WO2002072017A2

WO2002072017A2 - Method of drug target validation

Info

Publication number: WO2002072017A2
Application number: PCT/US2002/007294
Authority: WO
Inventors: Tito Andrew Serafini
Original assignee: Renovis, Inc.
Priority date: 2001-03-12
Filing date: 2002-03-12
Publication date: 2002-09-19
Also published as: AU2002306684A1; WO2002072017A9; WO2002072017A3

Abstract

A method of validating potential drug targets is provided. A potential drug target (or an inhibitor thereof) is introduced into animal lines such that the expression of the target is spatially or temporarily restricted. The animal lines are transgenic for a 'key gene' which encodes a 'key protein' that activates or inhibits expression of the potential target. Key protein expression is regulated by regulatory sequences from a 'characterizing gene,' whose expression is spatially or temporarily restricted. For target validation, a 'modulating construct' containing a nucleotide sequence encoding the target, or a product that modulates the expression of the target, is introduced into a transgenic cell line with a desired key gene expression pattern. Expression of the target, or modulator thereof, is regulated by the presence of the key protein, so that only cells expressing the key protein can activate or inhibit expression of the drug target.

Description

METHOD OF DRUG TARGET VALIDATION

1. TECHNICAL FIELD

The present invention relates to methods for validating potential drug targets. The invention provides methods of screening numerous potential drug targets in one or more transgenic animal lines, but does not require the time-consuming production of a transgenic line for each potential drug target to be validated.

2. BACKGROUND OF THE INVENTION An important goal in the design and development of new therapies for human diseases and disorders is the identification of potential drug targets. A drug target is a gene, or the protein product of a gene, that is related to a particular indication, disease, or disorder, and that serves as a target for drug development. A potential drug target is a product of an endogenous gene, the expression of which has been observed to increase or decrease in a particular disease state. When a gene or its protein product is validated as a drug target, an alteration in the activity of the gene (up-regulation or down-regulation) or its pattern of expression, or an alteration in the activity of its protein product, is demonstrated to treat, prevent, or ameliorate the indication.

A significant advance in the last decade has been the identification and generation of numerous genetic polymorphisms and mutations that perturb normal mammalian cellular and physiological functions. Based on this work, numerous transgenic animal models, in particular mouse models, have been developed for mammalian diseases and disorders. The analysis of transgenic animal models carrying genetic polymorphisms and mutations has shed light on the molecular mechanisms underlying mammalian (particularly human) diseases and disorders, leading to the identification of potential drug targets. There are significant limitations, however, in the currently available methods for validating such potential drug targets in a transgenic animal model (or by any other method). One significant limitation is the amount of time (usually several months) required to produce a potential founder transgenic animal, such as a transgenic mouse, that bears a particular mutation or transgene and that exhibits a particular disease state. More time is then required to establish a stable line of transgenic individuals derived from the founder. It is only after several months of work to establish a transgenic animal line that the line is ready to be used to screen for and validate a potential drug target and/or to be tested as a model for potential therapeutic treatments. Thus there is an urgent need for methods that permit more rapid and efficient screening for potential drug targets in transgenic animal lines. Such a technology should permit the screening of numerous potential drug targets, but not require the time-consuming production of a transgenic line for each potential drug target to be validated. We describe such a technology here.

3. SUMMARY OF THE INVENTION

The invention relates to a method of validating potential drug targets, i.e., a gene or protein product of a gene that is potentially related to a particular indication (e.g., a particular disease or disorder) and that potentially serves as target for drug development, for example, where the inhibition, altered expression, or increase in activity of the gene or protein product thereof treats, prevents or ameliorates the indication or symptom thereof. In a specific embodiment, the potential drug target is the product of an endogenous gene, the expression of which has been observed to increase or decrease in a particular disease state. The drug validation system of the present invention allows the screening of numbers of potential drug targets using one or a collection of transgenic animal lines. The method, as detailed below, does not involve the production of transgenic lines for each potential drug target to be validated but, rather, involves introduction of a potential drug target (or an inhibitor thereof) into one or more existing animal lines transgenic for a transactivator or transinhibitor that is conditionally expressed and that activates or inhibits expression of the potential drug target such that the potential drug target is either expressed or inhibited only in a particular subset of cells (i.e., expression is spatially or temporally restricted). The drug validation system of the invention is more flexible, convenient and efficient than other existing drug validation systems because it uses one of a limited set of transgenic animal lines not necessarily specific for the particular target, instead of requiring the production of a transgenic animal line for each target to be validated.

In particular, the drug validation method of the invention uses one or more transgenic animal lines, preferably transgenic mouse lines, transgenic for a DNA sequence that encodes a "key protein." The key protein is a protein that can activate or inhibit expression of a gene under the control of an expression element that is turned off or on by the key protein (for example, but not limited to, promoters and/or enhancers whereby transcription is turned on or off by a specific transactivator; recombinase target sites for which recombination is effected by a recombinase, and recombination positions the target gene for expression or inhibition of expression). In a preferred embodiment, the expression of the key protein is regulated by regulatory sequences from a gene (herein a "characterizing gene") that is endogenously expressed in a particular subset of cells. The gene encoding the key protein (the "key gene") can be introduced (either by insertion or replacement) into a non-coding sequence or coding sequence of the characterizing gene (but preferably not into a regulatory sequence), (for example, by introduction of such a modified characterizing gene, i.e., a transgene, including all or a portion of the regulatory sequences into the genome of the animal), such that the expression of the key gene substantially reproduces the endogenous expression pattern of the characterizing gene. An individual line is selected based on its expression of the key gene in a desired cell or population of cells. Preferably, the expression pattern of the key protein delineates a select subpopulation of cells. In particular, each transgenic line expressing a particular key gene under the control of the regulatory sequences of a characterizing gene is created by the introduction, for example by pronuclear injection, or by non-homologous recombination in embryonic stem cells that are introduced into embryos, of a vector containing the transgene into a founder animal, such that the transgene is transmitted to offspring in the line. The transgene preferably randomly integrates into the genome of the founder, but in specific embodiments, may be introduced by directed homologous recombination. In a preferred embodiment, the transgene is present at a location on the chromosome other than the site of the endogenous characterizing gene. In a preferred embodiment, homologous recombination in bacteria is used for target-directed insertion of the key gene sequence into a genomic DNA fragment containing all or a portion of the characterizing gene, including sufficient characterizing gene regulatory sequences to promote expression of the characterizing gene in its endogenous expression pattern. In a preferred embodiment, the characterizing gene sequences are on a bacterial artificial chromosome (BAC). In specific embodiments, the key gene coding sequences are inserted as a 5' fusion with the characterizing gene coding sequence such that the key gene coding sequences are inserted in frame and directly 3' from the initiation codon for the characterizing gene coding sequences. In another embodiment, the key gene coding sequences are inserted into the 3' untranslated region (UTR) of the characterizing gene and, preferably, have their own internal ribosome entry sequence (IRES). The vector (preferably a BAC) comprising the key gene coding sequences and characterizing gene sequences is then introduced into the genome of a potential founder animal to generate a line of transgenic animals. Potential founder animals can be screened for the selective expression of the key gene sequence in the population of cells characterized by expression of the endogenous characterizing gene. Transgenic animals that exhibit appropriate expression (e.g., detectable expression of the key gene product having the same expression pattern within the animal as the endogenous characterizing gene) are selected as founders for a line of transgenic animals.

For target validation, a "modulating construct" containing a nucleotide sequence encoding the potential drug target, or a product that specifically modulates (e.g., inhibits) the expression of the potential drug target, is introduced into an appropriate transgenic animal cell line. The expression of the potential drug target, or modulator thereof, is regulated (either activated or inhibited) by the presence of the key protein. For example, if the key protein is a transcriptional activator, the potential drug target is operably linked to a promoter activated by the key protein transcriptional activator. With this approach, only those cells expressing the key protein are able to activate or inhibit expression of the target encoded by the modulating construct. The modulating construct can contain a nucleotide sequence that is homologous to a selected endogenous gene sequence in the transgenic animal line or that is orthologously related to the endogenous gene sequence. Alternatively, the modulating construct can encode an inhibitor, including, but not limited to, a catalytic nucleic acid such as a ribozyme, inhibitory RNA (RNAi), or an inhibitor protein of the endogenous gene sequence.

Preferably, the modulating construct is a viral vector that is used to infect a general type or population of cells (for example, the cells of a mouse in a global fashion) expressing the key protein in a select subpopulation of the general type or population of cells. In a specific embodiment, the viral vector comprising the modulating construct is directly injected into a particular tissue region, e.g., a brain region. In specific embodiments, the viral vector is replication proficient; in an alternative embodiment, the viral vector is replication deficient.

In a preferred aspect, the invention provides a method of determining whether the modulation of expression of a potential target gene in a particular cell type is causally linked to a desired effect, for example, expression of the potential target causes the expression of a certain cell or tissue phenotype associated with a particular disease or disorder or with the treatment, prevention or amelioration of that disease or disorder. The subject methods are advantageous because they enable the validation of drug targets to proceed rapidly and efficiently, limited only by the rate at which modulating constructs can be produced, and not by the rate at which a transgenic animal line can be produced. A collection of transgenic animal lines expressing key proteins, for example, where the lines express the key protein in different cell populations, can be used repeatedly to validate many potential drug targets introduced via modulating constructs. The invention also provides non-human transgenic animals that express one or more potential drug target proteins (or inhibitors thereof) in a specific subset of cells. A transgenic animal of the invention comprises, for each potential drug target, a vector (or in certain embodiments a transgene) comprising a first nucleotide sequence encoding the potential drug target protein (or inhibitor thereof). The expression of each potential drug target protein (or inhibitor thereof) is under the control of a conditional expression element. The transgenic animal further comprises a transgene containing a key gene that encodes an inducer or suppressor of the conditional expression element. The key gene is operably linked to regulatory sequences of a characterizing gene corresponding to an endogenous gene or homolog of an endogenous gene such that the key gene is expressed in the transgenic animal with an expression pattern that is substantially the same as the expression pattern of the endogenous gene in a non-transgenic animal of the same species. The potential drug target protein(s) (or inhibitor(s) thereof) is selectively expressed in the cells expressing the key gene.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B. A. DNA fingerprint gel showing putative co-integrate clones. Three different BAC clones containing the 5HT6 gene were used. B. Southern hybridization showing that all three clones were indeed co-integrates. Hindlll fragments containing the homology box were labeled and were duplicated in co-integrates. See . Section 6.9 for details.

FIG. 2. Restriction mapping using DNA pulse-field gel (CHEF mapping protocol, Section 6.4) showing that one of the 5HT6-containing BAC clones (clone2) had a sufficiently large DNA fragment upstream of the 5HT6 transcription start site. Y-axis, molecular weight in kilobases (kB). M=marker. See Section 6.9 for details.

FIGS. 3A and B. A. DNA fingerprint gel showing putative resolvant clones. B. Southern hybridization showing that two out of four clones tested were indeed resolvants; Hindlll fragments containing Emerald (GFP) were labeled; two copies of Emerald were present in co-integrate (col) and only one copy was left in the resolvants. See Section 6.9 for details.

FIGS. 4A and B. Fluorescence (A.) and light (B.) photomicrographs of a section through the cortex of a transgenic mouse expressing the 5HT6 receptor BAC. The section was immunohistochemically stained with an anti-GFP primary antibody and a fluorescently- conjugated secondary antibody. See Section 6.9 for details. FIG. 5. Fluorescence photomicrograph of a section of the hippocampus of a transgenic mouse expressing the 5HT6 receptor BAC. The section was immunohistochemically stained with an anti-GFP primary antibody and a fluorescently- conjugated secondary antibody. See Section 6.9 for details. FIG. 6. DNA fingerprint gel showing putative co-integrate clones. Seven different

BAC clones containing the 5HT2A gene were used. Y-axis, molecular weight in kilobases (kB). SN=shuttle vector. See Section 6.10 for details.

