), EGFPΓäó, d2-EGFP,

S6ST, T203Y and GFPmut or other GFP variant.

29. The method of claim 27, wherein said reporter gene expression is detected

by fluorescence activated cell sorting or fluorescence microscopy.

30. The method of claim 24, wherein said test promoter comprises at least a

portion of a cytokine promoter.

31. The method of claim 24, wherein said test promoter comprises at least a

portion of a promoter selected from the group consisting of an IL-8, a P

selectin. an ELAM-1 and a CD 1 1 promoter.

SUBSTTTUTE SHEET (RULE 26)

32. A method of identifying and isolating from a live cell a nucleic acid

encoding a molecule that modulates expression from a test promoter or

other gene regulatory sequence in response to one or more agents,

comprising the steps of :

a. transfecting a host cell with a nucleic acid comprising a test

promoter and/or other gene regulatory sequence coupled with a

nucleotide sequence encoding a detectable reporter protein, to

produce transfected cells;

b. transfecting said transfected cells with a library of cDNA

molecules, to produce a co-transfection library;

c. providing to said co-transfection library one or more agents;

d. detecting the expression of said reporter protein in said co-

transfection library;

e. sorting the co-transfection library to create an enriched co-

transfection library, wherein said co-transfected cells are selected

for inclusion of cells expressing said reporter protein at levels that

are different from those found in unsorted co-transfected cells;

f. purifying, transforming, amplifying and repurifying the cDNA

molecules from step (b) until a sufficient level of enrichment for

cDNA molecules that modulate the expression of said reporter is

obtained; and isolating said cDNA molecules.

33. The method of claim 32, wherein said host cell is a mammalian cell.

34. The method of claim 33, wherein said mammalian cell is a COS or a Hela

cell.

35. The method of claim 32, wherein said reporter protein is fluorescently,

radioactively, biologically and/or enzymatically labeled.

36. The method of claim 35, wherein said reporter protein is selected from the

group consisting of green fluorescent protein (GFP), EGFPΓäó, d2-EGFP,

S6ST, T203 Y, GFPmut or other GFP variant.

37. The method of claim 35, wherein said reporter gene expression is detected

by fluorescence activated cell sorting or fluorescence microscopy.

38. The method of claim 32, wherein said test promoter comprises at least a

portion of a cytokine promoter.

SUBSTTTUTE SHEET (RULE 26)

39. The method of claim 38, wherein said test promoter comprises at least a

portion of a promoter selected from the group consisting of an IL-8, a P

selectin, an ELAM-1 and a CD1 1 promoter.

40. The method of claim 32, wherein said agent(s) participate in a cytokine

signal transduction pathway.

41. The method of claim 32, wherein said agent is a cytokine selected from the

group consisting of IL-1 or TNF╬▒.

42. The method of claim 32, wherein said expression levels are increased in

response to said agent(s).

43. The method of claim 32, wherein said expression levels are decreased in

response to said agent(s).

SUBSTTTUTE SHEET (RULE 26)