WO2005116070A2

WO2005116070A2 - Reporter system for detecting signal pathway activation in multiple cell types

Info

Publication number: WO2005116070A2
Application number: PCT/US2005/014199
Authority: WO
Inventors: Lewis T. Williams; Hongbing Zhang; Pierre Alvaro Beaurang; Srinivas Kothakota; Kristen Pierce
Original assignee: Five Prime Therapeutics, Inc.
Priority date: 2004-04-28
Filing date: 2005-04-27
Publication date: 2005-12-08
Also published as: WO2005116070A3; WO2005116070A9

Abstract

The invention provides a reporter system for studying signal transduction pathways. The reporter system includes a gene encoding a detectable gene product expressed in multiple cell types. A gene of interest can be used to target a specific locus in an embryonic stem cell, for example, the ROSA26 locus. The transformed stem cell is introduced into a blastocyst, which develops into a chimeric animal that produces the gene product of interest in multiple tissues, such that the effect of the gene product on the animal can be determined, both during development and in adulthood. Cell lines can be produced from cells or tissues obtained from the chimeric animal or its progeny. This reporter system can elucidate the components of signal transduction pathways when the reporter gene is activated under the regulatory control of a response element.

Description

X T ^φ^^g ϊ§^' r )R DETECTING SIGNAL PATHWAY ACTIVATION IN MULTIPLE CELL TYPES [001] This application claims the benefit (pursuant to 35 U.S.C. § 119(e)) of provisional applications 60/565,839, filed in the United States Patent and Trademark Office on April 28, 2004; 60/589,826, filed in the United States Patent and Trademark Office on July 22, 2004; and 60/642,604, filed in the United States Patent and Trademark Office on January 11, 2005. The disclosures of each are hereby incorporated by reference. INTRODUCTION FIELD OF THE INVENTION [002] This invention relates to applications 60/423 ,041 , filed November 1 , 2002, in the U.S. Patent and Trademark Office; 60/454,576, filed March 13, 2003, in the U.S. Patent and Trademark Office; PCT/US 03/34811, filed October 31, 2003, in the U.S. Patent and Trademark Office; 60/565,839, filed April 28, 2004, in the U.S. Patent and Trademark Office; and PCT/US 04/11270, filed April 30, 2004, in the U.S. Patent and Trademark Office, the contents of which are incoφorated herein in their entireties. [003] This invention relates to embryonic stem cells comprising reporter systems for detecting signal transduction pathway activation, inactivation, or inhibition. These reporter systems comprise reporter genes encoding heterologous polypeptides or antagonists thereto, under the control of elements responsive to activation, inactivation, or inhibition of a signal transduction pathway. The invention also relates to chimeric animals, tissues, and cells derived from embryonic stem cells that express the heterologous polypeptides or antagonists thereto. These animals, tissues, and cells can be used in research, for example, to determine the components of signal transduction pathways operating in health and disease. They can be used for therapeutic purposes, for example, to determine the efficacy, safety, toxicity, or pharmacokinetics of a potential drug candidate. They can also be used to determine the effects of therapeutic agents that affect signal transduction pathways. Additionally, these animals can be used to determine gene function in vivo and in vitro. This invention also relates to a DNA molecule comprising a liver-expressed promoter and a gene encoding a heterologous protein, which can be expressed to produce a functionally active protein in a chimeric reporter animal, as well as its tissues and cells. This invention further relates to the method of studying the effects of the introduced protein encoded by this DNA molecule on the chimeric reporter animal, its tissues, or its cells. BACKGROUND OF THE INVENTION [004] Signal transduction molecules form pathways that convert extracellular signals into cellular responses. Cells use myriads of signal-transduction pathways to regulate _ttseβulatlfJ SWism£fu|iβti^fi'iBrιd development. Signal transduction pathways comprise many different classes of proteins, among them ligands, receptors, kinases, phosphatases, proteases, transcription factors, GTPase switch proteins, which assume an active conformation only when bound to GTP, and adapter proteins, which connect multiprotein signaling pathways. The ability of cells to respond appropriately to extracellular and intracellular signals is dependent on proper regulation of signal transduction pathways. Abnormalities in signal transduction underlie many diseases, including the majority of cancers and many inflammatory conditions. [005] Stimuli that activate intracellular signal transduction pathways activate specific, known promoters. Promoter activation can be measured by reporter systems linked to the promoters, each of which can be further linked to a "readout" signal. The "readout" signal may be achieved by the expression of an easily detectable protein or a selectable marker. In current practices, reporter systems like these are generally introduced into cells by transfecting the cells with DNA molecules encoding the promoters and the "readout" signal, one cell type at a time. [006] One limitation of the current reporter systems is that some cell types are difficult to transfect with reporter systems. Another limitation is that the reporter system introduced into cells or animals is usually affected by the integration site and may be affected in a manner adverse to reporter detection. It is also difficult to introduce a reporter system into multiple cell types because transfections and selections are typically performed on one cell type at a time. Furthermore, it is cumbersome to introduce the reporter systems into intact animals, which typically necessitates generating a transgenic animal, and in most cases, the expression of the reporter systems is substantially hindered by the integration site. It would thus be advantageous to place the reporter in an open locus for homologous recombination so as to avoid the effect of the integration site. It would also be advantageous to situate the cells expressing secreted pathway-modulating factors adjacent to those harboring the reporters. Along those lines, then, it would be highly advantageous to express one or more secreted factors in the same cells with the reporter system. [007] Even if a reporter system can be effectively introduced into multiple cell types of an animal, it is currently difficult to study the modulating or therapeutic effect of heterologous proteins or polypeptide fragments on the chimeric reporter animal, its tissues, or its cells. This is largely due to the limitations associated with the methods of gene delivery. In vitro gene-delivery methods have been widely used to generate useful information about the function of the delivered proteins within the host cells, but these _mg ho4# iϊC ®efaIy£Bθ: u#elul in vivo. (Luo & Saltzman, 2000; Rochlitz, 2001). Viral in vivo DNA transfer methods are potentially powerful, but are often associated with low transfer efficiency, inability to sustain gene expression, danger to the host organism, or a combination of these drawbacks. (Nyuyen & Ferry, 2004). The efficiency of non- viral DNA transfer methods, including polylysine conjugates, various polymers, liposomes and naked DNA, is usually even lower than that of viral methods. This inefficiency has historically rendered the non- viral methods prohibitive and unpracticed. (Young & Dean, 2002; Wolff et al., 1990; Ascadi et al, 1991; Kobayashi et al, 2000). More recently, non- viral methods, including the intravascular delivery of naked plasmid DNA, in particular the hydrodynamic tail- vein method, have improved the delivery efficiency but yet to achieve sustainable long-term expression of the introduced gene. (Liu et al., 1999; Zhang et al., 1999; Nguyen & Ferry, 2004; Kobayashi et al, 2001). There is thus a need to find simple, inexpensive, and routine techniques that would allow efficient transfer as well as sustained expression of the transferred gene into the chimeric reporter animals so that the modulating or therapeutic effect of the introduced proteins or polypeptides on the animals may be measured and studied in vivo. SUMMARY OF THE INVENTION

[008] The invention provides a reporter system for studying signal transduction pathways. The reporter system includes a gene encoding a detectable gene product expressed in multiple cell types. A gene of interest can be used to target a specific locus in an embryonic stem cell, for example, the ROSA26 locus. The transformed stem cell is introduced into a blastocyst, which develops into a chimeric animal that produces the gene product of interest in multiple tissues, such that the effect of the gene product on the animal can be determined, both during development and in adulthood. Cell lines can be produced from cells or tissues obtained from the chimeric animal or its progeny. This reporter system can elucidate the components of signal transduction pathways when the reporter gene is activated under the regulatory control of a response element. [009] The invention provides reporter systems for signal transduction pathways. These systems comprise pathway-specific promoters linked to "readout" signal that are, for example, easily detectable proteins or selectable markers. The systems can be produced efficiently after they are introduced into non-human animals. The reporter systems are introduced into embryonic stem (ES) cells, which can be incorporated into one or more blastocysts, which can, in turn, be implanted into pseudo-pregnant non-human animals to produce chimeric animals expressing the reporter in a broad range of tissues and cell -!^t£y esr!!Iϊιl ^^^ provides an ES cell mouse expression system (ESpresso mouse). [010] Through this approach, transfecting a single ES cell can produce multiple transfected cell types, some of which may otherwise be difficult to transfect in their differentiated state. Substantially all the tissues in the resulting chimera have the potential to activate the reporter system in response to specific exogenous signals. The reporter systems can be specific for a single signal transduction pathway or can be expressed upon activation of any of a number of pathways. The reporter systems can also be specific for multiple integrated signaling pathways if relevant combination of pathway components such as transcription factor binding sites are included. The reporter systems in the different cell types of the chimeric animals can be used to detect pathway activation, or pathway deactivation following their activation, for example, by measuring the growth or differentiation factors that bind to cell-surface receptors during the activation or subsequent deactivation. The reporter systems in these cells can also be used in vivo and in vitro to measure the effect of signal transduction modulators, such as small molecules, secreted factors, or antibody agonists or antagonists of the pathway. [011] The invention also provides a DNA molecule with the promoter of a liver- expressed gene operably linked to a gene encoding a heterologous protein, which can be expressed in vivo to produce a functionally active secreted protein in a chimeric reporter animal, its tissues, or its cells. The invention further provides methods for sustained expression of DNA molecules of this kind in the chimeric animal, its progeny, and tissues or cells thereof. The protein or polypeptide fragment encoded by this DNA molecule, as well as the methods of sustained expression, may be used as tools to study the in vivo effect of the introduced secreted protein or polypeptide fragment on the signal transduction pathways in the chimeric reporter animal. [012] The invention provides a method of determining a function of a gene by introducing the gene into an embryonic stem cell, then introducing the embryonic stem cell into a blastocyst comprising an ES cell with exogenous DNA; allowing the blastocyst to develop into an animal; and observing the animal to determine gene function. Methods of testing the animal for functional alterations are known in the art. This invention also encompasses a method of determining gene function by introducing the gene into an embryonic stem cell, then introducing that embryonic stem cell into a blastocyst obtained from the progeny of a chimeric animal of the invention. [013] In its various embodiments, the invention also provides methods for evaluating the function of secreted proteins, tagging secreted proteins and identifying their target §ϊls^,ϊMK ^I^εx ιffes' ύ! ^lllo e than one molecule such s functionally interacting molecules, studying and modulating in vivo protein dynamics such as fusion protein function, and study and modulating antibody function, such as single chain antibody function. BRIEF DESCRIPTION OF THE TABLES AND FIGURES [014] Table l. Liver-expressed genes containing promoters. The liver-expressed genes containing promoters in this list are useful for introducing secreted protein or polypeptide factors into a chimeric reporter animal. Each is identified by an internal reference number (FP ID); a Reference ID that can be used to access information on the gene in the National Center for Biotechnology Information (NCBI) database; and annotation from the NCBI database (Genes Containing Useful Promoters). This list is not intended to be exhaustive and may have not included other promoters suitable for the stated purposes. [015] Table 2. Internal identification numbers (FP ID) and sequence identification numbers (SEQ ID NOS.) for the promoter Sequences. These promoter sequences are useful for introducing secreted protein or polypeptide factors into a chimeric reporter animal. It includes SEQ ID NOS: 1 - 122, each of which sequence provides the 5' untranslated region (utr), which contains the transcription start sites (TSS) and the genomic region about 1000 bp upstream of the utr for the identified gene; SEQ ID NOS: 123 - 244, each of which sequence provides the 5' utr of the identified gene; and SEQ ID NOS: 245 - 392, each of which sequence provides the 1500 bp upstream of the translation start site and includes the 5' utr of the identified gene. Each sequence is also associated with a Reference ID of Table 1. Table 2 further includes SEQ ID NOS: 393 and 394, which are introns useful for introducing secreted factors into the chimeric reporter animals of the present invention.

[016] Table 3. Coordinates of selected intronic sequences. These selected intronic sequences may become associated with the promoters as exemplified in Tables 1 and 2 when they are used to introduce secreted factors into a chimeric reporter animal. It includes the Source JD, the human chromosome on which the gene is located, a designation as to whether the intron belongs to the plus or the minus strand, as well as a designation of the intron' s genomic coordinates within the untranslated region (UTR Intron Coordinates).

[017] Figure 1. Design of targeting vectors for secreted molecules and gene targeting to the ROSA26 locus. The targeting vector PGKneobpA is a combination of PGKneo from New England Biolabs (Beverly, MA) and bovine growth hormone poly A (bpA) from BD Biosciences Clontech (Palo Alto, CA). It comprises a 5' homologous arm of fjffi" i®^t!aτgέu | etfi|ri^,i$n adenovirus major late transcript splicing acceptor (SA) that was PCR amplified from adenovirus genomic DNA, two gateway sites, a PGKneobpA neomycin selection marker (PGKneobpA), a 3' homologous arm of the ROSA26 targeting vector, as well as a non-TK negative selection marker. The 5' and 3' homologous arms were PCR amplified and cloned from genomic DNA according to information available in public genomic databases. A secreted factor of interest can be cloned into the ROSA26 target vector by cloning a secreted factor gene into a Gateway entry vector, followed by cloning it into the ROSA 26 targeting vector using the Gateway cloning technology (Invitrogen, Carlsbad, CA). [018] Figure 2. Design of response element vectors. The response element vectors of the invention comprise a pSK (Stratagene, La Jolla, CA) plasmid backbone, a 5' homologous arm of the ROS A26 targeting vector, a poly A site, a regulatory region of interest such as the FGF inducible response element (FiRE), a bpA site, a PGKneobpA site, a 3' homologous arm of the ROSA26 targeting vector, or a non-TK negative selection marker. [019] Figure 3. Schematic representation of one of the DNA constructs that may be used to introduce secreted factors into a chimeric reporter animal. "3A4 promoter" denotes the cytochrome P450 3A4 promoter. "EPO" is the gene encoding erythropoietm, and "lacZ" is the gene encoding β-galactosidase. "bPolyA" denotes a polyadenylation sequence. DETAILED DESCRIPTION OF THE INVENTION [020] The invention provides a novel model system for studying the molecular components of signal transduction pathways and the roles of these components in health and disease. It uses methods well known for incorporating exogenous nucleic acid sequences into the genome of animals, for example, intravascular injection of nucleic acids, to establish long term gene expression. Definitions [021] A "stem cell" is a pluripotent or multipotent cell with the abilities to self-renew, to remain undifferentiated and to become differentiated. Stem cells can divide without limit, for at least the lifetime of the animal in which they naturally reside. Stem cells are not terminally differentiated in that they are not at the end of a differentiation pathway. When a stem cell divides, each daughter cell can either remain a stem cell or embark on a course that would lead to terminal differentiation. [022] An "embryonic stem cell" is a stem cell that is present in or isolated from an embryo. It can be pluripotent, with the capacity to differentiate into each and every cell ipC f entan tlS