FIG. 7. Southern hybridization was used to verify duplication of A boxes in cointegrate clones. SN=shuttle vector. See Section 6.10 for details. FIG. 8. CHEF mapping used to determine that one of the BACs was constructed such that one of the 5HT2A BAC clones had a sufficiently large DΝA fragment upstream of the 5HT2A transcription start site. Y-axis, molecular weight in kilobases (kB). Asc = Asc- 1. restriction enzyme; Νot= Not-1 restriction enzyme. See Section 6.10 for details.

FIG. 9. DNA fingerprint gel showing putative resolvant clones (arrows). SV=shuttle vector; U=unmodifιed; CoI=co-integrate. See Section 6.10 for details.

FIG. 10. Southern hybridization showing that the two clones tested (arrows) were indeed resolvants. See Section 6.10 for details. U=unmodifιed; C=co-integrate. See Section 6.10 for details.

FIG. 11. Fluorescence photomicrograph of a section of brain tissue showing that the 5HT2A transgene was indeed expressed in subsets of neurons in transgenic animals (arrows point to two fluorescent cells). See Section 6.10 for details.

FIG. 12. A pLD53 shuttle vector designed to insert IRES-Emerald at the position specified by the A box, which is cloned into the vector using the indicated Ascl and Smal sites. The PCR product of the A box is cloned by digesting it with Ascl and then ligating with Ascl/Smal digested pLD53.

FIG. 13. A pLD53 shuttle vector designed to insert Emerald at the position specified by the A box (normally, at the 5' end of the gene, such that Emerald is produced from the transcribed mRNA instead of the gene into which the insertion occurs). The A box is shown cloned into the vector.

5. DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections set forth below. 5.1. TRANSGENIC ANIMAL LINES FOR DRUG VALIDATION

The drug validation system of the present invention allows the screening of numbers of potential drug targets using one or more transgenic animal lines. The drug validation system of the invention is more flexible, convenient and efficient than other existing drug validation systems because it uses one of a limited set of transgenic animal lines that is not necessarily specific for the particular drug target, instead of requiring the production of a transgenic animal line for each drug target to be validated.

Each transgenic line is created by the introduction of a transgene into a founder animal, such that the transgene is transmitted to offspring in the line. Methods for producing transgenic animal lines and collections of transgenic animal lines are described in Serafmi, U.S. Patent Application Serial No. 09/783,487 entitled "Collections of Transgenic Animal Lines (Living Library)" filed February 14, 2001, and Serafmi, U.S. Patent Application Serial No. (to be assigned) (Attorney Docket No. 10239-0036-999) entitled "Collections of Transgenic Animal Lines (Living Library)" filed February 14, 2002, both of which are incorporated herein by reference in their entireties.

A line may include transgenic animals that are derived from more than one founder animal but that contain the same transgene, preferably in the same chromosomal position and/or exhibiting the same level and pattern of expression within the animal. For example, in certain circumstances, it may be preferable to use more than one founder animal to maintain or rederive a line. In each transgenic animal line, a subset of cells of the transgenic animal that is characterized by expression of a particular endogenous gene (a "characterizing gene") also expresses the key gene, either constitutively or conditionally. The transgenic animal lines, collections of transgenic animal lines, and collections of vectors of the invention may be used for pharmacological, behavioral, physiological, electrophysiological, or drug discovery assays, for target validation, for gene expression analysis, etc.

Each transgenic animal line of the invention contains a transgene that comprises key gene coding sequences under the control of the regulatory sequences for a characterizing gene, such that the key gene has substantially the same expression pattern as the endogenous characterizing gene. The expression of the key gene permits activation or inhibition of a gene comprised in the modulating construct that encodes a potential drug target.

A transgene is a nucleotide sequence that has been or is designed to be incorporated into a cell, particularly a mammalian cell, that in turn becomes or is incorporated into a living animal such that the nucleic acid containing the nucleotide sequence is expressed (i e. , the mammalian cell is transformed with the transgene). The characterizing gene sequence is preferably endogenous to the transgenic animal, or is an ortholog of an endogenous gene, e.g., the human ortholog of a gene endogenous to the animal to be made transgenic. A transgene may be present as an extrachromosomal element in some or all of the cells of a transgenic animal or, preferably, stably integrated into some or all of the cells, more preferably into the germline DNA of the animal (i.e., such that the transgene is transmitted to all or some of the animal's progeny), thereby directing expression of an encoded gene product (i.e., the key gene product) in one or more cell types or tissues of the transgenic animal. Unless otherwise indicated, it will be assumed that a transgenic animal comprises stable changes to the chromosomes of germline cells. In a preferred embodiment, the transgene is present in the genome at a site other than where the endogenous characterizing gene is located. In other embodiments, the transgene is incorporated into the genome of the transgenic animal at the site of the endogenous characterizing gene, for example, by homologous recombination. Such a transgenic animal is created by introducing a transgenic construct of the invention into the animal's genome using methods routine in the art, for example, the methods described in Section 5.4 and 5.5, infra, and using the vectors described in Section 5.3, infra. A construct is a recombinant nucleic acid, generally recombinant DNA, generated for the purpose of the expression of a specific nucleotide sequence(s), or to be used in the construction of other recombinant nucleotide sequences. A transgenic construct of the invention includes at least the coding region for a key gene operably linked to all or a portion of the regulatory sequences, e.g. a promoter and/or enhancer, of the characterizing gene. The transgenic construct optionally includes enhancer sequences and coding and other non-coding sequences (including intron and 5' and 3' untranslated sequences) from the characterizing gene such that the key gene is expressed in the same subset of cells as the characterizing gene in the same transgenic animal or in a comparable (e.g., same species, strain, gender, age, genetic background, etc. (e.g., a sibling) non-transgenic animal, i.e., an animal that is essentially the same but for the presence of the transgene).

The key gene coding sequences and the characterizing gene regulatory sequences are operably linked, meaning that they are connected in such a way so as to permit expression of the key gene when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the characterizing gene regulatory sequences. Preferably the linkage is covalent, most preferably by a nucleotide bond. The promoter region is of sufficient length to promote transcription, as described in Alberts et al. (1989) in Molecular Biology of the Cell, 2d Ed. (Garland Publishing, Inc.). In one aspect of the invention, the regulatory sequence is the promoter of a characterizing gene. Other promoters that direct tissue-specific expression of the coding sequences to which they are operably linked are also contemplated in the invention. In specific embodiments, a promoter from one gene and other regulatory sequences (such as enhancers) from other genes are combined to achieve a particular temporal and spatial expression pattern of the key gene.

In a specific embodiment, the key gene coding sequences code for a protein that activates, enhances or suppresses the expression of a gene encoding a potential drug that is comprised in the modulating construct. More particularly, the transgene comprises the key gene coding sequences operably linked to characterizing gene regulatory sequences. The modulating construct comprises sequences encoding a potential drug target operably linked to an expression control element that is activatable or suppressible by the protein product of the key gene coding sequences. In particular embodiments, the sequences encoding the potential drug target operably linked to sequences that activate or suppress expression of the marker in the presence of the key gene protein product are present on a second transgene introduced into the transgenic animal containing the transgene with the key gene operably linked to the characterizing gene regulatory sequences, for example, but not by way of limitation, by injection of a viral vector, by random integration directly into the genome of the transgenic animal, or by breeding with a transgenic animal of the invention.

Methods that are well known to those skilled in the art can be used to construct vectors containing key gene coding sequences operatively associated with the appropriate transcriptional and translational control signals of the characterizing gene (see Section 5.1.2, infra). These methods include, for example, in vitro recombinant DNA techniques and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al, 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N.Y.; and Ausubel et al, 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., both of which are hereby incorporated by reference in their entireties.

The key gene coding sequences may be incorporated into some or all of the characterizing gene sequences such that the key gene is expressed in substantially the same expression pattern as the endogenous characterizing gene in the transgenic animal, or at least, in the anatomical region or tissue of the animal (by way of example, in the brain, spinal chord, heart, skin, bones, head, limbs, blood, muscle, peripheral nervous system, etc.) containing the population of cells to be marked by expression of the key gene coding sequences, so that tissue can be dissected from the transgenic animal, which tissue contains only cells of interest expressing the key gene coding sequences. By "substantially the same expression pattern" is meant that the key gene coding sequences are expressed in at least 80%, 85%, 90%, 95%, and preferably 100% of the cells shown to express the endogenous characterizing gene by in situ hybridization. Because detection of the key gene expression product (or a marker expressed therewith) may be more sensitive than in situ hybridization detection of the endogenous characterizing gene messenger RNA, more cells may be detected to express the key gene product in the transgenic mice of the invention than are detected to express the endogenous characterizing gene by in situ hybridization or any other method known in the art for in situ detection of gene expression.

For example, the nucleotide sequences encoding the key gene protein product may replace the characterizing gene coding sequences in a genomic clone of the characterizing gene, leaving the characterizing gene regulatory non-coding sequences. In other embodiments, the key gene coding sequences (either genomic or cDNA sequences) replace all or a portion of the characterizing gene coding sequence and the transgene only contains the upstream and downstream characterizing gene regulatory sequences. In a preferred embodiment, the key gene coding sequences are inserted into or replace transcribed coding or non-coding sequences of the genomic characterizing gene sequences, for example, into or replacing a region of an exon or of the 3' UTR of the characterizing gene genomic sequence. Preferably, the key gene coding sequences are not inserted into or replace regulatory sequences of the genomic characterizing gene sequences. Preferably, the key gene coding sequences are also not inserted into or replace characterizing gene intron sequences.

In a preferred embodiment, the key gene coding sequence is inserted into or replaces a portion of the 3' untranslated region (UTR) of the characterizing gene genomic sequence. In another preferred embodiment, the coding sequence of the characterizing gene is mutated or disrupted to abolish characterizing gene expression from the transgene without affecting the expression of the key gene. Preferably, the key gene coding sequence has its own internal ribosome entry site (IRES). For descriptions of IRESes, see, e.g., Jackson et al, 1990, Trends Biochem Sci. 15(12):477-83; Jang et al, 1988, J. Virol. 62(8):2636-43; Jang et al, 1990, Enzyme 44(l-4):292-309; and Martinez-Salas, 1999, Curr. Opin. Biotechnol. 10(5):458-64.

In another embodiment, the key gene is inserted at the 3' end of the characterizing gene coding sequence. In a specific embodiment, the key coding sequences are introduced at the 3' end of the characterizing gene coding sequence such that the transgene encodes a fusion of the characterizing gene and the key gene sequences. Preferably, the key gene coding sequences are inserted using 5' direct fusion wherein the key gene coding sequences are inserted in-frame adjacent to the initial ATG sequence (or adjacent to the nucleotide sequence encoding the first two, three, four, five, six, seven or eight amino acids of the characterizing gene protein product) of the characterizing gene, so that translation of the inserted sequence produces a fusion protein of the first methionine (or first few amino acids) derived from the characterizing gene sequence fused to the key gene protein. In this embodiment, the characterizing gene coding sequence 3' of the key gene coding sequences are not expressed. In yet another specific embodiment, a key gene is inserted into a separate cistron in the 5¹ region of the characterizing gene genomic sequence and has an independent IRES sequence.