with the ability to differentiate into more than one cell type. A pluripotent ES cell can also be said to be totipotent, with the ability to develop and differentiate, by cell division, into the whole organism. Embryonic stem cells derived from the inner cell mass of the embryo can act as pluripotent cells when placed into host blastocysts. [023] The term "gene" refers to a nucleic acid sequence that comprises coding sequences necessary for the production of a polypeptide or polypeptide precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties of the full-length polypeptide or a fragment thereof are retained. The term also encompasses the coding region of a gene and the sequences located adjacent to the coding region on both the 5' and 3' ends, such that the gene corresponds to the length of the full-length mRNA. The term "gene" encompasses both cDNA and genomic forms. [024] A "gene locus" is the position of a DNA segment, such as a gene, on a chromosome. For example, the G3BP (BT-5) locus is the position of the ras-GTPase- activating protein-binding protein (G3BP) in the BT-5 mouse cell line. The Rosa 26 locus is the position at which the ROSAβgeo retrovirus integrated into the genome of the ROSAβgeo26 (ROSA26) mutant strain of mice (Zambrowicz et al, 1997). [025] The terms "gene expression," and to "express" a nucleotide, used interchangeably herein, refer to the conversion of the information contained in a gene into a gene product such as an RNA or a protein. [026] An "intron" is a non-coding region of a gene which is transcribed into nuclear RNA. They are removed, or spliced out, from the nuclear RNA transcript, and are therefore absent in the mRNA transcript. Introns may contain regulatory elements such as enhancers. "Heterologous introns" are those derived from any source that does not naturally occur at the location at which the heterologous intron occupies or are derived from a cell type other than the one in which the heterologous intron is found. Heterologous introns may be derived, for example, from a tissue other than the source of the intron, or from an animal of a different species. [027] A "promoter" is a region of DNA that binds RNA polymerase before initiating the transcription of DNA into RNA. It directs RNA polymerase to bind to DNA, to open the DNA helix, and to begin RNA synthesis. Within the promoter sequence is a transcription initiation site, as well as RNA polymerase binding domains. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Some promoters are "constitutive" and initiate transcription in the absence of regulatory influences. Some

initiate transcription exclusively or selectively in one or a few tissue types. Some promoters are "inducible" and initiate gene transcription under the influence of an inducer. Induction can occur, for example, as the result of a physiologic response, a response to outside signals, or artificial manipulation. A promoter may be operably linked to a coding sequence and may be capable of effecting the expression of the coding sequence when proper factors are present. The promoter need not be contiguous with the coding sequence, but functions to direct the expression of the coding sequence. Thus, for example, intervening transcribed but untranslated sequences can be present between the promoter sequence and the coding sequence, as can translated introns. Such a promoter sequence, though interrupted and not naturally contiguous, is considered operably linked to the coding sequence. [028] A "response element" is a regulatory nucleic acid sequence that receives input from a physiologic or pathologic signal and contributes to the organism's response to that signal. Response elements may comprise specific binding sites for regulatory molecules. Response elements may increase or decrease transcription. They may act in a cell type- specific manner, but may also respond to one or more stimuli. Some response elements were found to integrate the input of multiple signal transduction pathways (Haremaki et al, 2003). [029] A "reporter system" is a group of interrelated elements including a reporter gene, its gene products and agents used to detect the gene products. A reporter gene typically encodes a gene product, that can be easily assayed. A reporter gene is typically operably linked to the upstream sequence of another gene and is typically introduced into cells via transfection. The assay detects and/or measures the gene product as a "readout signal." An example of a reporter gene is the lacZ gene, and an example of its gene product is β- galactosidase. The reporter gene is used to identify the active signal transduction pathways in the cell type, to which the reporter gene was introduced, and to determine the effects of test agents on response elements of a gene of interest. [030] "Signal transduction" is the conversion of a signal from one physical or chemical form into another. It can refer in particular to the sequential process initiated by interaction of an extracellular signal, such as a hormone, a growth factor, or a neurotransmitter, with a receptor, which causes a change in the level of an intracellular second messenger, such as calcium or cyclic AMP, and in turn culminate into one or more specific cellular responses, often mediated by the activation of a transcription factor. A "signal transduction pathway" is either or both the collection of molecules or the cascade of processes by which signal transduction takes place. CIOft^ & in.tia^^-'ris^piogressive developmental change to a more specialized form or function. Cell differentiation is the process a cell undergoes as it matures to become an overtly specialized cell type. Differentiated cells have distinct characteristics, perform specific functions, and are typically less likely to divide than their less differentiated counterparts. An undifferentiated cell, such as an immature, embryonic, or primitive cell, typically has a non-specific appearance. It may perform multiple, nonspecific activities, and may perform poorly, if at all, the functions typically performed competently by differentiated cells. [032] A "blastocyst" is an embryo at an early stage of development in which the fertilized ovum has already undergone cleavage, and the trophectoderm and a spherical layer of cells surrounding a fluid-filled cavity is forming or has formed. Inside the trophectoderm is a cluster of cells termed the inner cell mass. The trophectoderm is the precursor of the placenta, and the inner cell mass is the precursor of the embryo. Cells of the early mammalian embryo are pluripotent. [033] A "chimeric animal" or a "chimera" is an animal comprised of elements derived from genetically distinct individuals. When an inner cell mass blastomere of one animal is transferred into the embryo of a second animal, a donor cell of the blastomere can contribute genetic elements to every organ of the host embryo. The transferred inner cell mass blastomere may comprise recombinant DNA. [034] "Progeny" are those born of or derived from another. Progeny include all descendents of the first, second and all subsequent generations. Progeny include those organisms taken, received, or obtained from a parent organism. [035] A "cell line" is a population of cultured cells that has undergone a change that allows the cells to grow and proliferate in culture. [036] A "disease" is a pathological, abnormal and/or harmful condition of an organism. The term includes conditions, syndromes, and disorders. [037] A "therapeutic" or a "therapeutic agent" is an agent that is palliative, curative, or otherwise useful in treating or ameliorating a disease. A "prophylactic" is an agent that prevents the occurrence or recurrence of a disease. Examples of prophylactics include, but are not limited to, drugs or vaccines. [038] "Modulators," "modulatory agents," or "modulating agents" include substances that bind to and/or modulate a level or activity of a polypeptide or a level of mRNA encoding a polypeptide or nucleic acid, or substances that modulate the activity of a cell containing a polypeptide or nucleic acid. They may act as agonists, mimicking the function of an active molecule. Agonists include, but are not limited to, hormones, > ³C Bbo ;p^ as well as analogues and fragments thereof. They may also act as antagonists, competing for binding sites with an agonist but failing to induce an active response. Antagonists include, but are not limited to, hormones, antibodies, neurotransmitters, soluble receptors, as well as analogues and fragments thereof. [039] The terms "protein," "peptide" and "polypeptide," used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include naturally- occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or designer amino acids, amino acid analogs, peptidomimetics, depsipeptides, and polypeptides having modified, cyclic, bicyclic, depsicyclic, or depsibicyclic peptide backbones. The terms include single-chain proteins as well as multimers. The terms also include conjugated proteins; fusion proteins, which include, but are not limited to fetuin fusion proteins; glutathione S-transferase (GST) fusion proteins; fusion proteins with a heterologous amino acid sequence; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without N- terminal methionine residues; pegolyated proteins; and immunologically tagged, or his- tagged proteins. Also included in these terms are variations of naturally-occurring proteins, where such variations are homologous or substantially similar to the naturally- occurring protein, as well as the corresponding homologs from different species. Variants of polypeptide sequences include sequences comprising insertions, additions, deletions, or substitutions. [040] A "secreted" protein or polypeptide is a protein or polypeptide produced by a cell and exported extracellularly. Secreted proteins or polypeptides include extracellular fragments of transmembrane proteins that are proteolytically cleaved, as well as extracellular fragments of cell surface receptors, the fragments of which may be soluble. [041] The term "host cell" refers to an individual cell or cell culture, which can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide of the invention. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. [042] A "vector" is a nucleic acid molecule originating from a virus, a plasmid, a synthetic source, or a cell, into which another nucleic acid fragment of appropriate size can be integrated without losing the molecule's capacity for self-replication. Vectors can introduce nucleic acids into host cells, where they may be reproduced.

are those having an origin from a virus, or more generally an origin with a particle of nucleic acid (RNA or DNA) enclosed in a protein coat and generally capable of replicating within a host cell and spreading from cell to cell. [044] A "transcription initiation site" is the location of the first DNA nucleotide transcribed to RNA. The nucleotide at which transcription begins can be designated +1, and nucleotides numbered from this reference point. Negative numbers can indicate upstream nucleotides and positive numbers indicate downstream nucleotides. [045] "Transfection agents" bind to or complex with oligonucleotides or polynucleotides, and in doing so, mediates their entry into cells. Examples of transfection agents include, but are not limited to, cationic liposomes, lipids, polyamines, polyethylenimine and polylysine complexes. [046] The term "intravascular" refers to a route of administration in which a composition such as the nucleic acid composition of the present invention is placed within a vessel that is connected to a tissue or organ within the body of an animal. Within the cavity of the vessel, a bodily fluid flows to or from a body part. Examples of bodily fluids include blood, lymphatic fluid and bile. Examples of vessels include arteries, veins, lymphatics and bile ducts. The intravascular route includes delivery of nucleic acids or other agents through the tail vein of a mouse or a non-human animal. [047] A "liposome" is an artificial phospholipid bilayer vesicle formed from an aqueous suspension of phospholipid molecules. It may comprise one or more concentric phospholipid bilayers. Liposomes may be used medically, especially to convey vaccines, drugs, enzymes, or other substances to targeted cells or organs. [048] A "functionally active" or "biologically active" entity, or an entity having "biological activity," is one having at least one structural, regulatory, or biochemical function of a naturally-occurring molecule, or any function related to or associated with a metabolic or physiologic process. For example, an entity is functionally active when it participates in a molecular interaction with another molecule, when it has therapeutic value in alleviating a disease condition, or when it has prophylactic value, such as when it induces an immune response to the molecule. The functional activity of the present invention can include an improved desired activity, or a decreased undesirable activity. For example, the protein eryfhropoietin is functionally active when it stimulates the production of red blood cells. [049] As used herein, "extracellular" refers to the region outside a cell. The extracellular fragment of a transmembrane protein extends to the cell exterior.

to the region of the cell contained within its plasma membrane. The intracellular fragment of a transmembrane protein extends into the cell interior. [051] A "transmembrane protein" is a protein that extends into or through a lipid bilayer. Transmembrane proteins can span the membrane once, or more than once. They can function on one or both sides of a lipid bilayer, or transport molecules across the bilayer. [052] The term "operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their desired function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper transcription factors and other elements required for transcription are present. Such a promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated-yet-transcribed sequences can be present between a promoter sequence and a coding sequence, as can translated introns, but the promoter sequence can still be considered "operably linked" to the coding sequence. [053] The terms "liver-expressed gene" and "gene expressed in the liver," as used interchangeably herein, refer to a gene that is expressed in the liver. A "liver-expressed gene" may also be expressed in tissues other than liver. [054] A "naturally-occurring" molecule is one that exists in nature and without artificial aid. It can exist in any species, and includes all allelic and splice variants. [055] "Overexpression" includes any measurable increase over expressions at normal or baseline levels. [056] A "pharmaceutically acceptable carrier or excipient" is a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any conventional type. A pharmaceutically acceptable carrier or excipient is not toxic to the recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation. For example, a pharmaceutically acceptable carrier for a formulation containing polynucleotides does not include nucleases or other compounds known to be deleterious to polynucleotides. Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the effectiveness of the formulation. Other materials such as anti-oxidants, humectants, viscosity stabilizers, or the like can also be added as necessary.