In certain embodiments, an IRES is operably linked to the key gene coding sequence to direct translation of the key gene. The IRES permits the creation of polycistronic mRNAs from which several proteins can be synthesized under the control of an endogenous transcriptional regulatory sequence. Such a construct is advantageous because it allows marker proteins to be produced in the same cells that express the endogenous gene (Heintz, 2000, Hum. Mol. Genet. 9(6): 937-43; Heintz et al, WO 98/59060; Heintz et al, WO 01/05962; which are all incorporated herein by reference in their entireties).

Shuttle vectors containing an IRES, such as the pLD55 shuttle vector (see Heintz et al, WO 01/05962), may be used to insert the key gene sequence into the characterizing gene. The IRES in the pLD55 shuttle vector is derived from EMCV (encephalomyocarditis virus) (Jackson et al, 1990, Trends Biochem Sci. 15(12):477-83; and Jang et al, 1988, J. Virol. 62(8):2636-43, both of which are incorporated herein by reference in their entireties). The common sequence between the first and second IRES sites in the shuttle vector is shown below. This common sequence also matches pIRES (Clontech) from 1158-1710. TAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATAT GTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGG CCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATG CAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGA CAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGA CAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGG CACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGG CTCTCCTAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACTCCATT GTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGA GGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAA AAACACCATGATA (SEQ ID NO: 1) In a specific embodiment, the EMCV IRES is used to direct independent translation of the key gene coding sequences (Gorski and Jones, 1999, Nucleic Acids Research 27(9):2059-61).

In another specific embodiment the shuttle vectors pLD53-5' IRES-Em (FIG. 12) and pLD53-3' IRES-Em (FIG. 13) may be used.

In another embodiment, more than one IRES site is present in the transgene to direct translation of more than one coding sequence. However, in this case, each IRES sequence must be a different sequence.

In certain embodiments in which a key gene is expressed conditionally, the key gene coding sequence is embedded in the genomic sequence of the characterizing gene and is inactive unless acted on by a transactivator or recombinase, whereby expression of the key gene can then be driven by the characterizing gene regulatory sequences.

In other embodiments, a marker gene is expressed conditionally, through the activity of a key gene that is an activator or suppressor of gene expression. In this case, the key gene encodes a transactivator, e.g., tetR, or a recombinase, e.g., FLP, whose expression is regulated by the characterizing gene regulatory sequences. The marker gene is linked to a conditional element, e.g., the tet promoter, or is flanked by recombinase sites, e.g., FRT sites, and may be located anywhere within the genome. In such a system, expression of the key gene, as regulated by the characterizing gene regulatory sequences, activates the expression of the marker gene. In this way, cells expressing the key gene, and with the potential to activate or inhibit expression of the drug target when the modulating construct is present, can be marked. In one embodiment, the marker gene is contained in the modulating construct. In another embodiment, it is not contained in the modulating construct. In certain embodiments, exogenous translational control signals, including, for example, the ATG initiation codon, can be provided by the characterizing gene or some other heterologous gene. The initiation codon must be in phase with the reading frame of the desired coding sequence of the key gene to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al, 1987, Methods in Enzymol. 153: 516-544).

As detailed below in Section 5.3, the transgene construct comprising the key gene can also comprise one or more genes encoding selectable markers that enable identification and/or selection of recombinant vectors. The selectable marker may be the key gene product itself or an additional selectable marker not necessarily tied to the expression of the characterizing gene.

Preferably, the transgene comprises all or a significant portion of the genomic characterizing gene, preferably, at least all or a significant portion of the 5' regulatory sequences of the characterizing gene, most preferably, sufficient sequence 5' of the characterizing gene coding sequence to direct expression of the key gene coding sequences in the same expression pattern (temporal and/or spatial) as the endogenous counterpart of the characterizing gene. In certain embodiments, the transgene comprises one exon, two exons, all but one exon, or all but two exons, of the characterizing gene. Nucleic acids comprising the characterizing gene sequences and key gene coding sequences can be obtained from any available source. In most cases, all or a portion of the characterizing gene sequences and/or the key gene coding sequences are known, for example, in publicly available databases such as GenBank, UniGene and the Mouse Genome Informatic (MGI) Database to name just a few (see Section 5.2, infra, for further details), or in private subscription databases. With a portion of the sequence in hand, hybridization probes (for filter hybridization or PCR amplification) can be designed using highly routine methods in the art to identify clones containing the appropriate sequences (preferred methods for identifying appropriate BACs are discussed in Sections 5.3 and 6, infra) for example in a library or other source of nucleic acid. If the sequence of the gene of interest from one species is known and the counterpart gene from another species is desired, it is routine in the art to design probes based upon the known sequence. The probes hybridize to nucleic acids from the species from which the sequence is desired, for example, hybridization to nucleic acids from genomic or DNA libraries from the species of interest. By way of example and not limitation, genomic clones can be identified by probing a genomic DNA library under appropriate hybridization conditions, e.g., high stringency conditions, low stringency conditions or moderate stringency conditions, depending on the relatedness of the probe to the genomic DNA being probed. For example, if the probe and the genomic DNA are from the same species, then high stringency hybridization conditions may be used; however, if the probe and the genomic DNA are from different species, then low stringency hybridization conditions may be used. High, low and moderate stringency conditions are all well known in the art.

Procedures for low stringency hybridization are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA are pretreated for 6 hours at 40°C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10⁶ cpm ³²P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 hours at 40°C, and then washed for 1.5 hours at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM

EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 hours at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68°C and reexposed to film.

Procedures for high stringency hybridizations are as follows: Prehybridization of filters containing DNA is carried out for 8 hours to overnight at 65 °C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, ^" and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C for 1 hour in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1 X SSC at 50°C for 45 minutes before autoradiography.

Moderate stringency conditions for hybridization are as follows: Filters containing DNA are pretreated for 6 hours at 55°C in a solution containing 6X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution and 5-20 X 10⁶ CPM ³²P-labeled probe is used. Filters are incubated in the hybridization mixture for 18-20 hours at 55°C, and then washed twice for 30 minutes at 60°C in a solution containing 1 X SSC and 0.1% SDS.

With respect to the characterizing gene, all or a portion of the genomic sequence is preferred, particularly, the sequences 5' of the coding sequence that contain the regulatory sequences. A preferred method for identifying BACs containing appropriate and sufficient characterizing gene sequences to direct the expression of the key gene coding sequences in substantially the same expression pattern as the endogenous characterizing gene is described in Section 6, infra.

Briefly, the characterizing gene genomic sequences are preferably in a vector that can accommodate significant lengths of sequence (for example, 10 kb's of sequence), such as cosmids, YACs, and, preferably, BACs, and encompass at least 50, 70, 80, 100, 120, 150, 200, 250 or 300 kb of sequence that comprises all or a portion of the characterizing gene sequence. The larger the vector insert, the more likely it is to identify a vector that contains the characterizing gene sequences of interest. Vectors identified as containing characterizing gene sequences can then be screened for those that are most likely to contain sufficient regulatory sequences from the characterizing gene to direct expression of the key gene coding sequences in substantially the same pattern as the endogenous characterizing gene. In general, it is preferred to have a vector containing the entire genomic sequence for the characterizing gene. However, in certain cases, the entire genomic sequence cannot be accommodated by a single vector or such a clone is not available. In these instances (or when it is not known whether the clone contains the entire genomic sequence), preferably the vector contains the characterizing gene sequence with the start, i.e., the most 5' end, of the coding sequence in the approximate middle of the vector insert containing the genomic sequences and/or has at least 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 80 kb or 100 kb of genomic sequence on either side of the start of the characterizing gene coding sequence. This can be determined by any method known in the art, for example, but not by way of limitation, by sequencing, restriction mapping, PCR amplification assays, etc. In certain cases, the clones used may be from a library that has been characterized (e.g., by sequencing and/or restriction mapping) and the clones identified can be analyzed, for example, by restriction enzyme digestion and compared to database information available for the library. In this way, the clone of interest can be identified and used to query publicly available databases for existing contigs correlated with the characterizing gene coding sequence start site. Such information can then be used to map the characterizing gene coding sequence start site within the clone. Alternatively, the key gene sequences (or any other heterologous sequences) can be targeted to the 5' end of the characterizing gene coding sequence by directed homologous recombination (for example as described in Sections 5.3 and 6) in such a way that a restriction site unique or at least rare in the characterizing gene clone sequence is introduced. The position of the integrated key gene coding sequences (and, thus, the 5' end of the characterizing gene coding sequence) can be mapped by restriction endonuclease digestion and mapping. The clone may also be mapped using internally generated fingerprint data and/or by an alternative mapping protocol based upon the presence of restriction sites and the T7 and SP6 promoters in the BAC vector, as described in Section 6, infra.

In certain embodiments, the key gene coding sequences are to be inserted in a site in the characterizing gene sequences other than the 5' start site of the characterizing gene coding sequences, for example, in the 3'-most translated or untranslated regions. In these embodiments, the clones containing the characterizing gene are preferably mapped to insure that the clone contains the site for insertion in as well as sufficient sequence 5' of the characterizing gene coding sequences library to contain the regulatory sequences necessary to direct expression of the key gene sequences in the same expression pattern as the endogenous characterizing gene.

Once such an appropriate vector containing the characterizing gene sequences is constructed, the key gene can be incorporated into the characterizing gene sequence by any method known in the art for manipulating DNA. In a preferred embodiment, homologous recombination in bacteria is used for target-directed insertion of the key gene sequence into the genomic DNA encoding the characterizing gene and sufficient regulatory sequences to promote expression of the characterizing gene in its endogenous expression pattern, which characterizing gene sequences have been inserted into a BAC (see Section 5.4, infra). The BAC comprising the key gene and characterizing gene sequences is then introduced into the genome of a potential founder animal for generating a line of transgenic animals, using methods well known in the art, e.g. , those methods described in Section 5.5, infra. Such transgenic animals are then screened for expression of the key gene coding sequences that mimics the expression of the endogenous characterizing gene. Several different constructs containing transgenes of the invention may be introduced into several potential founder animals and the resulting transgenic animals then screened for the best expression (e.g. , highest level) and most accurate expression (i.e., best mimicking expression of the endogenous characterizing gene) of the key gene coding sequences.

The transgenic construct can be used to transform a host or recipient cell or animal using well known methods, e.g., those described in Section 5.4, infra. Transformation can be either a permanent or transient genetic change, preferably a permanent genetic change, induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell). Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. In one aspect of the invention, a vector is used for stable integration of the transgenic construct into the genome of the cell. Vectors include plasmids, retroviruses and other animal viruses, BACs, YACs, and the like. Vectors are described in Section 5.3, infra.

5.1.1. CHARACTERIZING GENE SEQUENCES

A characterizing gene is endogenous to a host cell or host organism (or is an ortholog of an endogenous gene) and is expressed or not expressed in a particular select population of cells of the organism. The population of cells comprises a discernable group of cells sharing a common characteristic. Because of its selective expression, the population of cells may be characterized or recognized based on its positive or negative expression of the characterizing gene. Accordingly, all or some of the regulatory sequences of the characterizing gene are incorporated into transgenes of the invention to regulate the expression of key gene coding sequences, as discussed above. Any gene which is not constitutively expressed, (/. e. , exhibits some spatial or temporal restriction in its expression pattern) can be a characterizing gene.

Preferably, the characterizing gene is a human or mouse gene associated with an adrenergic or noradrenergic neurotransmitter pathway, e.g., one of the genes listed in Table 1; a cholinergic neurotransmitter pathway, e.g., one of the genes listed in Table 2; a dopaminergic neurotransmitter pathway, e.g., one of the genes listed in Table 3; a GABAergic neurotransmitter pathway, e.g., one of the genes listed in Table 4; a glutaminergic neurotransmitter pathway, e.g., one of the genes listed in Table 5; a glycinergic neurotransmitter pathway, e.g., one of the genes listed in Table 6; a histaminergic neurotransmitter pathway, e.g., one of the genes listed in Table 7; a neuropeptidergic neurotransmitter pathway, e.g., one of the genes listed in Table 8; a serotonergic neurotransmitter pathway, e.g., one of the genes listed in Table 9; a nucleotide receptor, e.g., one of the genes listed in Table 10; an ion channel, e.g., one of the genes listed in Table 11 ; markers of undif erentiated or not fully differentiated cells, preferably nerve cells, e.g., one of the genes listed in Table 12; the sonic hedgehog signaling pathway, e.g., one of the genes in Table 13; calcium binding, e.g., one of the genes listed in Table 14; or a neurotrophic factor receptor, e.g., one of the genes listed in Table 15.

In certain embodiments, an ion channel encoded by or associated with a characterizing gene is preferably involved in generating and modulating ion flux across the plasma membrane of neurons, including, but not limited to voltage-sensitive and/or cation- . sensitive channels, e.g., a calcium, sodium or potassium channel.

In Tables 1-15 that follow, the common names of genes are listed, as well as their GeneCards identifiers (Rebhan et al, 1997, GeneCards: encyclopedia for genes, proteins and diseases, Weizmann Institute of Science, Bioinformatics Unit and Genome Center (Rehovot, Israel). GenBank accession numbers, UniGene accession numbers, and Mouse Genome Informatics (MGI) Database accession numbers where available are also listed.

GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences (Benson et al, 2000, Nucleic Acids Res. 28(1): 15-18). The GenBank accession number is a unique identifier for a sequence record. An accession number applies to the complete record and is usually a combination of a letter(s) and numbers, such as a single letter followed by five digits (e.g., U12345), or two letters followed by six digits (e.g., AF123456). Accession numbers do not change, even if information in the record is changed at the author's request. An original accession number might become secondary to a newer accession number, if the authors make a new submission that combines previous sequences, or if, for some reason, a new submission supercedes an earlier record.

UniGene (National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD; Schuler, 1997, J. Mol. Med. 75(10),694-698; Schuler et al, 1996, Science 274, 540-546; Boguski and Schuler, 1995, Nature Genetics 10, 369-371) is an experimental system for automatically partitioning GenBank sequences into a non- redundant set of gene-oriented clusters for cow, human, mouse, rat, and zebrafish. Within UniGene, expressed sequence tags (ESTs) and full-length mRNA sequences are organized into clusters that each represent a unique known or putative gene. Each UniGene cluster contains related information such as the tissue types in which the gene has been expressed and map location. Sequences are annotated with mapping and expression information and cross-referenced to other resources. Consequently, the collection may be used as a resource for gene discovery.

The Mouse Genome Informatics (MGI) Database is sponsored by the Jackson Laboratory (Bar Harbor, Maine). The MGI Database contains information on mouse genetic markers, mRNA and genomic sequence information, phenotypes, comparative mapping data, experimental mapping data, and graphical displays for genetic, physical, and cytogenetic maps.

TABLE 1

TABLE 2

TABLE 3

TABLE 4

TABLE 5

TABLE 6

TABLE 7

TABLE 8

35

53-

TABLE 9

TABLE 10

TABLE 11

TABLE 12

TABLE 13

TABLE 14

TABLE 15

All of the sequences identified by the sequence database identifiers in Tables 1-15 are hereby incorporated by reference in their entireties.

In yet another aspect of the invention, the characterizing gene sequence is a promoter that directs tissue-specific expression of the key gene coding sequence to which it is operably linked. For example, expression of the key gene coding sequences may be controlled by any tissue-specific promoter/enhancer element known in the art. Promoters that may be used to control expression include, but are not limited to, the following animal transcriptional control regions that exhibit tissue specificity and that have been utilized in transgenic animals: elastase I gene control region, which is active in pancreatic acinar cells . (Swift et al, 1984, Cell 38:639-646; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); enolase promoter, which is active in brain regions, including the striatum, cerebellum, CA1 region of the hippocampus, or deep layers of cerebral neocortex (Chen et al, 1998, Molecular Pharmacology 54(3): 495-503); insulin gene control region, which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-22); immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al, 1984, Cell 38:647-58; Adames et al, 1985, Nature 318:533-38; Alexander et al, 1987, Mol. Cell. Biol. 7:1436-44); mouse mammary tumor virus control region, which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-95); albumin gene control region, which is active in liver (Pinkert et al, 1987, Genes and Devel. 1:268-76); alpha-fetoprotein gene control region which is active in liver (Krumlauf et al , 1985, Mol. Cell. Biol. 5:1639-48; Hammer et al , 1987, Science

235:53-58); alpha 1-antitrypsin gene control region, which is active in the liver (Kelsey et al, 1987, Genes and Devel. 1:161-71); β-globin gene control region, which is active in myeloid cells (Mogram et al, 1985, Nature 315:338-40; Kollias et al, 1986, Cell 46:89-

94); myelin basic protein gene control region, which is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48:703-12); myosin light chain-2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314:283-86); and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al. ,

- 5: 1986, Science 234:1372-78).

In other embodiments, the characterizing gene sequence is protein kinase C, gamma (GenBank Accession Number: Z15114 (human); MGI Database Accession Number: MGI.97597); fos (UniGene No. MM5043 (mouse)); TH-elastin; Pax7 (Mansouri, 1998, The role of Pax3 and Pax7 in development and cancer, Crit. Rev. Oncog. 9(2): 141-9); Eph receptor (Mellitzer et al. , 2000, Control of cell behaviour by signalling through Eph receptors and ephrins; Curr. Opin. Neurobiol. 10(3):400-08; Suda et al, 2000, Hematopoiesis and angiogenesis, Int. J. Hematol. 71(2):99-107; Wilkinson, 2000, Eph receptors and ephrins: regulators of guidance and assembly, Int. Rev. Cytol. 196:177-244; . Nakamoto, 2000, Eph receptors and ephrins, Int. J. Biochem. Cell Biol. 32(1):7-12;

Tallquist et al, 1999, Growth factor signaling pathways in vascular development, Oncogene 18(55):7917-32); islet- 1 (Bang et al., 1996, Regulation of vertebrate neural cell fate by transcription factors, Curr. Opin. Neurobiol. 6(l):25-32; Ericson et al, 1995, Sonic hedgehog: a common signal for ventral patterning along the rostrocaudal axis of the neural tube, J. Dev. Biol. 39(5):809-16; β-actin; thy-1 (Caroni, 1997, Overexpression of growth-associated proteins in the neurons of adult transgenic mice, J. Neurosci. Methods 71(l):3-9).

As discussed above in Section 5.1, the transgenes of the invention include all or a portion of the characterizing gene genomic sequence, preferably at least all or a portion of the upstream regulatory sequences of the characterizing gene genomic sequences are present in the transgene, and at a minimum, the characterizing gene sequences that direct expression of the key gene coding sequences in substantially the same pattern as the endogenous characterizing gene in the transgenic mouse or anatomical region or tissue thereof are present on the transgene. In certain cases, genomic sequences and/or clones or other isolated nucleic acids containing the genomic sequences of the gene of interest are not available for the desired species, yet the genomic sequence of the counterpart from another species or all or a portion of the coding sequence (e.g., cDNA or EST sequences) for the same species or another species is available. It is routine in the art to obtain the genomic sequence for a gene when all or a portion of the coding sequence is known, for example, by hybridization of the cDNA or EST sequence or other probe derived therefrom to a genomic library to identify clones containing the corresponding genomic sequence. The identified clones may then be used to identify clones that map either 3' or 5' to the identified clones, for example, by hybridization to overlapping sequences present in the clones of a library and, by repeating the hybridization, "walking" to obtain clones containing the entire genomic sequence. As discussed above, it is preferable to use libraries prepared with vectors that can accommodate and that contain large inserts of genomic DNA (for example, at least 25 kb, 50 kb, 100 kb, 150 kb, 200 kb, or 300 kb) such that it is likely that a clone can be identified that contains the entire genomic sequence of the characterizing gene or, at least, the upstream regulatory sequences of the characterizing gene (all or a portion of the regulatory sequences sufficient to direct expression in the same pattern as the endogenous characterizing gene). Cross- species hybridization may be carried out by methods routine in the art to identify a genomic sequence from all species when the genomic or cDNA sequence of the corresponding gene in another species is known. As also discussed above, methods are known in the art and described herein for identifying the regulatory sequences necessary to confer endogenous characterizing gene expression on the key gene coding sequences (see Sections 5.3 and 6, infra). In specific embodiments, the characterizing gene sequences are on BAC clones from a BAC mouse genomic library, for example, but not limited to the CITB Bac Resources (ResGen, an Invitrogen Corporation, Huntsville AL) or RPCI-23 (BACPAC Resources, Children's Hospital Oakland Research Institute, Oakland, California) libraries, or any other BAC library.

In certain embodiments, the subset of cells of the transgenic animal that express the key gene also expresses an additional "marker gene" that encodes a detectable or selectable marker, or expresses a protein product that specifically induces or suppresses the expression of the detectable or selectable marker. For example, in specific embodiments, the transgene also contains a nucleotide sequence encoding a detectable or selectable marker also operatively linked to the characterizing gene sequences or activated by the key gene protein product such that the marker gene is expressed in the same cells as the key gene. In a specific aspect, the invention provides collections of transgenic animal lines for use in the drug validation methods of the invention. In preferred embodiments, a collection of such transgenic animal lines comprises at least two individual lines, more preferably at least three individual lines, and most preferably, at least five individual lines. In specific embodiments, a collection of transgenic animal lines comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 500, 1000, or 2000 individual lines. In other embodiments, a collection of transgenic animal lines comprises between 2 to 10, 10 to 20, 10 to 50, 10 to 100, 100 to 500, 100 to 1000, or 100 to 2000 individual lines. In the collections, each line of transgenic animals has a different characterizing gene and may or may not have different key gene coding sequences. In particular embodiments, each transgenic animal line of a collection of the invention has the same key gene coding sequences and in other embodiments, each transgenic animal line has a different key gene coding sequence.

In other preferred embodiments, the invention provides a collection of vectors for producing transgenic animal lines of the invention comprising at least two vectors, more preferably at least three vectors, and most preferably, at least five vectors. In specific embodiments, a collection of vectors comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 500, 1000, or 2000 vectors. In other embodiments, a collection of vectors comprises between 2 to 10, 10 to 20, 10 to 50, 10 to 100, 100 to 500, 100 to 1000, or 100 to 2000 individual vectors. In the collection of vectors of the invention, the characterizing gene for each vector is different and each vector may or may not have different key gene coding sequences. In particular embodiments, each vector has the same key gene coding sequences and in other embodiments, each vector has a different key gene coding sequence.

Each individual line or vector is selected for the collection of transgenic animals lines and/or vectors based on the identity of the subset of cells in which the key gene is expressed. In a preferred embodiment, the characterizing genes for the lines of transgenic animals in such a collection consist of (or comprise), for example but not by way of limitation, a group of functionally related genes (i.e., genes encoding proteins that serve analogous functions in the cells in which they are expressed such as proteins that function in the cell as biosynthetic and/or degradative enzymes for a cellular component, transporters, intracellular or extracellular receptors, and signal transduction molecules), a group of genes . in the same signal transduction pathway, or a group of genes implicated in a particular physiological or disease state, or expressed in the same or related tissue types.