tends to maintain a constant pH when a given concentration increment of hydrogen ion or hydroxide ion is added. At pH values outside the buffer zone, the capacity to resist changes in pH is less. The buffering power is at its maximum at the pH where the concentration of the proton donor (acid) equals that of the proton acceptor (base). Buffered solutions typically contain conjugate acid-base pairs. A buffered solution will demonstrate a lesser change in pH than an unbuffered solution in response to addition of an acid or a base. Any conventional buffer, including but not limited to Tris, phosphate and bicarbonate, can be used with the compositions herein. [058] An "autocrine process" denotes a process where the substance secreted from one type of cells acts on or otherwise affects the cell-surface receptors of the same type of cells. This is in contrast with the "paracrine process" where the substance secreted from one type of cells acts on or otherwise affects the cell-surface receptors of adjacent or neighboring cells of different type. This can be further compared to a "endocrine process" where the secreted substance from one type of cells passes through the bloodstream and acts on or otherwise affects cell-surface receptors of remote cells of different types. [059] The term "sustained expression" refers to the expression of a gene product for more than 2 to 4 days. For example, the expression of a protein for 5 to 7 days, 8 to 10 days, 11 to 13 days or more than 13 days constitutes sustained expression. Reporter Systems [060] The invention provides reporter systems for signal transduction pathways. In one of those systems, promoters specific to a signal transduction pathway are linked to a reporter gene product, which can be efficiently produced by introducing the reporter system into non-human animals. The reporter systems are introduced into ES cells, which are in turn introduced into one or more blastocysts. The blastocysts are then implanted into pseudo-pregnant non-human animals to produce chimeric animals that express the reporter systems. The resulting animals are called chimeric reporter animals. [061] Reporter systems typically comprise vectors with a reporter gene downstream of the cloning site. The reporter gene is usually chosen to be a protein that is not found in humans and is simple to assay for a readout signal. Reporter genes of the invention include, but are not limited to, those commonly used to examine the control of eukaryotic gene expression. One is the bacterial chloramphenicol acetyl transferase (CAT) gene. Another commonly used reporter is β-galactosidase, the product of the lacZ gene, which encodes an enzyme that hydrolyses the beta galactoside linkage in lactose to produce glucose and galactose. β-galactosidase also hydrolyses the chromogenic substrate p^;2s¥pj6p lttfogaIactδ'sMe:!pP.f^fβ). Another reporter gene, firefly luciferase, encodes a gene product that catalyses the reaction between luciferin and ATP, which produces photons of light detectable in a chemiluminescent bioassay for ATP. Yet another commonly used reporter gene encodes alkaline phosphatase, which catalyses the cleavage of inorganic phosphate non-specifically from a wide variety of phosphate esters, with a pH optimum greater than about 8. Green fluorescent protein, a jellyfish protein that, upon excitation with ultraviolet light, fluoresces with green visible light, is encoded by another commonly used reporter gene. The reporter systems of this invention also comprises humanized versions of GFP, wherein codons of the naturally-occurring nucleotide sequence are changed to more closely match the human codon bias, and GFP may be derived from a variety of species including Aequoria Victoria, Renilla reniformis, Renilla mulleri, and Ptilosarcus guernyi. The invention also includes derivatized versions of GFP, including but not limited to Enhanced Green Fluorescent Protein and other similar derivatives (U.S. Patent Nos. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445; 5,874,304; WO 99/49019; Peelle et al, 2001; Ma ∑ etal, 1999). Signal Transduction Pathway Modulation In Vitro and In Vivo [062] The reporter systems of the invention can provide information regarding the components, functions and effects of cellular signal transduction pathways. They are also useful for identifying pathway components that are rationally targeted in drug development and for measuring the effect of therapeutic agents on the target pathway components. The reporter systems of the invention can provide this information for many cellular signal transduction pathways. For example, they can be used to provide information about the vascular endothelial growth factor (VEGF) and the NF-κB transcription factor pathways, as well as therapeutic agents that act upon these pathways. The therapeutic agents, on the other hand, can include, but are not limited to, small molecules, secreted factors, or antibody agonists or antagonists of the pathways. [063] VEGF is a member of a family of homodimeric glycoproteins that are structurally related to the platelet-derived growth factors. Members of the VEGF family include placenta growth factor (PIGF), VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, colony stimulating factor 1 (CSF-1) and stem cell factor (SCF). Some members of the VEGF family, such as VEGF 121, VEGF 165, or others, are known to be secreted, while others, such as VEGF 189 or VEGF 206, are known to bind to cell-surface heparin-like molecules of the producing cells. (Bast et al, 2000). Known components of VEGF signal transduction pathways also include VEGF receptors, such as VEGF receptor- 1 |*!δ 7EGrøa^E ,ό¹ te^fJ 'pllSe tyrosine kinase 1 (Flt-1); VEGFR-2, also called kinase insert-domain containing receptor (KDR) in humans, or fetal liver kinase 1 (Flk-1) in rodents, respectively; VEGFR-3, also called Flt-4; neuropilin-1; and neuropilin-2 (Eurekah Bioscience Collection). [064] VEGF is known to activate multiple signaling pathways (Eurekah Bioscience Collection). VEGF has been described to activate the src pathway. Distinct members of the src kinase family, such as Src, Yes, or Fyn, are involved in distinct VEGF-mediated processes (Eurekah Bioscience Collection). VEGF, mediated by the phosphoinositol 3 (PI₃) kinase-AKT signaling pathway has been reported to be a survival factor for endothelial cells (Eurekah Bioscience Collection). [065] VEGF is a selective endothelial cell mitogen that promotes angiogenesis, the process of forming new blood vessels. New blood vessels may be formed by sprouting endothelial cells from pre-existing vessels, by intravascular subdivision and by remodeling, a process that transforms relatively uniformly sized vasculature into the small and large vessels network that undergoes maturation by recruiting smooth muscle cells and pericytes. Pathological neovascularization by angiogenesis plays a role in the progression of major diseases such as cancer, psoriasis, diabetes, ischemic disorders, or rheumatoid arthritis (Shibuya, 2003). VEGF has been described to induce not only tumor angiogenesis, but also blood vessel-dependent metastasis (Shibuya, 2003). VEGF has been described to bind and activate two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), and stimulate endothelial cell growth, survival and vascular permeability (Shibuya, 2003). Endothelial proliferation in angiogenesis has also been described to be mediated via the ras-raf-mitogen activated protein (MAP) pathway, while protein kinase C (PKC) activation has been described to be involved in endothelial migration and vascular permeability (Eurekah Bioscience Collection). [066] Knowledge of the components and function of the signal transduction pathways activated by VEGF is useful for developing VEGF antagonists. Current strategies include the development of receptor tyrosine kinase inhibitors, such as the small molecule tyrosine kinase inhibitor SU5416, the small molecule aniline-phthalazine PTK787, or the monoclonal antibodies directed against VEGF or its receptors as exemplified by the VEGF-specific antibody bevacizumab (Noble et al, 2004). Some of these agents have demonstrated antitumor activity in vivo in clinical trials (Hasan & Jayson, 2001). [067] The transcription factor NF-κB controls a wide range of genes and is involved in disease processes. The signal transduction pathways that utilize NF-κB-regulated F" OrϊήsefipCΦffiftia idjδgr l fti ^nes that encode proteins involved with immune and inflammatory responses as well as those involved with cell growth control (Baldwin, 1996). Many different intracellular signals induce activation of NF-κB. These signals may include hormones, stress, bacterial or viral infection, UV irradiation, B or T-cell activation, lipopolysaccharide, and certain cytokines, such as TNF or IL-1. [068] NF-κfi is a heterodimer of two related proteins of 65 kDa and 50 kDa (p65 and p50) that share a region of homology at their N-termini. That N-termini homologous region is required for DNA binding and dimerization. In resting cells, NF-κB is found in the cytoplasm. In response to an extracellular signal, NF-κB translocates to the nucleus, where it binds to specific sites in the DNA and regulates transcription (Lodish et al, 2000). In its inactive state, NF- B is sequestered in the cytoplasm by direct binding to its inhibitor I- B, which masks the nuclear localization sequences. In response to an extracellular signal, I-κB is phosphorylated at two N-terminal serine residues, targeted for ubiquitination, and degraded in the proteasome. Upon activation, NF-κB is released from I-κB and translocated to the nucleus where it would regulate gene expression. NF- KB then binds to specific DNA sequences in the nucleus and regulates gene expression. For example, NF-κB has been described to activate the transcription of immunoglobulin kappa light chains in B lymphocytes (Lodish et al, 2000). [069] Both peptide hormones and steroid hormones can regulate signal transduction pathways that utilize NF-κB. For example, peptide hormones may activate protein kinase C via the inositol pathway, releasing NF-κB from I-κB, thus permitting its translocation to the nucleus. Steroid hormones may inhibit NF-κB-regulated gene transcription. As an example, an anti-inflammatory glucocorticoid can bind to its receptor and induce a heat shock protein, which mediates the translocation of NF-κB to the nucleus but prevents the transcription of certain pro-inflammatory proteins, such as interleukin-6. Response Elements [070] Suitable response elements of the invention include fibroblast growth factor inducible response element (FiRE), cAMP response element (CRE), NF-κB response element (NF-κBRE), antioxidant response element (ARE), xenobiotic response element (XRE), serum response element (SRE), hypoxia response element (HRE), peroxisome proliferator response element (PPRE), glucocorticoid response element (GRE), activator protein response element (AP-1 RE), estrogen response element (ERE), interferon stimulatory response element (ISRE), interferon gamma activated sequence (GAS), tonicity-responsive enhancer/osmotic response element (TonEBP/ORE), retinoic acid ^ffeϊβon^ et M βl ^^ιi n§f5'( T)GATA(A/G)-3' (GATA) response element (GATA-RE). [071] The FiRE on the syndecan-1 gene has binding sites for activator protein 1, fibroblast growth factor (FGF)-inducible nuclear factor and upstream stimulatory factor (Jaakkola et al, 1998; Jaakkola & Jalkanen, 1999). It can induce gene expression in multiple cell types, as well as respond to multiple growth factors and affect transcription in a cell type-specific manner. FiRE responds to FGF-1, FGF-2 and FGF-4 in fibroblasts, but displays a different response pattern in keratinocytes than in fibroblasts. [072] Fibroblast growth factors (FGF) comprise a multigene family that exhibits mitogenic activity toward a wide variety of cells of mesenchymal, neuronal, and epithelial origin. The family includes, but is not limited to, acidic FGF ( FGF, FGF-1), basic FGF (bFGF, FGF-2), int-2 (FGF-3), hst/KS3 (FGF-4), FGF-5, FGF-6, keratinocyte growth factor (FGF-7), androgen-induced growth factor (AIGF or FGF-8), and glia activating factor (GAF or FGF-9). [073] Both acidic and basic FGF are angiogenic in vivo and comprise single-chain polypeptides of about 17,000 daltons. They share approximately 55% amino acid sequence identity. FGF-3 is a mitogen for mammary epithelial cells, normally expressed only in embryonic tissues. It has been shown to cause mammary gland hyperplasia in female mice and benign epithelial prostate hyperplasia in male mice (Bast et al, 2000). FGF-4 is a demonstrated mitogen for vascular endothelial cells, human melanocytes and mouse NIH/3T3 fibroblasts (Bast et al, 2000). FGF-5 is mitogenic for mouse fibroblasts and bovine heart endothelial cells (Bast et al, 2000). FGF-6 is a demonstrated mitogen for NIH/3T3 cells. FGF-7 was observed to be an epithelial cell mitogen, the expression of which has been documented in stromal, but not epithelial, cells of most epithelial tissues where it participates in epithelial renewal during wound repair and acts as a stromal mediator for epithelial cell proliferation and/or differentiation (Bast et al, 2000). FGF-8 is expressed during reproductive tract development and in the adult testes, acting in an autocrine manner to stimulate mammary carcinoma cell proliferation. FGF-9 has been implicated in oncogenic transformation in diseases such as colon or ovarian cancers (Bast et αZ., 2000). [074] Activation of the CRE is one of the later steps in those signal-transduction pathways initiated upon ligand binding to G_s protein-coupled receptors. Ligand binding activates adenylyl cyclase, which in turn stimulates cAMP production. The increase in intracellular cAMP induces the dissociation of protein kinase A catalytic subunits from their regulatory subunits. The catalytic subunits then move into the nucleus, where they

including the transcription factor cAMP response element binding protein (CREB). CREB has been reported to activate genes with the exacting palindromic CRE consensus sequence TGACGTCA in the promoter region, and CREB is ubiquitously expressed in the brain (Siegel et al, 1999). [075] NF-κB is a transcription factor found in cells that transcribe immunoglobulin light chain genes, and it is involved in various aspects of the immune response. In its inactive state, NF-κB is retained in the cytoplasm while complexed with an inhibitory subunit. The inhibitory subunit can be targeted for degradation by protein kinase C phosphorylation. After degradation, NF-κB dissociates from the inhibitory subunit and translocates to the nucleus. There, it activates various target genes by interacting with NF-T BRE. Inducing the NF-κB signal transduction pathway in pre-B cells enables endogenous NF-κB to bind its enhancer element, thus activating gene transcription. The NF- B response element has been sequenced (pNFκB-d2EGFP, Clontech, Palo Alto, CA), and NF-κB binding to its response element can, inter alia, lead to stem cell differentiation (Taub, 1996). [076] Genes that are activated by antioxidant and xenobiotic stress have promoters with one or more ARE and/or XRE. Many transcription factors, including Nrf, Jun, Fos, Fra, Maf, YABP, ARE-BPl, aromatic hydrocarbon receptor and estrogen receptor have been reported to bind to either or both ARE or XRE. For example, activated cytosolic factors catalyze the modification of Nrf and/or Jun proteins, both of which bind to ARE located within the promoter regions of multiple detoxifying and defensive genes, increasing their transcription (Dhakshinamoorthy et al, 2000). [077] Growth factors stimulate signaling pathways, resulting in the rapid transcriptional induction of approximately 100 immediate-early genes. This primary response to growth factor stimulation is mediated by SRE. SRE can be activated by multiple serum growth factors. It can then bind to transcription factors, including the serum response factor. Multiple signaling pathways, such as those involving cAMP-dependent protein kinase or MAP kinase, can act on multiple functional sequences within SRE (Lodish et al, 2000). [078] HREs are present in cells that respond to low oxygen concentrations. They may bind factors induced by a hypoxic state. For example, hypoxia induced factor- 1 has been reported to bind to the HRE on the erythropoietm gene, resulting in the marked enhancement of erythropoeitin transcription (Coulet et al, 2003). The human endothelial nitric oxide synthase (heNOS) gene is constitutively expressed in endothelial cells, with increasing expression during hypoxia. The HRE at position -5375 to -5366 of the heNOS promoter preferentially binds HIF-2 under hypoxic conditions, thus stimulating or . _ _ otherwise regula1ing^∞the%tnicfi tion of heNOS (Coulet et /., 2003). The vascular endothelial growth factor mediates angiogenesis in, for example, tumor cells, by interacting with the HRE (Tsuzuki et al, 2000).

[079] PPREs have been reported to bind to the peroxisome proliferator-activated receptor (PPAR) family of nuclear hormone receptors (Dreyer et al, 1993). For example, PPARs have been reported to bind a PPRE within the promoter region of the aconitase gene, and ligands for PPARs, such as certain anti-hyperlipidemia or diabetic drugs, can control the aconitase gene's transcriptional activity (Dreyer et al, 1993). [080] GREs are DNA sequences that serve as binding sites for a hormone-inducible transcription-activating complex. Glucocorticoid hormones bind to glucocorticoid receptors in the cytoplasm and the ligand/receptor complexes translocate to the nucleus, where they would bind GREs and activate transcription of cellular genes. GREs have been reported to be palindromes (Kupfer et al, 1990). A GRE can be specific for glucocorticoids, but can also serve as a common response element for glucocorticoids, progestins, mineralocorticoids and androgens (Beato, 1989).

[081] Protein kinase C regulates gene transcription by phosphorylating adaptor protein (AP)-l. AP-1 transcription factors, such as Jun or Fos, bind to a seven-base pair cis- regulatory element termed the tetradecanoyl-phorbol acetate (TPA) response element. When AP-1 is bound, the TRE confers sensitivity to TPA upon the cell. AP-1 binding to TRE has been implicated in the regulation of cell differentiation and proliferation. For example, AP-1 has been correlated with an undifferentiated state in osteoblasts. This undifferentiated state can be countered by a glucocorticoid response that promotes differentiation (Yaumauchi et al, 1999).

[082] Estrogens can exert their regulatory potential on gene expression by interacting with EREs, which are sometimes perfect and sometimes imperfect palindromes. Estrogen-induced gene expression has been reported to be influenced by differences in ERE sequences and by the estrogen receptor subtype bound to the ERE (Gruber et al, 2004).

[083] In response to one or both of interferon alpha or interferon beta, transcription factors such as STATs would bind to ISREs. In response to interferon gamma, a ST ATI homodimer binds GAS (Wolffe, et al, 2003). Interferon regulatory factors (IRF), such as IRF-1, IRF-2, or ISGF-3 gamma, modulate the action of these interferon response elements. IRFs can comprise transcriptional activators with tumor suppressor activity and transcriptional repressors. IRFs can also promote or suppress tumor activity (Gongora et al, 2000). [08 ]^' bRE/TόnE^' partifeipatδs n the osmotic regulation of many types of cells including but not limited to renal medullary cells. In response to increased tonicity, a tonicity- responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) binds to ORE/TonE, resulting in the altered expression of several genes, including those encoding aldose reductase, betaine/γ-aminobutyric acid transporter and sodium-myo- inositol cotransporter. The ORE/TonE has been reported to play a role in osmotic stimulation of cytokine gene transcription and integrin-mediated carcinoma metastasis (Ferraris et al, 2002). Hypertonicity increases the activity of TonEBP/OREBP and protects renal cells by enhancing transcription of genes that contribute to the accumulation of organic osmolytes. Some inhibitors of protein kinases reduce this tonicity-dependent activation. For example, protein kinase A mediates tonicity-dependent increases in transactivation, the activity of ORE/TonE and the induction of aldose reductase and betaine transporter mRNAs (Ferraris et al, 2002). [085] Retinoic acid receptors bind to DNA through their respective RAREs. These hormone response elements are arranged on the gene as two half-sites in tandem repeats. Their specificity is conveyed by the consensus sequence AGGTCA spaced by 1, 2, or 5 base pairs (Oosterveen, 2003). RARE binding is co-operative in that receptor binding to one of the half-sites facilitates receptor binding to the second site. RAREs have been reported to mediate growth and arrest of growth in breast cancer cells. For example, all- trans retinoic acid has been reported to bind to retinoic acid receptors and modulate gene transcription via RAREs (Oosterveen, 2003).