Additionally, the collection may consist of lines of transgenic animals in which the characterizing genes represent a battery of genes having a variety of cell functions, are expressed in a variety of tissue or cell types (e.g., different neuronal cell types, different immune system cell types, different tumor cell types, etc.), or are implicated in a variety of physiological or disease states (in particular, related disease states such as a group of different neurodegenerative diseases, cancers, autoimmune diseases or disorders of immune system function, heart diseases, etc.). The collection may also consist of lines of transgenic animals in which the characterizing genes represent a battery of genes expressed in particular neuronal cell types and circuits that control particular behaviors and underlie specific neurological or psychiatric diseases.

In preferred embodiments, the characterizing genes of the collection are a group of functionally related genes that encode the cellular components associated with a particular neurotransmitter signaling and/or synthetic pathway or with a particular signal transduction pathway, or the proteins that serve analogous functions in the cells in which they are expressed such as proteins that function in the cell as biosynthetic and/or degradative enzymes for a cellular component, transporters, intracellular or extracellular receptors, signal transduction molecules, transcriptional or translational regulators, cell cycle regulators, etc. Additionally, the group of functionally related genes that are characterizing genes can be associated with or implicated in known neuronal circuitry or in a particular physiological, behavioral or disease state. Such states or responses include pain, sleeping, feeding, fasting, sexual behavior, aggression, depression, cognition, emotion, etc.

In other embodiments, the characterizing genes can represent a battery of genes having a variety of cell functions, are expressed in a variety of tissue or cell types (e.g., different neuronal cell types, different immune system cell types, different tumor cell types, etc.), or are implicated in a variety of physiological or disease states.

In a preferred embodiment, a group of characterizing genes is a group of functionally related genes that encode a neurotransmitter, its receptors, and associated biosynthetic and/or degradative enzymes for the neurotransmitter. In other embodiments, the characterizing genes are groups of genes that are expressed in cells of the same or different neurotransmitter phenotypes, in cells known to be anatomically or physiologically connected, cells underlying a particular behavior, cells in a particular anatomical locus (e.g., the dorsal root ganglia, a motor pathway), cells active or quiescent in a particular physiological state, cells affected or spared in a particular disease state, etc.

In other embodiments, the characterizing genes are groups of genes that are : expressed in cells underlying a neuropsychiatric disorder such as a disorder of thought and/or mood, including thought disorders such as schizophrenia, schizotypal personality disorder; psychosis; mood disorders, such as schizoaffective disorders (e.g., schizoaffective disorder manic type (SAD-M); bipolar affective (mood) disorders, such as severe bipolar affective (mood) disorder (BP-I), bipolar affective (mood) disorder with hypomania and major depression (BP-II); unipolar affective disorders, such as unipolar major depressive disorder (MDD), dysthymic disorder; obsessive-compulsive disorders; phobias, e.g., agoraphobia; panic disorders; generalized anxiety disorders; somatization disorders and hypochondriasis; and attention deficit disorders.

In other embodiments, the characterizing genes are groups of genes that are expressed in cells underlying a malignancy, cancer or hyperproliferation disorder, including but not limited to the following: Leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute niyelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extrameduUary plasmacytoma; Waldenstrδm's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and cilliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to pappillary, nodular, and diffuse; lung cancers such as non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as . but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal, pelvic and/ or ureter); Wilms' tumor; bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma; and cancers including myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas.

In yet other embodiments, the characterizing genes are groups of genes that are expressed in cells underlying a malignancy, cancer or hyperproliferation disorder, including but not limited to the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic . myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and teratocarcinoma; cancers caused by aberrations in apoptosis, including but not limited to follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).

In another embodiment, the characterizing genes of the collection are all expressed in the same population of cells, e.g., motorneurons of the spinal cord, amacrine cells, astroglia, etc.

In another embodiment, the characterizing genes of the collection are expressed in different populations of cells.

In another embodiment, the characterizing genes of the collection are all expressed within a particular anatomical region, tissue, or organ of the body, e.g., nucleus within the brain or spinal cord, cerebral cortex, cerebellum, retina, spinal cord, bone marrow, skeletal muscles, smooth muscles, pancreas, thymus, etc.

In another embodiment, the characterizing genes of the collection are each expressed in a different anatomical region, tissue, or organ of the body. In another embodiment, the characterizing genes of the collection are all listed in only one of Tables 1-15, above.

In another embodiment, the characterizing genes of the collection are a group of genes where at least two, three, four, five, eight, ten or twelve genes are each from a different one of Tables 1-15, above. In another embodiment, at least one characterizing gene in the collection is listed in one of Tables 1-15, above.

In another embodiment, the characterizing genes of the collection comprise at least one gene from each of one, two, three, four or more of Tables 1-15, above.

In another embodiment, the characterizing genes of the collection are all expressed temporally in a particular expression pattern during an organism's development.

In another embodiment, the characterizing genes of the collection are all expressed during the display of a temporally rhythmic behavior, such as a circadian behavior, a monthly behavior, an annual behavior, a seasonal behavior, an estrous or other mating behavior, or other periodic or episodic behavior. In another embodiment, the characterizing genes of the collection are all expressed in cells of the nervous system that underlie feeding behavior. In a specific embodiment, the characterizing genes of the collection are all expressed in neuronal circuits that function as positive and negative regulators of feeding behavior and, preferably, that are located in the hypothalamus. In specific preferred embodiments, the invention provides vectors and lines of transgenic animals in which the characterizing gene is one of the genes listed in any of Tables 1-15, above.

In other embodiments, the invention provides lines of transgenic animals, wherein each transgenic animal contains two, four, five, six, seven, eight, ten, twelve, fifteen, twenty or more transgenes of the invention (i.e., containing key gene coding sequences operably linked to characterizing gene regulatory sequences). Each of the transgenes has a different characterizing gene. In a specific embodiment, all of the transgenes in the line of transgenic animals contain the same key gene coding sequences. In another embodiment, the transgenes in the line of transgenic animals have different key gene coding sequences (i.e., cells expressing differing characterizing genes express different key genes). Such lines of transgenic animals may be generated by introducing a transgene into an animal that is already transgenic for a transgene of the invention or by breeding two animals transgenic for a transgene of the invention. Once a line of transgenic animals containing two transgenes of the invention is established, additional transgenes can be introduced into that line, for example, by pronuclear injection or by breeding, to generate a line of transgenic animals transgenic for three transgenes of the invention, and so on.

5.1.2. KEY GENE SEQUENCES

A "key gene" encodes a key protein. A key protein is a protein that can activate or inhibit expression of a gene in another gene construct, which gene is under the control of an expression element that is turned off or on by the key protein (for example, but not limited to, promoters and/or enhancers whereby transcription is turned on or off by a specific transactivator; recombinase target sites for which recombination is effected by a recombinase and recombination positions the target gene for expression or inhibition of expression). In one aspect of the invention, the key protein specifically activates or represses expression of a gene in the modulating construct. In a preferred aspect of the invention, the gene activated or repressed by the key gene protein product encodes a potential drug target.

In other embodiments, the key gene encodes an RNA product that is an inhibitor such as a catalytic nucleic acid (e.g., a ribozyme or deoxyribozyme), an antisense RNA or double-stranded RNA that causes RNA interference (RNAi).

Preferably, the key gene product (and in certain embodiments, additionally, a marker gene turned on or repressed by the characterizing or key gene product) is not present in any cells of the animal (or ancestor thereof) prior to its being made transgenic. In other embodiments, the key gene product (and, in certain embodiments, a marker turned on or repressed by the characterizing or key gene product) is not present in a tissue in the animal (or ancestor thereof) prior to its being made transgenic, which tissue contains the subpopulation of cells to be isolated by virtue of the expression of the key gene coding sequences in the subpopulation and which can be cleanly dissected from any other tissues that may express the key gene product (and/or marker) in the animal (or ancestor thereof) prior to its being made transgenic.

In certain embodiments, the key gene product (and/or a marker turned on or repressed by the characterizing or key gene product) is expressed in the animal or in tissues neighboring and/or containing the subpopulation of cells to be isolated prior to the animal (or ancestor thereof) being made transgenic but is expressed at much lower levels, e.g., 2- fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold lower levels, than the key gene product (or marker transactivated thereby), i.e., than expression driven by the transgene. In a specific embodiment, the key gene coding sequences encode a fusion protein comprising or consisting of all or a portion of the key gene product that confers transcriptional activation or suppression properties on the fusion protein.

A key gene polypeptide, fragment, analog, or derivative may be expressed as a chimeric, or fusion, protein product (comprising a key gene encoded peptide joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein). Sequences encoding such a chimeric product can be made by ligating the appropriate nucleotide sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product as part of the transgene as discussed herein. In a specific embodiment, the chimeric gene comprises or consists of all or a portion of the characterizing gene coding sequence fused in frame to a key gene coding sequence. The key gene coding sequences can be present at a low gene dose, such as one copy of the key gene per cell. In other embodiments, at least two, three, four, five, seven, ten or more copies of the key gene coding sequences are present per cell, e.g., multiple copies of the key gene coding sequences are present in the same transgene or are present in one copy in the transgene and more than one transgene is present in the cell. In a specific embodiment in which BACs are used to generate and introduce the transgene into the animal, the gene dosage is one copy of the key gene per BAC and at least two, three, four, five, seven, ten or more copies of the BAC per cell. More then one copy of the key gene coding sequences may be preferable, in some instances, to achieve levels of the key gene protein product capable of activating or suppressing target expression from the modulating construct. In cases where the transgene is present at high copy numbers or even in certain circumstances when it is present at one copy per cell, coding sequences other than the key gene coding sequences, for example, the characterizing gene coding sequence, if present, and/or any other protein coding sequences (for example, from other genes proximal to the characterizing gene in the genomic DNA) are inactivated to avoid over- or mis-expression of these other gene products.

In a specific embodiment, the key gene is expressed selectively in neural cells. In particular embodiments, the key gene encodes a transactivator, preferably a transcription factor that specifically activates or inhibits transcription, preferably by binding to a specific nucleotide sequence (which sequence maybe operably linked to the target gene sequence). Any transactivator (or transinhibitor) paired with its corresponding promoter or enhancer element may be used. Preferably, the transactivator and transcriptional expression element are heterologous to the transgenic animal, such that the transactivator only activates expression (or a particularly level of expression, in certain embodiments) of the target gene, but are compatible with the transgenic animal. Thus, for example, the transactivator is a viral, bacterial or yeast transcription factor, for example, but not limited, Lac operator, VP16, gal 4, etc.

5.1.2.1. CONDITIONAL TRANSCRIPTIONAL

REGULATION SYSTEMS In certain embodiments, the key gene encodes a component of a conditional transcriptional regulation system. A gene encoding a potential drug target may be expressed conditionally by operably linking all or a portion of the regulatory sequences from the characterizing gene to at least the coding region for the key gene, wherein the key gene encodes a conditional regulatory element which in turn induces or represses the expression of the gene encoding the potential drug target.

Transactivators in these inducible or repressible transcriptional regulation systems are designed to interact specifically with sequences engineered into a vector. Such systems include those regulated by tetracycline ("tet systems"), interferon, estrogen, ecdysone, Lac operator, progesterone antagonist RU486, and rapamycin (FK506) with tet systems being particularly preferred (see, e.g., Gingrich and Roder, 1998, Annu. Rev. Neurosci. 21 : 377- 405; incorporated herein by reference in its entirety). These drugs or hormones (or their analogs) act on modular transactivators composed of natural or mutant ligand-binding domains and intrinsic or extrinsic DNA binding and transcriptional activation domains. In certain embodiments, expression of the potential drug target can be regulated by varying the concentration of the drug or hormone in medium in vitro or in the diet of the transgenic animal in vivo.

The inducible or repressible genetic system can restrict the expression of the potential drug target either temporally, spatially, or both temporally and spatially.

In a preferred embodiment, the control elements of the tetracycline-resistance operon of E. coli is used as an inducible or repressible transactivator or transcriptional regulation system ("tet system") for conditional expression of the potential drug target. A tetracycline- controlled transactivator can require either the presence or absence of the antibiotic tetracycline, or one of its derivatives, e.g., doxycycline (dox), for binding to the tet operator of the tet system, and thus for the activation of the tet system promoter (Ptet). Such an inducible or repressible tet system is preferably used in a mammalian cell.

In a specific embodiment, a tetracycline-repressed regulatable system (TrRS) is used (Agha-Mohammadi and Lotze, 2000, J. Clin. Invest. 105(9): 1177-83; incorporated herein by reference in its entirety). This system exploits the specificity of the tet repressor (tetR) for the tet operator sequence (tetO), the sensitivity of tetR to tetracycline, and the activity of the potent herpes simplex virus transactivator (VP16) in eukaryotic cells. The TrRS uses a conditionally active chimeric tetracycline-repressed transactivator (tTA) created by fusing the COOH-terminal 127 amino acids of vision protein 16 (VP16) to the COOH terminus of the tetR protein (which may be the key gene). In the absence of tetracycline, the tetR moiety of tTA binds with high affinity and specificity to a tetracycline-regulated promoter (tRP), a regulatory region comprising seven repeats of tetO placed upstream of a minimal human cytomegalovirus (CMV) promoter or β-actin promoter (β-actin is preferable for neural expression). Once bound to the tRP,. the VP16 moiety of tTA transactivates the gene encoding the potential drug target by promoting assembly of a transcriptional initiation complex. However, binding of tetracycline to tetR leads to a conformational change in tetR accompanied with loss of tetR affinity for tetO, allowing expression of the potential drug target gene to be silenced by administering tetracycline. Activity can be regulated over a range of orders of magnitude in response to tetracycline.

In another specific embodiment, a tetracycline-induced regulatable system is used to regulate expression of a potential drug target, e.g., the tetracycline transactivator (tTA) element of Gossen and Bujard (1992, Proc. Natl. Acad. Sci. USA 89: 5547-51; incorporated herein by reference in its entirety).

In another specific embodiment, the improved tTA system of Shockett et al. (1995, Proc. Natl. Acad. Sci. USA 92: 6522-26, incorporated herein by reference in its entirety) is used to drive expression of a potential drug target. This improved tTA system places the tTA gene under control of the inducible promoter to which tTA binds, making expression of tTA itself inducible and autoregulatory.

In another embodiment, a reverse tetracycline-controlled transactivator, e.g., rtTA2 S-M2, is used. rtTA2 S-M2 transactivator has reduced basal activity in the absence doxycycline, increased stability in eukaryotic cells, and increased doxycycline sensitivity (Urlinger et al, 2000, Proc. Natl. Acad. Sci. USA 97(14): 7963-68; incorporated herein by . reference in its entirety).

In another embodiment, the tet-repressible system described by Wells et al. (1999, Transgenic Res. 8(5): 371-81; incorporated herein by reference in its entirety) is used. In one aspect of the embodiment, a single plasmid Tet-repressible system is used. Preferably, a "mammalianized" TetR gene, rather than a wild-type TetR gene (tetR) is used (Wells et al, 1999, Transgenic Res. 8(5): 371-81).

In another embodiment, the GAL4-UAS system (Ornitz et al, 1991, Proc. Natl. Acad. Sci. USA 88:698-702; Rowitch et al, 1999, J. Neuroscience 19(20):8954-8965; Wang et al, 1999, Proc. Natl. Acad. Sci. USA 96:8483-8488; Lewandoski, 2001, Nature Reviews (Genetics) 2:743-755) is used.

In a specific embodiment the key gene encodes a GAL4-VP16 fusion protein (Wang et al, 1999, Proc. Natl. Acad. Sci. USA 96:8483-8488) , and the expression of a GAL4-VP16 fusion protein is driven by characterizing gene sequences. This fusion protein contains the DNA binding domain of GAL4 fused to the transcription activation domain of VP-16. Animals expressing the GAL4-VP16 fusion protein in a specific population of cells are crossed to a transgenic line of mice that contains a modulating construct containing a potential drug target, wherein the potential drug target is under the control of multiple tandem copies of GAL4 UAS.

In other embodiments, conditional expression of a gene encoding a potential drug target is regulated by using a recombinase system that is used to turn on or off the gene's expression by recombination in the appropriate region of the genome in which the potential drug target gene is inserted. The gene encoding a potential drug target is flanked by recombinase sites, e.g., FRT sites. Such a recombinase system (in which the key gene encodes the recombinase) can be used to turn on or off expression of a potential drug target (for review of temporal genetic switches and "tissue scissors" using recombinases, see Hennighausen & Furth, 1999, Nature Biotechnol. 17: 1062-63). Exclusive recombination in a selected cell type may be mediated by use of a site-specific recombinase such as Cre, FLP- wild type (wt), FLP-L or FLPe. Alternatively, and in a preferred embodiment, the target to be validated is under the regulatory control of an inactive promoter that is activated by site-

5 specific recombination. The promoter may be tissue specific or a constitutively active promoter. The β-actin promoter is preferred for constitutive expression in neural tissue.

Recombination may be effected by any art-known method, e.g., the method of Doetschman et al. (1987, Nature 330: 576-78; incorporated herein by reference in its entirety); the method of Thomas et al, (1986, Cell 44: 419-28; incorporated herein by

10 reference in its entirety); the Cre-loxP recombination system (Sternberg and Hamilton, 1981, J. Mol. Biol. 150: 467-86; Lakso et al, 1992, Proc. Natl. Acad. Sci. USA 89: 6232- 36; which are both incorporated herein by reference in their entireties); the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman etal, 1991, Science 251: 1351-55); the Cre-loxP-tetracycline control switch (Gossen and Bujard, 1992, Proc. Natl.

15 Acad. Sci. USA 89: 5547-51, incorporated herein by reference in its entirety); and ligand- regulated recombinase system (Kellendonk et al, 1999, J. Mol. Biol. 285: 1.75-82; incorporated herein by reference in its entirety). Preferably, the recombinase is highly active, e.g., the Cre-loxP or the FLPe system, and has enhanced thermostability (Rodriguez et al, 2000, Nature Genetics 25: 139-40; incorporated herein by reference in its entirety).

20 In certain embodiments, the conditional expression element is composed of target sites for recombination positioned such that in the presence of an appropriate recombinase, . the orientation of the target is reversed, thereby operably linking the first nucleotide sequence to a promoter such that the potential drug target sequence is expressed, wherein the key gene encodes the appropriate recombinase.

25 In certain embodiments, a recombinase system can be linked to a second inducible or repressible transcriptional regulation system. For example, a cell-specific Cre-loxP mediated recombination system (Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. USA 89: 5547-51) can be linked to a cell-specific tetracycline-dependent time switch detailed above (Ewald et al, 1996, Science 273: 1384-1386; Furth et al Proc. Natl. Acad. Sci. U.S.A. 91:

30 9302-06 (1994); St-Onge et al, 1996, Nucleic Acids Research 24(19): 3875-77; which are all incorporated herein by reference in their entireties).

In one embodiment, an altered cre gene with enhanced expression in mammalian cells is used (Gorski and Jones, 1999, Nucleic Acids Research 27(9): 2059-61; incorporated herein by reference in its entirety).

35 In a specific embodiment, the ligand-regulated recombinase system of Kellendonk et al (1999, J. Mol. Biol. 285: 175-82; incorporated herein by reference in its entirety) can be used. In this system, the ligand-binding domain (LBD) of a receptor, e.g., the progesterone or estrogen receptor, is fused to the Cre recombinase to increase specificity of the recombinase. 5 In specific embodiments, the expression key gene is also conditionally expressed.

5.1.2.2. KEY GENE SEQUENCES ENCODING

CATALYTIC NUCLEIC ACIDS

In certain embodiments, the key gene sequence encodes a catalytic nucleic acid. A 10 gene encoding the potential drug target may be expressed conditionally by operably linking all or a portion of the regulatory sequences from the characterizing gene to at least the coding region for the key gene, wherein the key gene encodes a catalytic nucleic acid, e.g., a ribozyme or deoxyribozyme, which in turn induces or represses the expression of the gene encoding a potential drug target. (For a review of catalytic nucleic acids, see Sun et al, 15 2000, Pharmacol. Rev. 52: 325-47; for a review of ribozyme constructs in transgenic animals, see Sokol and Murray, 1996, Transgenic Res. 5: 363-71; all incorporated herein by reference in their entireties).

In one embodiment, a transgene encoding a RNA-cleaving RNA enzyme or ribozyme operably linked to a characterizing gene regulatory sequence is introduced. In 20 specific embodiments, the ribozyme is a "hammerhead" or a "hairpin" ribozyme, and is used to induce specific RNA cleavage from a small catalytic domain.

In another embodiment, the catalytic nucleic acid is a DNA enzyme or deoxyribozyme (Sun et al, 2000, Pharmacol. Rev. 52: 325-47; incorporated herein by reference in its entirety). The deoxyribozyme is used to induce specific RNA or DNA 5 cleavage and to induce or suppress the expression of the potential drug target gene.

A catalytic nucleic acid can be designed to cleave a specific target RNA, e.g., an RNA encoding a potential drug target (for methods of catalytic nucleic acid design, including ribozyme design, see, Welch et al, 1998, Curr. Opin. Biotechnol. 9: 486-96; Sun et al. 2000, Pharmacol. Rev. 52: 325-47; both of which are incorporated herein by reference 0 in their entireties) .

In another embodiment, a catalytic nucleic acid is used to induce or suppress the expression of the potential drug target such that the potential drug target is expressed in cells other than those expressing the characterizing gene.

Localization of the catalytic nucleic acid product of a key gene is controlled by the 5 regulatory sequences of the characterizing gene, e.g., a promoter (Welch et al, 1998, Curr. Opin. Biotechnol. 9: 486-96). In a specific embodiment, the U6 promoter is used to confer nuclear localization. In another embodiment, tRNA-driven ribozyme expression is directed towards the nucleus or the cytoplasm depending on whether the tRNA-ribozyme transcript is spliced. In another embodiment, the adenovirus VA1 promoter targets ribozyme transcript specifically to the cytoplasm. In another embodiment, cytoplasmic transcription and localization of ribozymes is achieved using, e.g., the Semliki Forest virus 26S RNA- dependent RNA promoter/viral replicase system or the bacteriophage T7 RNA polymerase/promoter system.

For use of catalytic nucleic acids in vivo, the catalytic nucleic acids must be fully functional in the intracellular environment. Not all catalytic nucleic acids (e.g., ribozymes) . selected in vitro are expected to work in vivo, whereas catalytic nucleic acids selected in the intracellular environment should retain their function in vivo. Hamada et al (1999, Nucleic Acids Symp. Ser. 1999(42): 285-86; incorporated herein by reference in its entirety) describes a selection system in mammalian cells for active ribozymes by targeting at a particular gene. In this system, the target gene-knockdown cells become malignant and form foci. In the mammalian system, selected cells harbor the active ribozyme, indicating that the positive selection systems in vivo are operational. Such a method is particularly useful for identifying hyperactive ribozyme.