[086] GATA-RE responds to the DNA-binding protein GATA- 1 , which in turn regulates transcription of erythroid-specific genes. GATA-RE therefore mediates the formation of mature erythroid cells. GATA-RE can be transactivated by the transcription factor GATA-1. The GATA sequence, or its reverse complement, is recognized by multiple erythroid-specific transcription factors. For example, the HS-40 -globin regulatory site, a locus control region for the α-globin gene cluster, comprises multiple recognition elements for erythroid-specific transcription factors (Strachan & Read, 1999). [087] Response elements of the invention may be promoters. The stem cells of the invention can express nucleic acids under the regulatory control of inducible promoters. An example of an inducible promoter is the tetracycline-inducible promoter, which is induced to initiate transcription by tetracycline. Using a luciferase reporter, this promoter has been demonstrated to activate and inactivate its target transgenes (Canete-Soler et al, 1998). Another example of an inducible promoter is the ecdysone-inducible promoter, which is induced to initiate transcription by ecdysone. Using a lacZ or a green H^" f s- 3 If ff Fit Of ^e* ■ ' f $^■ f «s Λ srJ_E fluorescent protein reported, ' is' promoter has also been shown as a useful tool for studying gene function (Luers et al, 2000). [088] The stem cells of the invention can also express nucleic acids under the regulatory control of tissue-specific promoters. Examples of suitable tissue-specific promoters include, but are not limited to, the brain-specific astrocyte-specific (CNS) promoter for glial fibrillary acidic protein (GFAP), the kidney-specific promoter for kidney androgen regulated protein (KAP), the adipocyte-specific promoter for adipocyte specific protein (ap2), the blood vessel endothelium-specific promoter for vascular endothelial growth factor receptor 2 (VEGFR2), the liver-specific promoter for albumin, the pancreas- specific promoter for pancreatic duodenal homeobox 1 (PDX1), the muscle-specific promoter for muscle creatine kinase (MCK), the bone-specific promoter for osteocalcin, the cartilage-specific promoter for type II collagen, the lung-specific promoter for surfactant protein C (SP-C), the cardiac-specific promoter alpha-myosin heavy chain (α— MHC), as well as the intestinal epithelial-specific promoter fatty acid binding protein (FABP). [089] The astrocyte-specific (CNS) promoter for glial fibrillary acidic protein (GFAP) has been described (Miura et al, 1990). The promoter sequence and transcriptional startpoint of the GFAP gene have been characterized. The cis elements for astrocyte specific expression were found within 256 base pairs from the transcription startpoint. DNase I footprinting showed three trans-acting factor binding sites, GFI, GFII and GFIU, which respectively have AP-2, NFI and cyclic AMP-responsive element motifs (Miura et al, 1990). [090] The kidney-specific promoter for kidney androgen regulated protein (KAP) has been described (Ding et al. 1997). An exogenous 1542-base pair fragment of the kidney androgen-regulated protein (KAP) promoter was reported to specifically target the inducible expression in the kidney of the transgenic mice, which has the fragment. In situ hybridization demonstrated that expression of KAP mRNA was restricted to proximal tubule epithelial cells in the renal cortex (Ding et al, 1997). [091] The adipocyte-specific promoter for adipocyte specific protein (ap2), which is dysregulated in various forms of obesity, is structurally similar to TNFalpha. AP2 is involved in whole body energy homeostasis. It has been described by Hunt et al. to contain sequence information necessary for differentiation-dependent expression in adipocytes (Hunt et al, 1986). [092] The blood vessel endothelium-specific promoter for vascular endothelial growth factor receptor 2 (VEGFR2) was also described (Ronicke et al, 1996). Using RNase F^T M^ ^ ^i Ϊ*O . ₊ , _{J •} , _* . protection and primer extension" analyses, they revealed a single transcriptional start site located 299 base pairs upstream from the translational start site in an initiator-like pyrimidine-rich sequence. The 5 '-flanking region was found to be rich in GC residues and lacking a typical TATA or CAAT box. A luciferase reporter construct containing a fragment from nucleotides -1900 to +299 demonstrated strong endothelium-specific activity in transfected bovine aortic endothelial cells. Deletion analyses revealed that endothelium-specific VEGFR2 expression was stimulated by the 5 '-untranslated region of the first exon, which contains an activating element between nucleotides +137 and +299. In addition, two endothelium-specific negative regulatory elements were identified between nucleotides -4100 and -623. Two strong general activating elements were observed to be present in the region between nucleotides -96 and -37, which contains one potential NFKB and three potential transcription factor binding sites. This study suggested that VEGFR expression in endothelial cells is regulated by an endothelium- specific activating element in the long 5 '-untranslated region of the first exon and by negative regulatory elements located further upstream (Ronicke et al, 1996). [093] The liver-specific promoter for albumin was described by the same researchers who cloned the bovine serum albumin (bSA) promoter (Power et al, 1994). It functions efficiently in the differentiated, but not in the dedifferentiated, liver cells. Footprint analysis of the promoter revealed seven sites of DNA protein interaction extending from - 31 to -213. The deletion of one of these sites, extending from -170 to -236, resulted in a four-fold increase in promoter activity (Power et al, 1994). [094] The pancreas-specific promoter for pancreatic duodenal homeobox 1 (PDXl) was described in 2002 (Melloul et al, 2002). Upstream sequences of the gene up to about -6 kb showed islet-specific activity in transgenic mice, and several distinct sequences that conferred beta-cell-specific expression were identified. A conserved region localized to the proximal promoter around an E-box motif was found to bind members of the upstream stimulatory factor family of transcription factors (Melloul et al, 2002). [095] The muscle-specific promoter for muscle creatine kinase (MCK) was described as having relatively small size, good efficiency and muscle specificity (Larochelle et al, 1997). The authors generated replication-defective adenovirus recombinants with luciferase or beta-galactosidase reporter genes driven by a truncated (1.35 kb) MCK promoter/enhancer region that demonstrated efficient and muscle-specific transgene expression after local injection into muscle (Larochelle et al, 1997). [096] The bone-specific promoter for osteocalcin was described by the research group who found protein-DNA interactions at the vitamin D responsive element of the rat dstedcalciu gen^a^'t rAcleotides^466 to -437 (Bortell et Z., 1992). They also found a vitamin D-responsive increase in osteocalcin gene transcription accompanied by enhanced non- vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which contains a potential glucocorticoid responsive element. An osteocalcin CCAAT box was found at nucleotides -99 to -76. [097] The cartilage-specific promoter for type II collagen was described in 2003 (Osaki et al, 2003). Luciferase reporter constructs containing sequences of the type LT collagen promoter spanning -6368 to +125 base pairs were reported to be inhibited by the type II collagen inhibitor interferon-gamma. The interferon-gamma response was retained in the type TJ collagen core promoter region, which spans from -45 to +11 base pairs and contains the TATA-box and GC-rich sequences.

[098] The intestinal epithelial-specific fatty acid binding protein promoter (FABP) was described as both cell-specific and exhibiting regional differences in its expression within continuously regenerating small intestinal epithelium. Sequences located within 277 nucleotides of the start site of intestinal FABP transcription were reported to be sufficient to limit the expression of the reporter gene (human growth hormone) to the intestine. Nucleotides -278 to -1178 of the intestinal FABP gene mediated its expression in the distal jejunum and ileum (Sweetser et al, 1988).

[099] The lung-specific promoter for surfactant protein C (SP-C) was described by researchers in 1990. They identified the transcriptional start site and a TATAA consensus element located 29 base pairs five prime to exon 1 (Glasser et al, 1990). /

[0100] The cardiac-specific promoter alpha-myosin heavy chain (α-MHC) was described in a 1996 report. According to the reports, sequences from -344 to -156, which included a CArG box, direct cardiac-muscle-specific expression from a heterologous promoter. They also reported that α-MHC sequences from -86 to +16 promoted activity from two heterologous enhancers in a muscle-specific fashion, and that mutational analysis of an E-box and a CArG box within the promoter revealed that they act as negative and positive regulatory elements, respectively (Molkentin et al, 1996). [0101] Promoters specific for expression in B-cells include the IgM promoter. Promoters specific for expression in T-cells include the CD2, CD4 and CD8 promoters. These promoters, characteristically of T-cell promoters, do not have a TATA box but use multiple sites to initiate transcription (Outram & Owen, 1994). Promoters specific for expression in NK cells include the NKG2D and natural cytotoxicity receptor promoters. Promoters specific for expression in macrophages include the Mac-1 and — & rf ••_'•" '? Ufi."ff βf f-at , ι . , • . . . myeloperoxidase romotetsr'ana the myeloperoxidase promoters comprise a minimal promoter region and multiple enhancer regions (Austin et al, 1995). [0102] Chen and Jackson reported that CD Id presents lipid antigens to a specific population of NK T cells which are involved in host immune defense, suppression of autoimmunity and rejection of tumor cells (Chen & Jackson, 2004). While the transcriptional regulation and tissue distribution of the expression of CD Id were not known, the authors identified dual promoters upstream of the open reading frame encoding the CD Id gene. The proximal promoter was found within the region -106 to +24, and the distal promoter in the region -665 to -202 with the A of the start codon located at position +1. The region covering the proximal promoter produced a much higher luciferase activity in Jurkat cells, which are immortalized T-cells, than in K562 cells, which are erythroleukemia cells, whereas a much lower luciferase activity was found with the distal promoter, indicating a cell type-specific activity of the two promoters. [0103] It has also been reported recently that although the gene products of the E2A gene are ubiquitously expressed, E2A mice display selective abnormalities in lymphocyte development, suggesting a requirement of an E2A gene product for lymphocyte development (Hata & Mizuguchi, 2004). The authors found that the promoter of the E2A gene lacked a TATA box, and that primer extension analysis showed several transcription initiation sites, a feature that is characteristic of TATA-less promoters. A positive regulatory segment was identified at -357 to -158 of the 5' flanking region, and a negative regulatory segment was identified at -831 to -358 of the 5' flanking region. The segment -257 to -238 was found to play a role in the basal promoter activity of the E2A gene. Gene Trap Vectors [0104] The invention provides gene trap vectors useful for identifying the discrete expression pattern of genes during signal transduction. Constructs with a reporter gene but lacking a promoter are designed so that activation of the reporter gene depends on its insertion within an active transcription unit. Following insertion, the tagged gene can be detected in space and time by assaying for the reporter gene product. Introduction of gene trap vectors into ES cells has led to the derivation of transgenic lines that show a variety of gene expression patterns (Coffin et al, 1997). [0105] The trap vectors contain a reporter gene that is not expressed unless it is integrated into an intron or exon of a transcription unit. Integration results in an expression pattern that reflects the pattern of the endogenous transcription unit. The reporter gene provides a molecular tag for cloning the "trapped" gene of the transcription unit. All of the above-described reporter systems can be used with the gene trap vectors described below.

[0106] Gene trap vectors can be constructed in retroviral vectors. One such vector, which maps to mouse chromosome 6, was constructed with the reporter gene in reverse orientation with respect to retroviral transcription, downstream of a splice acceptor sequence (Soriano & Robertson, 2002; Zambrowicz et al, 1997). Infecting ES cells with this ROSAβgeo retroviral vector resulted in mice comprising the ROSAβgeo26 (ROS A26) mouse strain. This strain was produced by random retroviral gene trapping in the ES cells.

[0107] The reporter gene of ROSA26 mice is ubiquitously expressed during embryonic development and in all hematopoietic cells, consistent with the trapping of an endogenous gene programmed to be ubiquitously expressed (Zambrowicz et al, 1997). It displays virtually complete penetrance and no variation of expression. Staining of ROSA26 mouse tissues and fluorescence-activated cell sorter analysis of hematopoietic cells from mice of the ROSA26 strain demonstrates the ubiquitous expression of the proviral βgeo reporter gene. Embryos demonstrated blue staining in all cells at 9.5 days. Ubiquitous staining was found in neonatal brain, bone marrow, cartilage, heart, intestine, kidney, liver, lung, pancreas, muscle (skeletal and smooth), skin (dermis and epidermis), spleen, submandibular gland, fhymus, trachea and urinary bladder (Zambrowicz et al, 1997). [0108] Bone marrow transfer experiments illustrate the general utility of this strain of mice for chimera and transplantation studies. The ROSA26 gene trap vector integrated into a region that produces three transcripts. Two of these transcripts, lost in ROSA26 homozygous animals, originate from a common promoter and share identical 5' ends, but neither contains a significant open reading frame. The third originates from the reverse strand (Zambrowicz et al, 1997).

[0109] ROSA26 heterozygotes do not display an overt phenotype and are recovered in expected numbers from heterozygous fathers bred to wild type. Significantly fewer than expected homozygotes can be recovered from crosses between two heterozygous parents, but these homozygotes do not display an overt phenotype and are fertile. [0110] Another ubiquitously expressed endogenous gene suitable for use in gene trapping to identify gene expression patterns during signal transduction is the ras-GTPase- activating protein (GAP)-binding protein (G3BP). The G3BP gene encodes a cytosolic 68 kDa protein that binds to the SH3 domain of a ras-GTPase activating protein and functions as a ras effector protein and a helicase. This gene was disrupted in the BT-5 mouse line, the expression of which was observed in a strong and ubiquitous manner at developmental stages from gastrulation to organogenesis. Embryonic Stem Cells

[0111] Embryonic stem cells can be pluripotent; they can differentiate into any of the cells present in the organism. When they divide in vivo, pluripotent stem cells can maintain their pluripotency while giving rise to differentiated progeny. Thus, stem cells can produce replicas of themselves that are pluripotent, but they are also able to differentiate into lineage-restricted committed progenitor cells. Stem cells can reproduce and differentiate in vitro. Embryonic stem cells have been directed to differentiate into cardiac muscle cells in vitro and, alternatively, into early progenitors of neural stem cells, and then into mature neurons and glial cells in vitro (Trounson, 2002). [0112] The embryonic stem cells of the invention can be derived from a variety of non- human animal species, including mammalian species such as mouse, rat, guinea pig, sheep, goat, bovine, rabbit, canine, feline, porcine, ovine, or equine. Embryonic stem cells of the invention transfected with erythropoietm or IL-5 have been demonstrated to stably express physiologically functional erythropoietm or IL-5, respectively (Zhang et αl., 2003). Transfected embryonic stem cells of the invention also have been demonstrated to express other secreted proteins that are involved in signal transduction, such as the parathyroid hormone-like protein, FrizB or sFRP3; myostatin; bone morphogenetic protein 4; insulin-like growth factor 1; neuropeptide Y; growth hormone; Wnt 2; or Wnt 11 (Zhang et αl, 2003).

[0113] Mouse embryonic stem cells are derived from the inner cell mass, the cells which give rise to the embryo. An inner cell mass of a donor mouse at the blastomere stage of development can be transferred into the embryo of a second mouse, and the donor mouse can contribute genes to every organ of the host embryo. Inner cell mass blastomeres can be isolated from the embryo and cultured in vitro to produce ES cell cultures and cell lines. ES cells retain their totipotency in vitro, and each of them can contribute to all the organs of a host embryo following injection into the host embryo. ES cell cultures and cell lines can incorporate new DNA as transgenes. ES cells can be transformed with nucleic acids encoding a protein or a fragment of a protein, which can be a secreted protein or an extracellular domain of a transmembrane protein. [0114] In an embodiment of the invention, the ES cells are transformed with gene trap vectors such as ROSA26 or G3BP, which are ubiquitously expressed and encode proteins involved in signal transduction. Examples of proteins involved in signal transduction that are suitable for use in the invention include, but are not limited to, extracellular ^l'

ell surface and intracellular receptors; protein kinases; phosphatases; lipases; proteases; and other enzymes that modulate the activity of other molecules; activator proteins; GTPase switch proteins; trimeric G-proteins; monomeric G-proteins; scaffold proteins; transcription factors; response factors; and response elements. [0115] The activity of the proteins or fragments thereof encoded by the nucleic acids used to transfect the stem cells can be assayed. In this aspect, the gene encoding the protein or fragment is expressed, and the modulation of proliferation and/or differentiation of the stem cells transformed with the gene are observed. Changes in the rate of proliferation, the lack of proliferation, and/or differentiation of the genetically- altered stem cells can be compared with the wild type, non-genetically-altered stem cells. [0116] For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown under culture conditions well known in the art. The vector is introduced into ES cells by transformation methods such as electroporation, liposome delivery, microinjection, or the like, which are also well known in the art. The vector can contain genes for a secreted protein, which can be, for example, growth factors, cytokines, or hormones. The endogenous mouse gene is replaced by the disrupted disease gene during cell division through homologous recombination and integration. The transformed ES cells are then selected for studying cell proliferation and differentiation. The differentiated embryonic stem cells can form various cell types and tissues in vitro, such as neural cells, hematopoietic lineages, or cardiomyocytes (Bain et al, 1995; Wiles & Keller, 1991; Klug et al, 1996). [0117] The vector can also comprise regulatory DNA, for example, one or more response elements. The vector can further comprise a reporter gene, as described above. When the vector comprises both regulatory DNA and a reporter gene, it can be used to detect and/or measure activity that effects the response elements. For example, a vector with a reporter gene operably linked to a response element can be used to report the in vivo activity of fibroblast growth factor. [0118] In an embodiment of the invention, a mouse ES cell comprises a reporter system under the regulatory control of a promoter responsive to the activation of a signal transduction pathway. In an embodiment of the invention, a mouse ES cell comprises a reporter gene operably linked to a response element such as FiRE. The reporter system is inserted into the ROSA26 or the G3BP(BT5) locus. The ES cell can then be cultured, induced to differentiate, incorporated into an ES cell library, or introduced into a blastocyst. [0119] Adding factors that promote differentiation to the transformed stem cells can induce the cells to differentiate into different cell types by factors that promote differentiation. The factors that promote differentiation can be lineage-specific or non- lineage-specific, and can be supplied individually, in a formulation containing a combination of factors, or by the addition of a cell or cells that are capable of providing the differentiation factors to the genetically-modified stem cell. The differentiated cell can be identified by markers on the surface of the cell or by its phenotype. For example, the transformed ES cells are selected, exposed to an exogenously added differentiation factor, and the proliferation and differentiation of the stem cell populations into various cell types and tissues in vitro, such as neural cells, hematopoietic lineages, or cardiomyocytes can be studied (Bain et al, 1995; Wiles & Keller, 1991; Klug et al, 1996).