5.1.2.3. KEY GENE SEQUENCES ENCODING

ANTISENSE RNA In certain embodiments, the key gene encodes an antisense RNA that is antisense to a sequence that encodes a potential drug target. In these embodiments, a potential drug target is expressed conditionally by operably linking all or a portion of the regulatory sequences from the characterizing gene to at least the coding region for the key gene, wherein the key gene encodes an antisense RNA that suppresses the expression of the gene encoding the potential drug target (see, e.g., Gudkov et al, 1994, Proc. Natl. Acad. Sci. USA 91 : 3744-48; incorporated herein by reference in its entirety; for a review of the design and uses of antisense constructs in transgenic animals, see Sokol and Murray, 1996, Transgenic Res. 5: 363-71, incorporated herein by reference in its entirety.)

5.1.2.4. KEY GENE SEQUENCES ENCODING

SEQUENCES THAT PRODUCE RNA INTERFERENCE (RNAi In certain embodiments, the key gene encodes a sequence that produces RNA interference (RNAi). A potential drug target may be expressed conditionally by operably linking all or a portion of the regulatory sequences from the characterizing gene to at least the coding region for the key gene, wherein the key gene encodes a sequence that produces RNAi, which in turn induces or represses the expression of the gene encoding the potential drug target.

RNA interference (RNAi) is defined as the ability of double-stranded RNA (dsRNA) to suppress the expression of a gene corresponding to its own sequence. RNAi is also called post-transcriptional gene silencing or PTGS. Since the only RNA molecules normally found in the cytoplasm of a cell are molecules of single-stranded mRNA, the cell has enzymes that recognize and cut dsRNA into fragments containing 21-25 base pairs (approximately two turns of a double helix). The antisense strand of the fragment separates enough from the sense strand so that it hybridizes with the complementary sense sequence on a molecule of endogenous cellular mRNA. This hybridization triggers cutting of the mRNA in the double-stranded region, thus destroying its ability to be translated into a polypeptide. Introducing dsRNA corresponding to a particular gene thus knocks out the cell's own expression of that gene in particular tissues and/or at a chosen time.

Double-stranded (ds) RNA can be used to interfere with gene expression in mammals (Wianny & Zernicka-Goetz, 2000, Nature Cell Biology 2: 70-75; incorporated herein by reference in its entirety). In one embodiment, dsRNA is used as inhibitory RNA (RNAi) of the function of a potential drug target gene to produce a phenotype that is the same as that of a null mutant of the potential drug target gene (Wianny & Zernicka-Goetz, 2000, Nature Cell Biology 2: 70-75).

5.1.3. MARKER GENE SEQUENCES In certain embodiments, the transgene construct comprising the characterizing gene and key gene sequences also comprises one or more sequences encoding selectable markers that, once the transgene is introduced into a vector, enables identification and/or selection of the recombinant vector. The selectable marker may be the key gene product itself or an additional selectable marker not necessarily tied to the expression of the characterizing gene.

The additional detectable or selectable marker can encode such proteins as a signal- producing protein, epitope, fluorescent or enzymatic marker, or inhibitor of cellular function or, in specific embodiments, encodes a protein product that specifically activates or represses expression of a detectable or selectable marker. The marker sequences may code for any protein that allows cells expressing that protein to be detected or selected (or O 02/072017

specifically activates or represses the expression of a protein that allows cells expressing that protein to be detected or selected). Preferably, the marker gene product (and in certain embodiments, a marker turned on or repressed by the characterizing or key gene) is not present in any cells of the animal (or ancestor thereof) prior to its being made transgenic; in other embodiments, the marker gene product (and, in certain embodiments, a marker turned on or repressed by the characterizing or key gene product) is not present in a tissue in the animal (or ancestor thereof) prior to its being made transgenic, which tissue contains the subpopulation of cells to be isolated by virtue of the expression of the marker gene coding sequences in the subpopulation and which can be cleanly dissected from any other tissues that may express the marker gene product in the animal (or ancestor thereof) prior to its being made transgenic.

In certain embodiments, the marker gene product is expressed in the animal or in tissues neighboring and/or containing the subpopulation of cells to be isolated prior to the animal (or ancestor thereof) being made transgenic but is expressed at much lower levels, e.g., 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold lower levels, than the key gene product, i.e., than expression driven by the transgene. In a specific embodiment, the marker coding sequences encode a fusion protein comprising or consisting of all or a portion of the key gene product that confer the detectable or selectable property on the fusion protein, for example, where the marker sequence encodes an epitope that is not detected elsewhere in the transgenic animal or that is not detected in or neighboring the tissue that contains the subpopulation of cells to be isolated.

In a specific embodiment, the detectable or selectable marker is expressed everywhere in the transgenic animal except where the key gene is expressed, for example, where the key gene codes for a repressor that represses the expression of the detectable or selectable marker which is otherwise constitutively expressed (e.g., is under the regulatory control of the β-actin promoter (preferred for neural tissue) or CMV promoter). In one aspect of the invention, expression of the marker gene coding sequences in a subpopulation of cells of the transgenic animal (or explanted tissue thereof or dissociated cells thereof) permits detection, isolation and/or selection of the subpopulation. In specific embodiments, the marker gene encodes a marker enzyme, such as lacZ or β-lactamase, or a reporter or signal-producing protein such as luciferase or GFP.

In one embodiment, the marker gene encodes a protein-containing epitope not normally detected in the tissue of interest by immunohistological techniques. For example, the marker gene could encode CD4 (a protein normally expressed in the immune system) and be expressed and detected in non-immune cells. O 02/072017

In another embodiment, the marker gene encodes a tract-tracing protein such as a lectin (e.g., wheat germ agglutinin (WGA)).

In another embodiment, the marker gene encodes a toxin.

A marker gene polypeptide, fragment, analog, or derivative may be expressed as a chimeric, or fusion, protein product (comprising a marker gene encoded peptide joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein). Sequences encoding such a chimeric product can be made by ligating the appropriate nucleotide sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product as part of the transgene as discussed herein. In a specific embodiment, the chimeric gene comprises or consists of all or a portion of the characterizing gene and/or the key gene coding sequence fused in frame to an epitope tag.

The marker gene coding sequences can be present at a low gene dose, such as one copy of the marker gene per cell. In other embodiments, at least two, three, four, five, seven, ten or more copies of the marker gene coding sequences are present per cell, e.g. , multiple copies of the marker gene coding sequences are present in the same transgene or are present in one copy in the transgene and more than one transgene is present in the cell. In a specific embodiment in which BACs are used to generate and introduce the transgene into the animal, the gene dosage is one copy of the marker gene per BAC and at least two, three, four, five, seven, ten or more copies of the BAC per cell. More then one copy of the marker gene coding sequences may be preferable, in some instances, to achieve detectable or selectable levels of the marker gene. In cases in which the transgene is present at high copy numbers or even in certain circumstances when it is present at one copy per cell, coding sequences other than the marker gene coding sequences, for example, the characterizing gene coding sequence, if present, and/or any other protein coding sequences (for example, from other genes proximal to the characterizing gene in the genomic DNA) are inactivated to avoid over- or mis-expression of these other gene products.

5.1.3.1. MARKERGENE SEQUENCES ENCODING MARKERENZYMES

A gene that encodes a marker enzyme (or a chimeric protein corhprising a catalytic or active fragment of the enzyme) is preferably selected for use as a marker gene. The marker enzyme is selected so that it produces a detectable signal when a particular chemical reaction is conducted. Such enzymatic markers are advantageous, particularly when used in vivo, because detection of enzymatic expression is highly accurate and sensitive. Preferably, 02/072017

a marker enzyme is selected that can be used in vivo, without the need to kill and/or fix cells in order to detect the marker or enzymatic activity of the marker.

In specific embodiments, the marker gene encodes β-lactamase (e.g., GeneBLAzer™ Reporter System, Aurora Biosciences), E. coli β-galactosidase (lacZ, InvivoGen), human placental alkaline phosphatase (PLAP, InvivoGen) (Kam et al. , 1985, Proc. Natl. Acad. Sci. USA 82: 8715-19), E. coli β-glucuronidase (gus, Sigma) (Jefferson et al, 1986, Proc. Natl. Acad. Sci. USA 83:8447-51), alkaline phosphatase, horseradish peroxidase, with β-lactamase being particularly preferred (Zlokarnik et al, 1998, Science 279: 84-88; incorporated herein by reference in its entirety). In other embodiments, the marker gene encodes a chemiluminescent enzyme marker such as luciferase (Danilov et al. , ^■ 1989, Bacterial luciferase as a biosensor of biologically active compounds. Biotechnology, 11 :39-78; Gould et al, 1988, Firefly luciferase as a tool in molecular and cell biology, Anal. Biochem.l75(l):5-13; Kricka, 1988, Clinical and biochemical applications of luciferases and luciferins, Anal. Biochem. 175(1): 14-21; Welsh et al, 1997, Reporter gene expression for monitoring gene transfer, Curr. Opin. Biotechnol. 8(5):617-22; Contag et al, 2000, Use of reporter genes for optical measurements of neoplastic disease in vivo, Neoplasia 2(1 -2):41-52; Himes et al, 2000, Assays for transcriptional activity based on the luciferase reporter gene, Methods Mol. Biol. 130:165-74; Naylor et al, 1999, Reporter gene technology: the future looks bright, Biochem. Pharmacol. 58(5):749-57, all of which are incorporated by reference in their entireties).

Cells expressing PLAP, an enzyme that resides on the outer surface of the cell membrane, can be labeled using the method of Gustincich et al. (1997, Neuron 18: 723-36; incorporated herein by reference in its entirety).

Cells expressing β-glucuronidase can be assayed using the method of Lorincz et al, 1996, Cytometry 24(4): 321 -29, which is hereby incorporated by reference in its entirety.

5.1.3.2. MARKER GENE SEQUENCES ENCODING

REPORTERS OR SIGNAL-PRODUCING PROTEINS The marker gene can encode a marker that produces a detectable signal. In one aspect of the invention, the marker gene encodes a reporter or signal-producing protein. In another embodiment, the marker gene encodes a signal-producing protein that is used to monitor a physiological state.

In one embodiment, the reporter is a fluorescent protein such as green fluorescent protein (GFP), including particular mutant or engineered forms of GFP such as BFP, CFP O 02/072017

and YFP (Aurora Biosciences) (see, e.g., Tsien et al, U.S. Patent No. 6,124,128, issued September 26, 2000, entitled Long Wavelength Engineered Fluorescent Proteins; incorporated herein by reference in its entirety), enhanced GFP (EGFP) and DsRed (Clontech), blue, cyan, green, yellow, and red fluorescent proteins (Clontech), rapidly degrading GFP-fusion proteins, (see, e.g., Li et al, U.S. Patent No. 6,130,313, issued

October 10, 2000, entitled Rapidly Degrading GFP-Fusion Proteins; incorporated herein by reference in its entirety), and fluorescent proteins homologous to GFP, some of which have spectral characteristics different from GFP and emit at yellow and red wavelengths (Matz et al, 1999, Nat. Biotechnol. 17(10): 969-73; incorporated herein by reference in its entirety). In a specific embodiment, the marker gene encodes a red, green, yellow, or cyan fluorescent protein (an "XFP"), such as one of those disclosed in Feng et al. (2000, Neuron, 28: 41-51; incorporated herein by reference in its entirety).