ES Cell Libraries

[0120] Libraries of transfected stem cells can be compiled to express selected polypeptides known or hypothesized to modulate selected in vivo or in vitro cellular functions. These libraries can also be used to screen, test, or compare potentially therapeutic or otherwise modulatory agents (Zhang et al, 2003). In one embodiment, the invention provides a library of transformed mouse ES cells comprising nucleic acid molecules encoding polypeptides, which are targeted to the ROSA 26 or the G3BP loci of the ES cells.

[0121] The libraries comprise a plurality of cells located in an addressable matrix. The matrix contains a plurality of spots or wells, each having an address. An example of such an address would be "column 2, row 4." The number of addressable spots in the matrix can range from between 5-50, 10-100, 20-200, 30-300, 40-400, 50-500, 60-600, 70-700, 80-800, 90-900, 100-1000, 250-2000, 350-3000, 450-4000, 550-5000, 650-6000, 750- 7000, 850-8000, 950-9000, 1050-10000, and 10000-50000, or more. At least one cell, and preferably more, is located in one or more spots or wells in the matrix. Each address in the matrix can contain either the same or a different number of cells. Each address can also contain the same or different type of cells. At least some of the stem cells are transformed with at least one, and optionally 2, 3, 4, 5, or more introduced nucleic acid molecules. The invention provides this cell library on any suitable substrate or support, such as a 96-well plate, a 384-well plate, a plate with larger number of wells, a glass slide containing depressions or wells in rows and columns, or a similar substrate or support that is suitable for high throughput analysis, or can be adapted for use in a robotics system. [0122] The nucleic acid molecules introduced into the stem cells of the library can encode secreted molecules, transmembrane molecules, or intracellular molecules, comprising, for example, transcription factors, nuclear receptors, kinases, phosphatases, proteases and ion channels. These molecules may have either a stimulatory or an inhibitory effect on the transformed cells. The stem cells of the library may exhibit a gain of function upon the introduction of the nucleic acid molecules, for example, the cells may acquire the ability to secrete certain proteins, the secretion of which is known to be mediated by particular signal transduction pathways. Cells exhibiting such a gain of function can be further transformed by introducing additional nucleic acid molecules that affect the gain of function, for example, by introducing nucleic acid molecules that knock out function. The stem cells of the library may also exhibit a loss of function, for example the cells may lose the ability to perform functions mediated by certain signal transduction pathways. This twice-transformed library can comprise inhibitory molecules, such as RNAi molecules useful for knocking out cellular functions. [0123] The stem cells of the library can thus serve as a source of secreted molecules; they can be placed in contact with other stem cells to determine the effect of the secreted molecules on the signal transduction pathways of the other stem cells. The invention also provides a library of stem cells that have differentiated into cells of different lineages, including but not limited to cardiomyocytes, T cells, B cells, leukocytes, other cells of the hematopoietic system, neurons, astrocytes, glia cells, other cells of the central nervous system, liver cells, bone cells, cartilage cells, pancreatic islet cells, kidney cells, muscle cells and other cells of the body.

[0124] The stem cell library can comprise a first stem cell transformed with a first nucleic acid molecule that encodes a first protein, a second stem cell transformed with a second nucleic acid molecule that encodes a second protein, a third stem cell transformed with a third nucleic acid molecule that encodes a third protein, a fourth stem cell transformed with a fourth nucleic acid molecule that encodes a fourth protein, and so on, up to tens, hundreds, thousands, or tens of thousands of stem cells, each transformed with a different nucleic acid molecule encoding a different protein. Each of the nucleic acid molecules of the library can encode a different polypeptide. It can also comprise stem cells transformed with nucleic acid molecules encoding proteins of the same family or proteins of different families. The families can comprise, for example, secreted proteins, transmembrane proteins, ligands, receptors, kinases, phosphatases, proteases, lipases, transcription factors, GTPase switch proteins, adapter proteins, as well as other molecules involved in signal transduction. it >!_ι. B * - . «- ^■ =. , , . [0125] The medium in which the stem cells are suspended can comprise added proteins or fragments of proteins, which may come into contact with the stem cells suspended therein. The added proteins or fragments may be present in the cells other than the stem cells in the medium. These non-stem cells can express the proteins or fragments on their cell surface, and/or secrete them. The added proteins or fragments may also be present in a group of stem cells that is different from the first group of stem cells containing the reporter systems, where the two groups of stem cells are suspended in the same medium. The cells in this second group of stem cells can also express the proteins or fragments on their cell surface, and/or secrete them. The medium can comprise (a) a stem cell transformed with a first different nucleic acid molecule; (b) two different stem cells: one transformed with a first different nucleic acid molecule and the other transformed with a second different nucleic acid molecule, respectively; (c) three different stem cells: one transformed with a first different nucleic acid molecule, a second transformed with a second different nucleic acid molecule, and a third transformed with a third different nucleic acid molecule, respectively; (d) four different stem cells: one transformed with a first different nucleic acid molecule, a second transformed with a second different nucleic acid molecule, a third transformed with a third different nucleic acid molecule, and a fourth transformed with a fourth different nucleic acid molecule, respectively; and/or (e) five or more different stem cells: one transformed with a first different nucleic acid molecule, a second transformed with a second different nucleic acid molecule, a third transformed with a third different nucleic acid molecule, a fourth transformed with a fourth different nucleic acid molecule, a fifth transformed with a fifth different nucleic acid molecule, and so on, up to ten, a hundred, a thousand and tens-of -thousands of different stem cells, each transformed with a different nucleic acid molecule, respectively. [0126] The library includes reporter cells, also referred to as readout cells, which are capable of exhibiting observable biological effects or phenotype changes when placed in contact with a biological molecule that has either stimulatory or inhibitory function. These reporter cells can be embryonic or adult stem cells, and the introduced nucleic acid molecules may encode factors that would cause these cells to differentiate into cells of different lineages. The stem cell reporter cells can be placed in contact with biologically active proteins or fragments of protein, such that the reporter cells can be used to determine the function and/or effect of one or more polypeptides on the signal transduction pathways as reflected in, for example, either or both the growth or differentiation of the stem cells. [0127] The cells can also be selected from T cells, B cells, cells of the central nervous system (CNS), cartilage cells, bone cells, pancreatic islet cells, fat cells and oocytes. Stem cell reporter cells may differentiate to produce cells selected from CNS cells including but are not limited to brain cells, neurons, astrocytes, and glial cells; T cells; B cells; cartilage cells; bone cells; pancreatic islet cells; fat cells; heart cells; liver cells; kidney cells; lung cells; muscle cells; and eye cells. The reporter cells can also be derived from animal species other than mice, such as, for example, frogs, rabbits, cows, pigs and the like.

[0128] The invention provides a combined library of transformed cells and reporter cells that are in physical contact with each other, and can be used, for example, to study molecular interaction. The transformed cells can express polypeptides involved in signal transduction. For example, the transformed cells can express a ligand, and a nucleic acid molecule introduced into the reporter stem cells can encode a receptor. [0129] The invention further provides a method of determining the function of a first protein encoded by a first nucleic acid molecule, where the method comprises: allowing a first transformed stem cell to grow, where the first transformed stem cell is transformed with a first nucleic acid molecule that is targeted to a first locus; and observing one or more signal transduction pathways of the first transformed stem cell to determine the function of the first protein. This method can also be used to determine the function of a library of proteins, where the method comprises: transforming a stem cell library with a plurality of nucleic acid molecules encoding a plurality of different polypeptides; allowing the stem cells in the library to grow or differentiate; and observing the signal transduction processes in the stem cells of the library. The invention also provides a method of massive parallel screening for signal transduction activities, where the method comprises: providing a combinatorial library comprising a plurality of stem cells in an addressable matrix, where the cells are transformed with a plurality of distinguishable nucleic acid molecules encoding a plurality of proteins or fragments; and monitoring the library of stem cells for signal transduction activities. Chimeras From ES Cells

[0130] An ES cell transformed with exogenous DNA can be injected into an early-stage mouse embryo. The ES cell can then integrate into the host embryo, resulting in a chimeric mouse. Some of the chimera's cells will be derived from the host's own ES cells, but some portion of its cells will be derived from the donor ES cell transformed with the exogenous gene. If the treated cells become part of the germ line of the mouse, some of its gametes will be derived from the donor cell. If such a chimeric mouse is "^ir i ra ."^" 1 «i 1 eg mated with a wild-type mouse;" some of its progeny will carry one copy of the inserted gene. When these heterozygous progeny are mated to one another, about 25% of the resulting offspring are predicted to carry two copies of the inserted gene in every cell of their bodies (Gossler et al, 1986). Thus, in three generations: the chimeric mouse, the heterozygous mouse and the homozygous mouse, an exogenous gene will be present in both copies of the chromosomes of the mouse genome. [0131] The invention provides a non-human animal implanted with the blastocyst comprising an ES cell transformed with exogenous DNA. It also provides a chimeric animal produced from such a blastocyst. The blastocyst can be obtained from any animal model of a human disease, such as a SCID mouse, a non-obese diabetic mouse, an RB-/- mouse, or a p53-/- mouse. [0132] Transfected ES cells can be used to make chimeric animals that express the reporter in various specified tissues, for example, when the expression is under the control of tissue-specific promoters. These animals are called chimeric reporter animals. The chimeric reporter animals may include both cells that express a factor such as a growth factor under the control of one or more signal transduction pathways and cells that express a response element selective for that factor, where both kinds of cells are linked to a reporter gene. These chimeric reporter animals can be used to test or determine which tissues respond to protein factors or small molecules administered to the animals. This in vivo reporter system can be used to test drug efficacy, toxicity, pharmacokinetics and metabolism. [0133] The invention provides the progeny of these chimeric reporter animals. The progeny can be produced by breeding the chimeric animals to obtain germ line transmission of the reporter system. The resulting progeny can be either heterozygous or homozygous for a reporter system comprising the exogenous DNA. The invention also provides a progeny blastocyst comprising a blastocyst from the progeny of the chimeric animals. This progeny blastocyst can further comprise an introduced ES cell, which contains a factor that regulates a gene response element. This progeny blastocyst can be implanted into an animal, and the invention provides an animal produced from said progeny blastocyst. [0134] In an embodiment, the invention provides an animal treated with a protein therapeutic or small molecule drug, or the progeny of the animal, wherein the heterologous protein therapeutic is introduced into the chimeric animal via a DNA molecule that comprises a first sequence operably linked to a second sequence, wherein the first sequence comprises a promoter of a liver-expressed gene and the second ...!' 11 if ..• IF it «..,,. jj ,. . „ „ " i ^,Jseqύeritie"feri^,ec)'dέ^'s at le'as one "heterologous protein therapeutic, wherein the DNA molecule is other than a naturally-occurring molecule and is free of viral-derived sequences, and the second sequence is other than a reporter gene, wherein the DNA molecule can be expressed in vivo in the chimeric animal to produce at least one protein therapeutic that is functionally active in the animal. [0135] Using gene trap vectors with reporter systems, a single ES cell transfection can result in the generation of multiple transfected cell types. When the transfected ES cell is introduced into a blastocyst that is then incorporated into a chimeric animal, all the tissues of the chimera have the potential to activate the reporter system upon responding to the appropriate exogenous signals. [0136] The transformed stem cells of the invention can be used to develop in vivo mouse models for human disease. In general, a gene construct encoding a polypeptide is inserted into the mouse ES cells to produce transfected stem cells. One or more polypeptides can be encoded by the construct. The polypeptide can be, for example a secreted protein, a fragment of a secreted protein, a transmembrane protein, an extracellular domain of a transmembrane protein, or a combination of these. The gene construct can be inserted in the ROSA26 or G3BP locus to allow gene expression in most or all tissues of the mouse. [0137] The resulting transfected stem cells are inserted into a blastocyst, for example, at the 64 cell stage, to form a chimeric blastocyst. Normal mice, knockout mice, or mouse models for human diseases can provide a source for these blastocysts. When implanted into a pseudo-pregnant mouse, the blastocysts can develop into chimeric embryos, fetuses and mice. The chimeric mice can also be produced by breeding, for example, by crossing a mouse carrying a gene of interest with a normal mouse, a knockout mouse, or a mouse model of human disease. [0138] The invention also encompasses a blastocyst comprising one or more response elements and reporter genes, as well as a chimeric reporter mouse developed from the blastocyst. The invention encompasses breeding these chimeric reporter mice so that the transgene is transmitted in the germ line, then generating another chimera that combines cells from the offspring carrying the transgene with cells transformed with one or more agents that affect the response element. For example, a chimera is formed from (1) a donor blastocyst from a mouse chimera bred to stably carry a transgene comprising FiRE operably linked to lacZ and (2) an ES cell comprising a vector with FGF. This chimera expresses both FGF and a response element to FGF. It can be used to identify and characterize FGF expression in both the developing and adult organism. It can also be

agents that alter FGF expression in vivo, such as those therapeutic and prophylactic agents useful for either or both treating or preventing diseases and conditions in which FGF expression is altered. [0139] Mouse models that are useful for practicing the invention include, but are not limited to, models for signal transduction disorders, such as cancers or inflammatory diseases. Examples of these models are mice that overexpress Aβ peptide or TGFβpeptide. Other useful mouse models include the severe combined immunodeficiency (SCTD) mouse, non-obese diabetic mouse, Rb-/- mouse, and p53 -/- mouse. These models provide opportunities to observe whether the inserted genes would correct the deficiencies associated with each signal transaction disorder. [0140] Specifically, the invention provides a system for conducting in vivo and in vitro testing the function, expression and manufacture of signal transduction proteins. The system targets a gene to a locus, for example, the ROSA 26 locus in mouse ES cells, and allows the transfected DNA to proliferate and differentiate in vitro. The ROSA 26 locus directs the ubiquitous expression of the heterologous gene (Zhang et al, 2003). For example, the effect of the transfected DNA on healthy or diseased cells can be monitored in vitro. Differentiation of cells such as cardiomyocytes, hepatocytes, or skeletal myocytes can be monitored by morphologic, histologic, or physiologic criteria. [0141] The tissues of the chimeric mice or their progeny can be isolated and studied. Alternatively, cells and or cell lines can be isolated from the tissues of the chimeric mice or their progeny and studied. Tissues and cells from any organs in the body, including heart, liver, lung, kidney, spleen, thymus, muscle, skin, blood, bone marrow, prostate, breast, stomach, brain, spinal cord, pancreas, ovary, testis, eye and lymph node, are suitable for use. [0142] For example, the invention includes the observation that ES cells transfected with interleukin-5 and incorporated into a blastocyst produced a chimeric mouse that expressed a greater than normal number of eosinophils in the liver. Eosinophilia expression is a previously observed effect of interleukin-5, and demonstrates that the ES cell mouse expression system (ESpresso mouse) can be used to determine the function of unknown and novel secreted polypeptides. Mice possessing phenotypic changes as a result of transgene expression may physically appear only slightly chimeric. [0143] The invention also includes a method for tracking the activity of a physiological modulator or a pathological agent in vivo. In an embodiment, the activity of a growth factor can be identified and characterized by assaying the activity of a reporter gene that is operably linked to a response element for that growth factor. For example, the activity ιr ιr""T' ,._" if IMI: J: I [ II:;:;: ^• -it \\ \i, »\\

" " δf " FG 'can be Identified' and characterized throughout a chimeric animal as described above by assaying a reporter gene linked to the vector shown in Figure 2. The signal transduction pathways that modulate the in vivo activity of FGF can be identified and characterized by assaying the response of the reporter genes to agent and conditions that perturb the pathways. In this manner, agents that modulate the in vivo activity of FGF can be identified and characterized by assaying their in vivo effects on their respective response elements. [0144] The reporter systems in the chimeric cells, both in vitro and in vivo, can be used to measure the effect of signal transduction modulators, such as small molecules, secreted proteins or polypeptide fragments thereof, or antibody agonists or antagonists of the pathway. These systems can be used to detect pathway activation, for example, by growth or differentiation factors that bind to cell surface receptors and activate a pathway. Examining reporter gene expression across different cell and tissue types provides information about the cell and tissue types that utilize a particular signal transduction system or systems. Examining reporter gene expression across the developmental spectrum of an organism provides information about the stages of development during which the organism uses the signal transduction system or systems. In another of these systems, a gene encoding a secreted factor may be used to introduce the particular factor directly into the chimeric animal, so that the effect of this factor on the signal transduction pathways of the chimeric animal may be observed and studied. The genes encoding the secreted factor may even be expressed in the same ES cells, before these cells are injected or otherwise introduced to the peudo-pregnant animals. These reporter systems may comprise more than one cloning site so that more than one secreted factors may be introduced into the chimera at one time. As a result, diverse effects on the signal transduction pathways in the chimera may be studied simultaneously. [0145] Transformed stem cells can also be used in the in vivo determination of gene function. For example, a gene of interest can be used to target a specific locus in an ES cell, such as the Rosa 26 or the G3BP locus. The transformed stem cell can be injected into an embryonic precursor, such as a blastocyst, using standard techniques, and the blastocyst can be implanted into the uterus of non-human animal such as a mouse by methods well known in the art. The blastocyst can then be allowed to develop into a chimeric embryo and chimeric fetus in vivo, and ultimately, a chimeric animal such as a chimeric mouse. Preferably, the chimeric embryo, fetus, or animal produces the product encoded by the gene of interest in multiple tissues, such that the effect of the gene iDπt J' ' J! c; ||" n:::;; - IL . n, _α

" ^"product On the^' embryo,' fetus of animal, can be determined. Cell lines can be produced from cells or tissues obtained from the chimeric embryo, fetus, or animal above. [0146] In addition, the ES cells can be used to make chimeric animals that have the reporter expressed in various specified tissues, such as by using tissue-specific promoters. Such chimeric animals are useful for testing or determining which tissues respond to protein factors or small molecules administered to the animals. In an embodiment, the invention provides a method for identifying the tissues that display physiologic or pathologic responses to known factors, such as growth factors like FGF, in a chimeric animal that expresses a response element for the known factor in most or all tissues. In this embodiment, the reporter gene linked to the response element can be assayed in a comparative manner among different tissues and/or cell types, and can be assayed in a comparative manner after treatment with known or hypothetical modulators of the factor. This embodiment provides a method of identifying modulators, including prophylactic and therapeutic modulators, of factors known or hypothesized to be involved in disease pathogenesis, such as fibroblast growth factor. Such modulators can include small molecule, secreted proteins or polypeptide fragments thereof, or antibody agonists or antagonists of the pathway. [0147] In another embodiment, the ES cells can be used to make chimeric animals that have both the reporter systems and the secreted factors expressed in various tissues by, for example, using tissue-specific promoters. These co-expressed reporter animals may also be used to determine the effect of the co-expressed secreted factors on the tissues and cells. The characteristics of these co-expressed reporter animals can be compared with their counterparts that do not express the secreted proteins. In Vivo and In Vitro Effects of Therapeutics [0148] The in vivo reporter systems of this invention can be used to test the efficacy, toxicity, pharmacokinetics, and metabolism of therapeutic agents. Examining reporter gene expression in cells, tissues and animals that have been treated with a candidate therapeutic agent provides information about the effect of the candidate agent on the signal transduction system or systems. [0149] Methods of expressing transgenes are known in the art. These methods include methods of expressing transgenes in vivo by targeting transgene cassettes into predetermined loci, as performed in WO 03/020743. In an embodiment, the invention incorporates the methods of WO 03/020743. In this embodiment, expression of a gene or genes of interest is determined by an exogenous promoter included in a transgene cassette and targeted to a predetermined locus where it directs the expression of the gene(s) of inte esfwlmbiϊt

promoter endogenous to the predetermined locus. The cassette may, however, be influenced by other elements present at the targeted locus, for example, enhancers and locus control regions.

[0150] A gene of interest can be used to target a specific locus in an ES cell, such as the Rosa 26 or the G3BP locus, and the transformed embryonic stem cell can be provided to a tissue of an animal, for example, an immunocompromised animal such as a nude mouse. The ES cell can then develop into a chimeric neoplasm, such as a teratoma, and the effect of the gene product on the neoplasm can be determined, thus providing information on the action of particular agents on the neoplasm, which is omnipresent in cancerous and precancerous cells. Cell lines can also be developed from such a chimeric neoplasm.

[0151] The chimeric reporter animals of the invention, their progeny, as well as the tissues and cells derived thereof, can be used in assays for screening, testing and comparing agents or libraries of agents. The agents can be genes, proteins, peptides, small molecules and the like. Any convenient multiplex testing configuration can be used. For example, the chimera can be used to study the effect of an agent on signal transduction. The chimeras, their progeny, and their tissues and cells can be used to detect combination effects, that is, the effect of the gene of interest as well as any additional factors or cells, on signal transduction. The additional factors include, for example, factors in solution and factors secreted by cells or present as extracellular portions of transmembrane proteins. In an aspect of the invention, the secreted factors may be expressed near the cells where the reporter systems are expressed. In another aspect, the secreted factors may be expressed in the same cells where the reporter systems are expressed, which may be accomplished by, for example, having multiple cloning sites on the reporter-system DNA molecule used to transfect the ES cells. [0152] In yet another aspect of the invention, the activity of the signal transduction polypeptides encoded by the nucleic acids that transfect the embryonic stem cell can be assayed. In this aspect, the gene encoding the protein is expressed, and the modulation of the chimera transformed with the gene are observed. Thus, changes in function can be detected by directly observing the signal transduction pathways, or by observing a phenotype such as one or more of the rate of proliferation, a lack of proliferation, or differentiation of the genetically-altered mice. This aspect provides a method of determining an in vivo effect of a therapeutic agent by administering the agent to a chimeric reporter animal or its progeny and determining the tissue or cell type in which the reporter system is activated by a signal transduction pathway. The therapeutic agent -can; f f 'example', be a rotein-therapeutic or a small molecule therapeutic. A protein therapeutic can be introduced into the chimeric animal or its progeny by viral or non- viral DNA transfection methods. A small molecule therapeutic can be introduced into the chimeric animal or its progeny via routine means of drug delivery. The invention also provides a method of determining one or more components of a signal transduction pathway by administering a molecule or compound to a chimeric animal or its the progeny followed by determining the one or more genes activated by the molecule or compound.

Administering Liver-Expressed Protein or Polypeptide Therapeutics [0153] DNA molecules encoding proteins or polypeptides modulators or therapeutics can be introduced into the chimeric reporter animals, their progeny, as well as their tissues or cells, through either viral or non- viral delivery. Viral methods utilize viral vectors or viral promoters. Viral vectors including adenovirus, adeno-associated virus, retrovirus or lentivirus vectors (Luo & Saltzman, 2000; Relph et al, 2004; Liu et al, 2001; Rochilitz., 2001). Non-viral methods include transferring DNA with the help of transfection reagents such as polylysine conjugates, various polymers, liposomes, or transferring "naked" DNA in the absence of transfecting reagents. Both viral and non-viral methods are potentially powerful tools of introducing exogenous DNA molecules, but the currently-known techniques are plagued by low transfer efficiency, inability to sustain gene expression, danger to the host, or a combination of these drawbacks (Nyuyen & Ferry, 2004). [0154] Viral vector gene delivery techniques utilize the powerful machinery viruses have acquired through evolution to transfer foreign DNA into cells (Luo & Saltzman, 2000). Viral vectors have been reported to efficiently transfer genes into cells or multi-cell organisms, leading to prolonged expression of the gene product upon integration into the mammalian genome (Luo & Saltzman, 2000; Relph et al, 2004). But these vectors are incapable of carrying a large amount of foreign DNA, and the integration into mammalian genome may inactivate the endogenous genes required for cell viability or activate proto-oncogenes (Liu et al., 2001). Retroviral vectors can only transfect proliferating cells (Rochlitz, 2001). Those viral vectors that fail to integrate into the host genome may harm the host by, for example, mutating to reacquire viral infectious ability (Nguyen & Ferry, 2004). This, plus the difficulty and cost associated with making large quantities of viral vectors, make the viral vector methods impractical for introducing and/or studying the effect of protein or polypeptide therapeutics on the chimeric reporter animals, their progeny, their tissues, or their cells.

use the viral derived cytomegalovirus (CMV) and Rouse sarcoma virus (RS V) promoters to express or overexpress proteins in the target cells or multi-cell organisms. Injection of DNA molecules containing genes driven by viral promoters have shown mixed results when it comes to long-term expression of the proteins or peptides of interest (Rizzuto et al, 1999; Lefesvre et al, 2002; Tripathy et al, 1996; Kameda et al, 2003; Jiang et al, 2001; He et al, 2004; Herwijer et al, 2001; Zhang et al, 2000). Injected viral promoters that failed to drive the expression of genes of interest could be expressed under certain other conditions, causing immune or various other harmful effects to the host (Nguyen & Ferry, 2004). [0156] Non- viral methods transfer purified DNA molecules either with or without the help of transfection reagents such as liposomes, lipids, or polyamines (Debs et al, 2003). When the DNA is transferred in the absence of transfection reagents, the DNA is said to be "naked." Many known advantages of naked DNA injections include, but are not limited to, the ease and low cost of making and purifying the DNA, the safety of delivering only the DNA sans the viral vectors, as well as the ability to express the foreign gene encoded by the DNA in the host (Young & Dean, 2002; Wolff et al, 1990; Acsadi et al, 1991). The drawbacks of currently known naked DNA methods are mostly related to either or both of the low transfer efficiency or the limited duration of expression (Kobayashi, et al, 2000). [0157] Efforts to improve non- viral naked DNA methods have led to the development of hydrodynamic tail vein methods, which can be performed in a variety of ways to achieve stable expression of the gene product. The hydrodynamic delivery of nucleic acids to the tissues and organs of animals in vivo is well known to those of skill in the art, and has been reviewed by Hodges and Scheule, 2003; and Hagstrom, 2003. Hydrodynamic pressure can, in some embodiments, contribute to the stable integration of naked nucleic acid. In an embodiment, approximately 25 ug of a plasmid of interest with a CMV promoter can be injected into the tail vein of 4-8 week old mice in a volume approximating 10% of body weight over a time span of approximately five seconds. Hydrodynamic tail vein methods, which entail injecting naked DNA under high pressure, with a high volume and in a short time frame into a blood vessel, particularly the tail vein of the host, have been demonstrated to be effective in achieving stable integration (Liu et al, 1999; Zhang et al, 1999; Nguyen & Ferry, 2004). The currently-known methods of tail- vein injection, however, have not been shown to sustain the expression of the foreign gene to a sufficiently long term so that the effect of the gene product on the host organism may be studied (Kobayashi et al, 2001; He et al, 2004). ., , , , ,,,, I ,,,, , „ .. , ,

" "'-[Ol58 lrr ή embodiment, tne'ϊnvention provides a method of gene expression by intravascular and intramuscular injection of naked plasmid DNA, as described by Zhang et al., 1999 and WO 00/50617. High levels of plasmid DNA expression can be obtained by tail vein injections, with the highest levels of expression achieved by rapidly injecting the plasmid DNA in large volumes, approximately 2.5 ml (Zhang et al. 1999; WO 00/50617). [0159] In an embodiment, the invention provides a method of gene expression by tail vein injection according to the method described by Monahan et al., U.S. Patent No. 6,627,616, for expressing a secreted gene product in a small animal model, thereby allowing assessment of its therapeutic utility and determination of its mode of action. [0160] In an embodiment, the invention provides a method of gene expression from minicircular DNAs devoid of bacterial sequences, by tail vein injection, as described by Chen et al., 2003; WO 04/020605; U.S. Application No. 2004/0214329. Bacterial DNA linked to a mammalian expression cassette can be effective in transcriptional silencing of the transgene in vivo. This method results in the expression of therapeutically relevant and persistent levels of therapeutic products in vivo (Chen et al., 2003; Liu et al., 1999; WO 04/020605; U.S. Application No. 2004/0214329). [0161] In an embodiment, pegolyated nucleic acids can be stably expressed following tail vein injection (Yant et al., 2004). Liver damage is sometimes observed after tail vein injection but is transient. [0162] The invention provides a DNA molecule with a promoter of a liver-expressed gene operably linked to a gene encoding a heterologous secreted protein, which can be expressed in vivo in the chimeric reporter animal. The DNA molecule comprises a first sequence operably linked to a second sequence wherein the first sequence comprises a promoter of a liver-expressed gene and the second sequence encodes at least one heterologous secreted protein or polypeptide, wherein the DNA molecule is other than a naturally occurring molecule and is free of viral-derived sequences, and the second sequence is not a reporter gene, wherein the DNA molecule can be expressed to produce at least one functionally active protein in the chimeric reporter animal. The DNA molecules of the invention are also free of transfection agents, which may otherwise bind to, complex with, or mediate the cell entry of oligonucleotides or polynucleotides. The promoter of the first sequence can comprise a transcription start site, which is included in the 5' untranslated region and may provide specificity to the expression of the functionally active protein. The heterologous protein encoded by the second sequence can be a secreted protein or a polypeptide fragment thereof, a transmembrane protein, an _,

'^"' '' -extøcellu at foteitf^ fragment of a transmembrane protein, or an intracellular protein, the expression of which is under the control of a liver-expressed gene promoter. Preferably, the heterologous protein encoded by the second sequence is a secreted protein. The heterologous protein may serve as modulating agents or modulators, which affect physiological processes in the host animal. The same protein may also serve as a therapeutic or a therapeutic agent. The DNA molecule can further comprise a third sequence that is operably linked to the first and second sequences, and that comprises an intron sequence. This third sequence can be a heterologous intron or an intron comprising a nucleotide sequence of the invention, and/or as further described in Table 3. [0163] The liver plays a central role in metabolism and production of seram proteins. It has two circulatory systems, a systemic circulation system that brings oxygenated blood directly from the heart and a portal circulation system that brings nutrients from the intestines. In addition, it has a system of ducts that transports metabolites, drugs, toxins and other materials out of the liver via bile into the small intestine. Particles injected into the blood circulation can quickly reach the liver, readily putting the particles in directly contact with the liver cells. Because of these features, liver-expressed gene of interest can be a powerful tool to study the effect of the gene product on the animal, both within the liver and throughout the body. For studying the chimeric reporter animals, their progeny, as well as their tissues and cells, secreted protein or polypeptide factors encoded by liver-expressed exogenous genes are particularly useful. Secreted proteins or polypeptide fragments capable of being modulators or therapeutics include, but are not limited to, hormones, cytokines, growth factors, clotting factors, anti-proteases, angiogenic proteins (e.g., VEGFs and FGFs), angiogenic proteins (e.g., endostatin sand angiostatin) and other proteins present in the blood. [0164] One large family of over a thousand proteins expressed in the liver is the cytochrome P450 protein family. The proteins of this family are heme-thilate monooxygenases that perform various oxidation reactions and participate in the body's disposal of harmful substances by enhancing the water-solubility of these substances. Operably linking the promoter sequence of genes expressed in the liver, for example the promoter sequence of any of the cytochrome P450 proteins, to a gene encoding the secreted protein or polypeptide of interest can lead to expression of that gene in the liver and other sites where the promoter is active. [0165] In another aspect, the invention provides a composition comprising the DNA molecule and a pharmaceutically acceptable carrier or excipient. The latter can be saline

a DNA complexing agent. This composition may also comprise a nucleotide sequence encoding a protein that serves to enhance either or both expression or folding of the exogenous protein modulator or therapeutic encoded by the second sequence of the DNA molecule. [0166] In yet another aspect, the invention provides a vector comprising the DNA molecule and an origin of replication. The vector may further comprise a nucleotide sequence encoding a reporter gene or a nucleotide sequence encoding an antibiotic resistance gene, such as a tatracyclin-resistance gene, a penicillin-resistance gene, a vancomycin-resistance gene, a chloramphenicol-resistance gene, a zwittermycin- resistance gene, or a kanamycin-resistance gene. [0167] In a further aspect, the invention provides a method of inducing sustained expression of a protein or a polypeptide in the chimeric reporter animal by providing the composition described above, injecting the composition into the animal and allowing the expression of the secreted protein or polypeptide. The composition can be injected under pressure. The animal can be injected with the composition intravenously, and the duration of the injection can be about 5 seconds. This method can be used to inject the composition into a reporter mouse, in which case the composition may have a volume of about 1, 1.5, or 2 ml. This method can be used to obtain expression and protein activity that are detectable on day 3, day 7, day 8, day 11, day 13, or any day thereafter. [0168] The invention further provides a method of inducing sustained expression of more than one protein or polypeptide by providing more than one of the DNA compositions described above, injected into the animal under pressure. This method can be used to obtain expression and activity information of multiple proteins on day 3, day 7, day 8, day 11, day 13, or any day thereafter. [0169] The invention also provides co-expression of more than one secreted protein or polypeptide. This is achieved by linking the liver-expressed promoter operably to multiple genes encoding a number of different proteins or polypeptides. Co-expressed molecules may interact with one another in the reporter animal, which may or may not result in a cumulative effect on the chimeric animal, its tissues, or its cells. For example, one may modulate the level of expression of another (Ahn et al, 2004). If the co- expressed molecules do not interact with one another, then the reporter animal co- expressing these molecules can be used to simultaneously evaluate the modulation or therapeutic effects of multiple modulators or protein therapeutics. r •>^■ if IF j i .. - " en ^,! Transpϋsabfe ElernWts'^{1 "ilf :!l!} [0170] Transposable elements can be used to incorporate nucleic acid sequences into the genome of model organisms. Transposable elements are particularly useful for inserting sequences into a gene of interest so that the encoded protein is not properly expressed, creating a "knock-out" animal having a loss-of-function phenotype. One of these, the sleeping beauty transposon system (Ivies et al., 1997), can stably integrate genes encompassed within a transposon up to approximately 6 kb in size, into the genome of vertebrates, including mice, in vivo (Yant et al., 2004; U.S. Patent No. 6,613,752; Karsi et al., 2001). The technique of integrating genes of interest into the mouse genome in vivo has been demonstrated to be safe and the expression has been demonstrated to be due to stable integration, since immune clearance was reported to have eliminated the transfected cells (Karsi et al., 2001). They have been used to achieve long term expression of therapeutics in mice, including mouse models of disease (Yant et al., 2000). [0171] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. [0172] It must be noted that, as used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a subject polypeptide" includes a plurality of such polypeptides and reference to "the agent" includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth. [0173] Further, all numbers expressing quantities of ingredients, reaction conditions, % purity, polypeptide and polynucleotide lengths, and so forth, used in the specification and claims, are modified by the term "about," unless otherwise indicated. Accordingly, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties of the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits, applying ordinary rounding techniques. Nonetheless, the numerical values set forth in the specific examples are reported as jranp-ir / ιι it cj; - , f MI ^li;"li oi • , precisely 'as possible: Any numerical value, however, inherently contains certain errors from the standard deviation of its experimental measurement. [0174] With respect to ranges of values, the invention encompasses each intervening value between the upper and lower limits of the range to at least a tenth of the lower limit's unit, unless the context clearly indicates otherwise. Further, the invention encompasses any other stated intervening values. Moreover, the invention also encompasses ranges excluding either or both of the upper and lower limits of the range, unless specifically excluded from the stated range. [0175] The specification is most thoroughly understood in light of the cited references, all of which are hereby incorporated by reference in their entireties. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. EXAMPLES [0176] The examples, which are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. The listing of these examples are not intended to suggest that the experiments described therein are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations are unavoidable and should always be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Example 1. Targetability as a Screening Method for Identifying Factors that Inhibit ES Cell Proliferation or Induce Differentiation [0177] It is currently difficult to study factors that will inhibit proliferation or induce differentiation because, using the available technologies, the cells expressing these factors will not grow. But these factors are likely to be potent modulators or therapeutics, which makes them classic and logical focal points of drag development efforts. The method of the current invention solves this unmet need. The ROS A26 locus can drive ubiquitous expression of genes that are targeted to this locus. The targeting frequency to the ROSA26 locus is high, up to about 50% (Zambrowicz et al, 1997). The same homologous arms are used for targeting all selected molecules to the ROSA 26 locus.

vecϊol-ror 'Secreted or other molecules targeted to the ROSA26 locus was constructed as shown in Figure 1. The PGKneobpA fragment was made by combining PGKneo from New England Biolabs (Beverly, MA) and bovine growth hormone poly A (bpA) from BD Biosciences Clontech (Palo Alto, CA). The adenovirus major late transcript splicing acceptor (SA) was PCR amplified from adenovirus genomic DNA. The 5' and 3' homologous arms were PCR amplified and cloned from genomic DNA according to a public genomic database such as the NCBI. As read from the 5' to 3' direction, the basic targeting vector without the gene of interest was made by inserting a fragment containing SA, the Gateway cassette (Invitrogen, Carlsbad, CA), poly A, and PGKneo between the 5' and 3' homologous arms of the ROSA 26 targeting arms. [0179] The master targeting vector was constructed by cloning the SA (PCR amplified) and bpA fragment into pBluescript. Then the PGKneobpA was cloned 3' to the bpA fragment. The Gateway conversion cassette (Invitrogen, Carlsbad, CA) was then cloned between SA and bpA. Then the whole fragment containing the SA, Gateway cassette, polyA, and PGKneo was cloned between the 5' and 3' homologous arms of the ROSA 26 targeting arms (the 5' and 3' homologous arms were PCR amplified and cloned from genomic DNA according to the public genomic database). Only the targeted clones were demonstrated to have a PCR product. [0180] As shown in Figure 1, a secreted factor of interest can be cloned into the ROSA26 targeting vector. A secreted factor gene is cloned into a Gateway entry vector then subsequently cloned into the ROSA 26 targeting vector by the Gateway cloning technology (Invitrogen, Carlsbad, California). The initial number of secreted molecules selected for targeting/expression will be about 100-200. The 'potent' factors that inhibit ES cell growth or induce differentiation can be identified by considering the fact that no targeted clones can be obtained solely for these clones, and adjusting the selection process accordingly. [0181] The endogenous ROSA26 promoter will drive the expression of the secreted factor. The ROSA26 promoter resides in the 5' homologous arm. The 5' and 3' homologous arms and the non-TK negative selection marker will be used to target the secreted factor onto the ROSA 26 locus. The PGKneobpA will be used as a selection marker for the targeting experiment. The S A and bpA are sequences that facilitate the expression of the secreted factor. The above targeting fragment is cloned into the multiple cloning site of a plasmid such as the pBluescript (Stratagene, La Jolla, California). _ιt

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Effects of Combinations of Secreted Factors in Proliferation and Differentiation [0182] Different ES clones from the ES cell library will be co-cultured in various combinations. These co-cultures will generate a pool of cells that secretes a number of molecules simultaneously. The secreted molecules can combine to affect signal transduction pathways. The effect can then be monitored by, for example, measuring or observing the proliferation and/or differentiation of the cells. Pools -of ES cells, with each pool expressing from about one to about 10 different secreted molecules, will be generated. The effects of these pools of secreted factors on the differentiation of ES cells (see examples that follow) or proliferation of other cell types will be tested. Example 3. Study of Secreted Factor Function in Hematopoietic Lineages [0183] ES cells can differentiate into mature hematopoietic cells under defined experimental conditions. For example, erythropoietin and/or interleukin la (EL- la) in the presence of IL 3 can induce this differentiation. A library of ES cells, each ES clone expressing a different secreted molecule, will be tested to monitor the ability of each ES clone's ability to differentiate (Zhang et al, 2003). Lineage marker(s) and morphology will be used to follow differentiation. Also, the synergistic effect of the combination of secreted molecules will be tested as described in Example 2. Example 4. Study of Secreted Factor Function in Pancreatic Beta Cell Differentiation [0184] There is intensive research on the differentiation of ES cells into pancreatic beta cells, yet without success. The multiple pools of ES cells generated as described in Example 2 will be used to test the ability of these secreted factor combinations to induce the ES cells' differentiation potential to develop into pancreatic beta cells. A number of beta cell markers, such as insulin, PDX-1, PAX-4, PAX-6, Nkx2.2 and Nkxδ.l, insulin I, insulin π, or glucose transporter 2, will be used to track the differentiation. Example 5. Chimeric FGF and FiRE Mice [0185] The response element vectors described in Figure 2 comprise a pSK (Stratagene, La Jolla, CA) plasmid backbone, a 5' homologous arm of the ROSA26 targeting vector, a poly A site to stop endogenous ROS A26 transcription, and a response element operably linked to a reporter gene. The response element can be the FGF inducible response element (FiRE), and the reporter gene can be lacZ. The vector also comprises bpA, PGKneobpA, and a 3' homologous arm of the ROSA26 targeting vector. [0186] The FGF inducible response element (FiRE) shown in Figure 2 was designed based on the publicly available sequence of the syndecan I gene, a heparin sulfate .v i ,.- i it > , . t irii irn

" ' ^rbteogiyc'at expressed ttode 'the regulation of FGF in a cell-type specific manner. Other elements responsive to other factors such as growth factors, metals, hormones, or other agents, are known to those of skill in the art, and can be substituted for FiRE. These vectors can be targeted to the ROSA26 or the G3BP loci of ES cells. [0187] The resulting FiRE-lacZ cells are injected into wild type C57B16 blastocysts to generate chimeras that are bred to obtain germ line transmission, then bred back to C57B16 to generate both heterozygous and homozygous C57B16 FiRE-lacZ mice using techniques known to those of skill in the art. These C57B16 FiRE-lacZ mice can then be used to identify and characterize the expression, function_? and regulation of the factors such as the FGF that drive their respective response elements. [0188] These reporter mice can also be used to generate chimeric reporter animals with one or more additional factors that drive their response elements. For example, ES cells expressing FGF can be introduced into C57B16 FiRE-lacZ mouse blastocysts to generate the reporter chimeras. The ES cells will contribute FGF and the C57B16 FiRE-lacZ mice will contribute the responsive reporter. The local action of FGF can then be observed in the resulting chimeras by in situ hybridization (ISH) or immunohistochemistry (IHC), using techniques known to those skilled in the art. ISH and ISC analyses can be performed to determine the cell and tissue expression patterns of various FGFs. The mice can be treated with agents that alter FGF signaling pathways, and the resulting effect on FGF expression can be determined by assaying the reporter gene. The agents can be proteins or peptide fragments, some if not all of which may be introduced into the mice via tail- vein injection of the nucleic acid encoding both a liver-expressed promoter and the desired agent. [0189] The cells that express the reporter gene, lacZ, can also be isolated by affinity and or cell sorting techniques such as fluorescent activated cell sorting, as they are known in the art, and these cells can be analyzed by microarray analysis for signal transduction pathway components of interest. Example 6. Constructs Containing Human Cytochrome P450 3A4 Promoter [0190] Two plasmid constructs containing the human cytochrome P450 3A4 promoter were made with a pBlueScript (Stratagene; La Jolla, CA) backbone. These constracts are represented schematically in Figure 3. The first contained the cytochrome P450 3A4 promoter operably linked to a monkey erythropoietin (EPO) gene. The second contained the same promoter operably linked to lacZ. Both constracts also contained poly A tails. Similar constructs may be made with an intron, for example, an exogenous intron, ID' |1" ^'if" ,,^■" B if If'lE iri"^* ." «j |t » ->« if»i_| ,««_t

" bet\ve6ϊϊ tBe^μprdmoter^l'ahd' ffie- unctional gene of interest. Suitable introns include those shown in Tables 2 and 3. The insertion of an intron can enhance in vivo gene expression. Example 7. DNA Delivery by Tail Vein Injection into the Reporter Animal [0191] The DNA constructs described in Example 6 can be purified using a Qiagen Plasmid Maxi Kit (Qiagen, Inc.; Valencia, CA). The constracts are then resuspended at a concentration of 25 μg/ml in saline. Two groups of reporter mice are injected with the naked DNA. Each mouse from the first group is injected with 2 ml of the EPO construct, and each mouse from the second group is injected with 2 ml of the lacZ constract (Figure 3). The duration of each injection is approximately 5-8 seconds. Example 8. Cytochrome P450 3A4 Promoter-driven EPO Expression [0192] On days 1, 3 and 7 following injection of the constructs described in Example 6, three mice from each group can be anesthetized and their blood can be drawn. Serum EPO levels of the individual mice are then determined by ELISA according to manufacturer's directions (R&D Systems; Minneapolis, MN). In a separate experiment, blood can be drawn from mice on day 8, 11 and 13 following injection of the EPO or lacZ constracts, as described above. Seram EPO level of individual mice may then be determined by ELISA. Example 9. Cytochrome P450 3A4 Promoter-driven lacZ Expression [0193] On day 11 after injection, the livers of mice injected with either the EPO construct or the lacZ constract can be harvested. As negative controls, the livers of wild-type (WT), uninjected, mice are also harvested. The livers are fixed in paraformaldehyde, then whole mount stained with 1 mg/ml X-gal using a kit from Specialty Media (Phillipsburg, N.J.). Example 10. Cytochrome P450 3A4 Promoter-driven EPO Expression [0194] On day 13, blood can be drawn from mice injected as described above with either the lacZ or the EPO construct, and sent to Quality Clinical Lab (Glendale, CA) for determination of hematocrit levels. Three mice injected as described above with either the lacZ or the EPO constract may be sacrificed on day 14 and their spleens may be isolated. Splenomegaly, as well as extramedullary hematopoeisis, may be observed if present. Example 11. DNA Molecules Containing the Promoter of a Liver Expressed Gene and a Gene Encoding a Protein or Peptide Therapeutic [0195] A nucleotide sequence suitable for expression of a protein or polypeptide modulator or therapeutic of interest may be prepared in accordance with standard recombinant DNA methodology. The nucleotide sequence may encode, for example, a , _{,, „} , ^ ^lr' ""Seire b bteifi a fttsiO^'n'pfdteln, a single-chain antibody, or a tagged protein. A DNA constract may be made comprising this nucleotide sequence operably linked to the promoter of a liver-expressed gene. Constructs of the invention may include 5' untranslated regions, including the transcription start site. They may include intron and or enhancer sequences. Gene expression and biodistribution may be monitored by, for example, methods described in the literature (Kobayashi et al, 2001). [0196] Constructs containing sequences encoding two or more proteins, or two or more constracts each containing sequences encoding a different protein, can be injected into the chimeric animals. The interaction between the two molecules can be studied in vivo. In addition, the differences in function of a protein injected alone and that of groups of proteins injected together can be determined. Example 12. Evaluating Protein Function with a Sequence of the Invention [0197] Constructs containing sequences encoding a protein or polypeptide modulator or therapeutic of interest may be injected into the chimeric animals. After allowing the protein to be expressed, the function of a protein, for example, a secreted protein, can be determined by observing changes in physiological markers or by histology of the organs such as liver, heart, lung, kidney, adrenal, lymph nodes, blood, brain, pancreas, spinal cord, or muscles. The function of a protein that interacts with the receptors of the liver cells via an autocrine process can be evaluated by looking at changes in liver function. The function of single-chain antibodies can be determined by studying immune markers. This technique can be used to inject mice with various constracts and quickly determine the function of numerous proteins.

Table 1. Annotation of Liver-Expressed Genes

Table 2. Sequence Identification Numbers

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o

ID'

Table 3. Intron Coordinates

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SEQUENCE LISTING

[0199] A sequence listing transmittal sheet, and a sequence listing in both computer readable and paper format accompanies this application.

Claims

^, „^„,^„ i. , ^l ' -ⁱ. n ^,..^,..' ,.' i 't,i 44 CLAIMS 1. An embryonic stem cell comprising a reporter system, wherein the reporter system comprises a reporter gene that is under regulatory control of a response element, wherein the responsive element is responsive to activation, inactivation, or inhibition by a signal transmitted through a signal transduction pathway, and the reporter system is situated at a gene locus of the embryonic stem cell that renders the reporter gene expressible in more than one cell type upon differentiation of the embryonic stem cell. 2. The embryonic stem cell of claim 1, wherein the reporter system further comprises a gene expressing a secreted factor. 3. The embryonic stem cell of claim 1, wherein the reporter system is selected from a luciferase reporter system, a green fluorescent protein reporter system, an alkaline phosphate reporter system, and a β-galactosidase reporter system. 4. The embryonic stem cell of claim 1, wherein the response element is selected from a fibroblast growth factor response element, NF-κB response element, vascular endothelial growth factor receptor response element, antioxidant response element, xenobiotic response element, seram response element, cAMP response element, hypoxia response element, retinoic acid response element, peroxisome proliferator response element, glucocorticoid response element, activator protein-1 response element, estrogen response element, interferon stimulatory response element, tonicity-responsive enhancer/osmotic response element, retinoic acid response element and a 5'(A/T)GATA(A/G)-3' response element. 5. The embryonic stem cell of claim 1, wherein the response element is an inducible response element. 6. The embryonic stem cell of claim 1, wherein the response element is a promoter. 7. The embryonic stem cell of claim 1, wherein the embryonic stem cell is a mouse embryonic stem cell. 8. The embryonic stem cell of claim 1, wherein the gene locus is selected from ROSA26 and G3BP(BT5). 9. A blastocyst comprising at least one introduced embryonic stem cell of claim 1. 10. A blastocyst comprising at least one introduced embryonic stem cell of claim 2. 11. A blastocyst comprising at least one introduced embryonic stem cell of claim 3. :-^lr-L ^{» ,}.^' f ;"^'i' '^•■"'^,Aⁱ'biaslocys rbc)rϋprising at least one introduced embryonic stem cell of claim 4. 13. A blastocyst comprising at least one introduced embryonic stem cell of claim 5. 14. A blastocyst comprising at least one introduced embryonic stem cell of claim 6. 15. A blastocyst comprising at least one introduced embryonic stem cell of claim 7. 16. A blastocyst comprising at least one introduced embryonic stem cell of claim 8. 17. The blastocyst of any of claims 9-16, wherein the blastocyst is obtained from an animal model of a human disease. 18. The blastocyst of any of claims 9-16, wherein the blastocyst is a mouse blastocyst. 19. An animal implanted with the blastocyst of any of claims 9-16. 20. A chimeric animal produced from the blastocyst of claims 19. 21. The chimeric animal of claim 20, wherein the blastocyst is obtained from an animal model of a human disease. 22. The chimeric animal of claim 20, wherein the blastocyst is a mouse blastocyst. 23. The chimeric animal of claim 22, wherein the blastocyst is obtained from a SCID mouse, a non-obese diabetic mouse, a RB-/- mouse, or a p53-/- mouse. 24. A progeny of the chimeric animal produced from the blastocyst of any of claims 9-16. 25. The progeny of claim 24, where the progeny is produced by breeding the chimeric animal of 20 to obtain germ line transmission of the reporter system. 26. The progeny of claim 24, wherein the progeny is heterozygous or homozygous for the reporter system. 27. A progeny blastocyst comprising a blastocyst obtained from the progeny of claim 24. 28. The progeny blastocyst of claim 27, further comprising an introduced embryonic stem cell, wherein the introduced embryonic stem cell comprises a factor that regulates the responsive element. 29. The progeny blastocyst of claim 27, wherein the blastocyst is implanted into an animal. _^ „_„„ ^ _ _ ^

^,'!^••'' , ⁱⁱ .^{•' l} ^^t ^«'A_n"^n l l^' produced from the progeny blastocyst of claim 28. 31. A progeny of the animal of claim 30. 32. A tissue obtained from the chimeric animal of claim 20 or its progeny. 33. A tissue obtained from the animal of claim 30 or its progeny. 34. The tissue of claim 32, wherein the tissue is selected from heart, liver, lung, kidney, spleen, thymus, muscle, skin, blood, bone marrow, prostate, breast, stomach, brain, spinal cord, pancreas, ovary, testis, eye, bone, cartilage, and lymph node. 35. The tissue of claim 33, wherein the tissue is selected from heart, liver, lung, kidney, spleen, thymus, muscle, skin, blood, bone marrow, prostate, breast, stomach, brain, spinal cord, pancreas, ovary, testis, eye, bone, cartilage and lymph node. 36. A cell obtained from the chimeric animal of claim 20 or its progeny. 37. A cell obtained from the animal of claim 30 or its progeny. 38. A cell line derived from the tissue of claim 32. 39. A cell line derived from the tissue of claim 33. 40. A cell line derived from the cell of claim 36. 41. A cell line derived from the cell of claim 37. 42. The chimeric animal of claim 20 or its progeny that has been treated with a heterologous protein therapeutic or a small molecule drag. 43. The treated animal of claim 42 or its progeny, wherein the heterologous protein therapeutic is introduced into the chimeric animal via a DNA molecule that comprises a first sequence operably linked to a second sequence, wherein the first sequence comprises a promoter of a liver-expressed gene and the second sequence encodes at least one heterologous protein therapeutic, wherein the DNA molecule is other than a naturally-occurring molecule and is free of viral-derived sequences, and the second sequence is other than a reporter gene, wherein the DNA molecule can be expressed in vivo in the chimeric animal to produce at least one protein therapeutic that is functionally active in the animal. 44. The DNA molecule of claim 43, wherein the DNA molecule further comprises a third sequence that is operably linked to the first and second sequence, wherein the third sequence comprises an intron sequence. 45. The DNA molecule of claim 44, wherein the intron is a heterologous intron. 46. The DNA molecule of claim 44, wherein the intron comprises a nucleotide sequence selected from SEQ ID NO.: 1 to SEQ ID NO.: 394. 47. The DNA molecule of claim 43, wherein the heterologous protein therapeutic is a secreted protein. _{i ^} _ ⁱ'-" ^I . ^ϊ ,'^''4^f8v-"^ι; "iϊle ' >N rtfjB(9le of claim 43, wherein the heterologous protein therapeutic is a transmembrane protein. 49. The DNA molecule of claim 43, wherein the heterologous protein therapeutic is an extracellular fragment of an intracellular protein. 50. The DNA molecule of claim 43, wherein the heterologous protein therapeutic is an intracellular protein or an intracellular fragment of a transmembrane protein. 51. The DNA molecule of claim 43 , wherein the heterologous protein therapeutic is a human protein. 52. The DNA molecule of claim 43, wherein the heterologous protein therapeutic is a mouse protein. 53. The DNA molecule of claim 43, wherein the promoter comprises a transcription start site, and the transcription start site comprises a start sequence selected from SEQ ID NO.: 1 to SEQ ID NO.: 394. 54. The DNA molecule of claim 53, wherein the promoter comprises a nucleotide sequence, and the nucleotide sequence is all or a part of a sequence selected from SEQ ID NO.: 1 to SEQ ID NO.: 394. The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 245 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 246 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 247 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 248 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 249 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 250 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 251 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 252, The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 253 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 254 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 255 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 256 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 257 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 258 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 259 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 260 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 261 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO. 262 F it ii'" c: ιr TΪeDN ϊMcile of claim 53, wherein the promoter is SEQ ID NO.: 263. 74 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 264. 75 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 265. 76 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 266. 77 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 267. 78 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 268. 79 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 269. 80 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 270. 81 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 271. 82 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 272. 83 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 273. 84 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 274. 85 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 275. 86 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 276. 87 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 277. 88 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 278. 89 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 279. 90 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 280. 91. The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 281. 92 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 282. 93 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 283. 94 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 284. 95 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 285. 96 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 286. 97 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 287. 98 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 288. 99 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 289. 100 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 290. 101 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 291. 102 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 292. 103 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 293. 104 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 294. 105 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 295. 106 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 296. 107 The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.: 297. # €tfϊe- D»r I@le of claim 53 , wherein the promoter is SEQ JD NO.: 298 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 299 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 300 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 301 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 302 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 303 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 304 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 305 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 306 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 307 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 308 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 309 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 310 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 311 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 312 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 313 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 314 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 315 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 316 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 317 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 318 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 319 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 320 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 321 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 322 The DNA molecule of claim 53 wherein the promoter is SEQ ID NO.: 323 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 324 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 325 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 326 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 327 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 328 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 329 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 330 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 331 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 332 "?" 1AQ if" TSe^'DN ϊ ϊcile of claim 53 , wherein the promoter is SEQ ID NO.: 333. 144 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 334. 145 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.: 335. 146 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 336. 147 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 337. 148 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 338. 149 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 339. 150 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 340. 151 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 341. 152 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 342. 153 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.: 343. 154 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.:344. 155 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.:345. 156 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.-.346. 157 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.:347. 158 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.:348. 159 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.:349. 160 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.:350. 161 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.:351. 162 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.:352. 163 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.:353. 164 The DNA molecule of claim 53 , wherein the promoter is SEQ ID NO.:354. 165 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:355. 166 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:356. 167 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:357. 168 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:358. 169 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:359. 170 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:360. 171 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:361. 172 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:362. 173 The DNA molecule of claim 53 wherein the promoter is SEQ JD NO.:363. 174 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:364. 175 The DNA molecule of claim 53 , wherein the promoter is SEQ D NO.:365. 176 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:366. 177 The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO.:367. P rT 0$$s1&}$ϊ b8he of claim 53, wherein the promoter is SEQ ID NO.:368. 179. The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.:369. 180. The DNA molecule of claim 53 , wherein the promoter is SEQ JD NO. : 370. 181. The DNA molecule of claim 53, wherein the promoter is SEQ JD NO.:371. 182. The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.:372. 183. The DNA molecule of claim 53, wherein the promoter is SEQ JD NO.:373. 184. The DNA molecule of claim 53, wherein the promoter is SEQ JD NO.:374. 185. The DNA molecule of claim 53 wherein the promoter is SEQ JD NO. : 375 186. The DNA molecule of claim 53 wherein the promoter is SEQ JD NO. : 376 187. The DNA molecule of claim 53 wherein the promoter is SEQ JD NO.:377 188. The DNA molecule of claim 53 wherein the promoter is SEQ ID NO.:378 189. The DNA molecule of claim 53 wherein the promoter is SEQ ID NO.:379 190. The DNA molecule of claim 53 wherein the promoter is SEQ ID NO.:380 191. The DNA molecule of claim 53 wherein the promoter is SEQ JD NO.:381 192. The DNA molecule of claim 53 wherein the promoter is SEQ JD NO.:382 193. The DNA molecule of claim 53 wherein the promoter is SEQ ID NO.:383 194. The DNA molecule of claim 53 wherein the promoter is SEQ ID NO.:384 195. The DNA molecule of claim 53 wherein the promoter is SEQ JD NO.:385 196. The DNA molecule of claim 53, wherein the promoter is SEQ JD NO.:386 197. The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.:387 198. The DNA molecule of claim 53, wherein the promoter is SEQ JD NO.:388 199. The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.:389 200. The DNA molecule of claim 53, wherein the promoter is SEQ JD NO.:390 201. The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.:391 202. The DNA molecule of claim 53, wherein the promoter is SEQ ID NO.:392 203. The DNA molecule of claim 53, wherein the promoter is SEQ JD NO.:393 204. The DNA molecule of claim 53, wherein the promoter is SEQ JD NO.:394 205. The DNA molecule of claim 53, wherein the second sequence encodes a protein other than alpha- 1 antitrypsin. 206. A heterologous protein therapeutic composition comprising a DNA molecule of claim 43 and a pharmaceutically acceptable carrier or excipient. 207. The heterologous protein therapeutic composition of claim 206, further comprising a third sequence that is operably linked to the first and the second sequence, wherein the third sequence comprises an intron sequence selected from SEQ ID NO.: 1 to SEQ JD NO.: 394, and a pharmaceutically acceptable carrier or excipient. ^{!h» ,i,},^, ,. „^•

206, wherein the carrier or excipient is other than a liposome or a DNA complexing agent. 209. The composition of claim 207, wherein the carrier or excipient is other than a liposome or a DNA complexing agent. 210. The composition of claim 206, wherein the carrier or excipient is saline, phosphate buffer saline, or a buffer. 211. The composition of claim 207, wherein the carrier or excipient is saline, phosphate buffer saline, or a buffer. 212. The composition of claim 206, further comprising a nucleotide sequence that comprises a sequence encoding a protein that enhances either or both the expression or folding of the heterologous protein therapeutic encoded by the sequence of the DNA molecule. 213. The composition of claim 207, further comprising a nucleotide sequence that comprises a sequence encoding a protein that enhances either or both the expression or folding of the heterologous protein therapeutic encoded by the sequence of the DNA molecule. 214. The treated animal of claim 42, wherein the protein therapeutic is introduced into the chimeric animal via a vector comprising the DNA molecule of 53 and an origin of replication. 215. The vector of claim 214, further comprising a nucleotide sequence encoding a reporter gene. 216. The vector of 214, further comprising a nucleotide sequence encoding an antibiotic resistance gene. 217. The treated animal of claim 42, wherein a reporter system is activated by a signal transduction pathway. 218. A method of determining an in vivo effect of a therapeutic, comprising: (a) administering the therapeutic to the chimeric animal of claim 20 or its progeny; and (b) identifying the tissue or cell type in which the reporter system is activated by a signal transduction pathway. 219. The method of claim 218, wherein the therapeutic is a protein therapeutic or a small molecule therapeutic. 220. The method of claim 219, wherein the protein therapeutic administered to the chimeric animal of claim 20 or its progeny comprising: [l-^ιr I^',. ( .^•■ ^^"3i -U'(aⁱ) ro'vιαiiϊ ⁱ'a ιy^! of the composition of comprising a DNA molecule that comprises first sequence operably linked to a second sequence, wherein the first sequence comprises a promoter of a liver-expressed gene selected from all or part of SEQ JD NO.: 1 to SEQ ID NO.: 394, and the second sequence encodes at least one heterologous protein therapeutic, wherein the DNA molecule is other than a naturally occurring molecule and is free of viral derived sequences, and the second sequence is other than a reporter gene; (b) injecting the composition into the animal; and (c) allowing expression of the protein therapeutic. 221. The method of claim 220, wherein the composition is injected under pressure. 222. The method of claim 220, wherein the composition is injected intravenously. 223. The method of claim 220, wherein the duration of the injecting step is about 5 seconds. 224. The method of claim 220, wherein the composition has a volume of about 1, 1.5, or 2 ml. 225. The method of claim 220, wherein the expression and protein activity of the protein therapeutic are detectable on day 3. 226. The method of claim 220, wherein the expression and protein activity of the protein therapeutic are detectable on day 7. 227. The method of claim 220, wherein the expression and protein activity of the protein therapeutic are detectable on day 8. 228. The method of claim 220, wherein the expression and protein activity of the protein therapeutic are detectable on day 11. 229. The method of claim 220, wherein the expression and protein activity of the protein therapeutic are detectable on day 13. 230. A method of identifying one or more components of a signal transduction pathway comprising: (a) administering a molecule or compound to the chimeric animal of claim 20 or its progeny; and (b) identifying the one or more genes activated by the molecule or compound. 231. A method of determining a function of a gene encoding a factor comprising: (a) introducing the gene into an embryonic stem cell; i- ^v X ., ^{, !}LH .»l !.:^;^ ^» ' i'n oiduiing the embryonic stem cell of (a) into a blastocyst, wherein the blastocyst is the blastocyst of any one of claims 9-16 or its progeny; (c) allowing the blastocyst of (b) to develop into an animal; and (d) observing the animal of (c) to determine gene function.