In a specific embodiment, the marker gene encodes E. coli β-glucuronidase (gus), and intracellular fluorescence is generated by activity of β-glucuronidase (Lorincz et al, 1996, Cytometry 24(4): 321-29; incorporated herein by reference in its entirety). In another specific embodiment, a fluorescence-activated cell sorter (FACS) is used to detect the activity of the E. coli β-glucuronidase (gus) gene (Lorincz et al, 1996, Cytometry 24(4): 321-29). When loaded with the Gus substrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis by flow cytometry. This assay can be used to FACS-sort viable cells based on Gus enzymatic activity (see Section 5.7, infra), and the efficacy of the assay can be measured independently by using a fluorometric lysate assay. In another specific embodiment, the intracellular fluorescence generated by the activity of both β-glucuronidase and E. coli β-galactosidase enzymes are detected by FACS independently. Because each enzyme has high specificity for its cognate substrate, each reporter gene can be measured by FACS independently.

In another embodiment, the marker gene encodes a fusion protein of one or more different detectable or selectable markers and any other protein or fragment thereof. In particular embodiments, the fusion protein consists of or comprises two different detectable or selectable markers or epitopes, for example a lacZ-GFP fusion protein or GFP fused to an epitope not normally expressed in the cell of interest. Preferably, the markers or epitopes are not normally expressed in the transformed cell population or tissue of interest.

In another embodiment, the marker gene encodes a "measurement protein" such as a protein that signals cell state, e.g., a protein that signals intracellular membrane voltage, such as SBFI and PBFI (Molecular Probes, Eugene, OR). 5.2. MODULATING CONSTRUCT

5.2.1. SEQUENCES ENCODING POTENTIAL DRUG TARGETS

The invention relates to a method of validating potential drug targets, i.e., a gene or protein product of a gene that is potentially related to a particular indication (e.g. , a particular disease or disorder) and that potentially serves as target for drug development, for example where the inhibition, altered expression, or increase in activity of the gene or protein product thereof treats, prevents or ameliorates the indication or symptom thereof.

In a specific embodiment, a human gene is validated in a transgenic mouse. In other embodiments, an ortholog of an endogenous gene in a transgenic animal is validated. In another specific embodiment, the potential drug target is the product of an endogenous gene, the expression of which has been observed to increase or decrease in a particular disease state.

In other specific embodiments, the potential drug target is the product of an endogenous gene, the expression of which has been observed to increase or decrease during the activation of a particular neurotransmitter pathway, a cell signaling pathway, a disease state, known neuronal circuitry, or a physiological or behavioral state or response. Such states or responses include pain, sleeping, feeding, fasting, sexual behavior, aggression, depression, cognition, emotion, etc. In certain embodiments, a potential drug target-encoding gene encodes a receptor, transporter or uptake molecule, synthetic enzyme or degradative enzyme of a diffusible intercellular signaling molecule such as a neurotransmitter, e.g., 5HT, dopamine, acetylcholine, norepinephrine, GABA, glutamate/ AMP A/NMD A, glycine, or histamine; an intercellular signaling peptide, e.g.,opioid peptide, neurokinin, CCK, CRF, galanin, GRH, interferon, interleukin, motilin, neuroimmunophilin, neurotensin, NPY, angiotensin, bradykinin, Substance P, TRH, or vasopressin; an intercellular signaling fatty acid, e.g., prostaglandin, Cox-2, or anandamide; a small intercellular signaling molecule, e.g., adenosine; an adhesion molecules; or a permease.

In other embodiments, a potential drug target-encoding gene encodes a cell surface receptor or protein that interacts with the extracellular matrix or with another cell surface protein such as ICAM, myelin basic protein, or receptor tyrosine kinase.

In other embodiments, a potential drug target-encoding gene encodes an ion channel such as a sodium, potassium, or calcium channel.

In other embodiments, a potential drug target-encoding gene encodes an ion-binding protein such as a calcium-binding protein or an iron-binding protein. In other embodiments, a potential drug target-encoding gene encodes a molecule that is a component of a second messenger or other signal transduction system such as a signaling system using a lipase, a cyclic nucleotide, e.g., cAMP, a phospholipase, a phosphatase, a kinase, PKC, a SH2/SH3 -containing protein, or NO. In other embodiments, a potential drug target-encoding gene encodes a trophic factor, e.g., a cytokine or NT4/5.

In other embodiments, a potential drug target-encoding gene encodes an intracellular receptor such as a steroid receptor, e.g., an epalon, vomeropherin, or estrogen receptor.

In other embodiments, a potential drug target-encoding gene encodes an enzyme or a by-product such as a protease, an ATPase, aldose reductase or an enzyme that has a free radical substrate.

In other embodiments, a potential drug target-encoding gene encodes a component of an amyloid processing system such as amyloid or a presenilin.

In other embodiments, a potential drug target-encoding gene encodes a component of a system for blood clotting or for blood-clotting metabolism such as glycoprotein lib, thrombin, or a platelet aggregation mediator.

In other embodiments, a potential drug target-encoding gene encodes a component of a vesicle cycling system such as a tetanus target.

In other embodiments, a potential drug target-encoding gene encodes a cytoskeletal protein.

In certain embodiments, the potential drug target is not a bacterial gene.

The expression of a potential drug target, or modulator thereof, is regulated (either activated or inhibited) by the presence of the key protein. For example, if the key protein is a transcriptional activator, the potential drug target is operably linked to a promoter activated by the key protein transcriptional activator. For target validation, a "modulating construct" containing a nucleotide sequence encoding the potential drug target, or a product that modulates (e.g., inhibits) the expression of the potential drug target, is introduced into the cells of an appropriate transgenic mouse line.

Only cells expressing the key protein are able to activate or inhibit expression of the potential drug target. The sequence encoding the potential drug target can be a nucleotide sequence that is homologous to a selected endogenous gene sequence in the transgenic animal line or that is orthologously related to the endogenous gene sequence. Alternatively, it can encode an inhibitor, including, but not limited to, inhibitory RNA (RNAi) or an inhibitor protein of an endogenous gene sequence encoding a potential drug target. In a preferred aspect of the invention, the gene sequence encoding the potential drug target is expressed conditionally, using any type of inducible or repressible system available for conditional expression of genes known in the art, e.g., a system inducible or repressible by tetracycline ("tet system"); interferon; estrogen, ecdysone, or other steroid inducible system; Lac operator, progesterone antagonist RU486, or rapamycin (FK506). For example, a conditionally expressible transgene can be created in which the coding region for the potential drug target gene is operably linked to a genetic switch, such that expression of the potential drug target gene can be further regulated. One example of this type of switch is the tetracycline-based switch. In a specific embodiment, the key gene product is the conditional enhancer or suppressor which, upon expression, enhances or suppresses expression of a gene encoding a potential drug target present either in a modulating construct or elsewhere in the genome of the transgenic animal.

Two separate plasmids can be introduced sequentially that contain the genetic sequences that allow reversible induction of expression of the potential drug target on the modulating construct in response to tetracycline (tet) (Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. USA 89, 5547-51). Alternatively, a single autoregulatory cassette can be used that allows reversible induction of expression of the potential drug target in the modulating construct in response to tetracycline (tet) (Hofmann et al, 1996, Proc. Natl. Acad. Sci. USA 93, 5185-90, incorporated herein by reference in its entirety).

In one embodiment, the target under control of the inducible or repressible conditional regulatory elements is introduced using a retrovirus. To avoid potential interference of the strong retroviral long terminal repeat enhancer and promoter elements with the function of the tet-regulated cytomegalo virus (CMV) minimal promoter, the vector can be self-inactivating, eliminating transcription from the long terminal repeat after infection of target cells (Hofmann et al, 1996, Proc. Natl. Acad. Sci. USA 93, 5185-90). Tandem tet operator sequences and the CMV minimal promoter can be used to drive expression of a bicistronic mRNA, leading to transcription of the gene of interest (e.g., the drug target gene) and the internal ribosome entry site (IRES)-controlled transactivator (e.g., Tet repressor-VP16 fusion protein). In the absence of tet, there is a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of tet, gene expression is prevented (Hofmann et /.,1996, Proc. Natl. Acad. Sci. USA 93, 5185- 90).]

In another embodiment, an inducible lentiviral vector system can be used to conditionally express the potential gene target (Kafri et al, 2000, Molecular Therapy 1(6), 516-21, incorporated herein by reference in its entirety). In a specific embodiment, the inducible lentiviral vector system contains the entire tet-regulated system developed by Gossen and Bujard (1992, Proc. Natl. Acad. Sci. USA 89, 5547-51). The lentiviral vector comprises a potential drug target gene and the tetracycline transactivator under the control of the tetracycline-inducible promoter and the human CMV promoter, respectively. By adding or withdrawing doxycycline from the drinking water of an animal transgenic for the tet system, induction and suppression of potential drug target gene expression can be regulated in vivo.

In specific embodiment, the recombinant lentiviral vector is used to transform neurons, and doxycycline is used to regulate potential drug target gene expression in the neurons (Kafri et al, 2000, Molecular Therapy 1(6), 516-21; incorporated herein by reference in its entirety). Using such a vector, terminally differentiated neurons can be made to express the drug target gene.

A reverse tetracycline-controlled transactivator (rtTA) system can be combined with a promoter (Mansuy et al, 1998, Neuron 21, 257-65, incorporated herein by reference in its entirety). Expression can be reversed by removal of doxycycline. In another embodiment, the Cre-loxP recombination system is combined with a tetracycline-dependent genetic switch and tissue-specific control elements (Utomo et al. , 1999, Nat. Biotechnol. 17, 1091-96; incorporated herein by reference in its entirety). Using the methods of Utomo et al, a gene in a specific tissue can be targeted. The characterizing gene sequence drives the expression of the reverse tetracycline-controlled transactivator (rtTA). Placed in cis configuration to the rtTA transcription unit, the rtTA-inducible^' promoter directs expression of Cre recombinase. In another specific embodiment, the Cre recombinase gene is under control of a tet gene switch.

In a specific embodiment, a mouse strain is generated in which regulatory sequences from a characterizing gene drive FLPe expression (Rodriguez et al , 2000, Nature Genetics 25, 139-40; incorporated herein by reference in its entirety). For assessing recombinase activity, a FLP indicator strain is generated in which cells that have undergone a site- specific recombination event, or their daughter cells, are marked by a gain of β- galactosidase (β-gal) activity. The indicator transgene (HmgcπFRTZ) is composed of an FRT-disrupted lacZ reporter gene driven by mouse Hmgcr (encoding hydroxymethylglutaryl- coenzyme A reductase) promoter/enhancer sequences. To profile FLP activity, recombinase mice are crossed to this indicator strain. Offspring carrying both the recombinase and the indicator transgenes are analyzed for FLP -mediated lacZ activation by histochemical detection of β-gal in tissue sections. A nuclear localization signal may be appended to the amino terminus of β-gal to enable visualization of individual cells and to increase sensitivity by concentrating β-gal activity in the nucleus. The usefulness of the Hmgcr.FRTZ indicator strain can be evaluated by generating a fully recombined derivative strain. Hmgcr:FRTZ mice may be crossed to produce F2 Hmgcr:FRTZ-A mice that are fully transgenic for the recombined indicator, making lacZ expression dependent only on the combined activity of the Hmgcr promoter and surrounding chromosomal DNA. Accordingly, in a particular embodiment, the FLP recombinase can be expressed as the key gene and used to regulate expression of the target gene using site specific recombination. In one embodiment, an altered cre gene with enhanced expression in mammalian cells is used as the key gene (Gorski and Jones, 1999, Nucleic Acids Research 27(9), 2059- 61; incorporated herein by reference in its entirety). Preferably, a cre gene having a mutated splice acceptor site is preferably used to reduce the risk of undesired mRNA splicing event. A conditionally expressible transgene can be site-specifically inserted into an untranslated region (UTR) of genomic DNA of the gene encoding the potential drug target, e.g., the 3' UTR or the 5' region, so that expression of the transgene via the conditional expression system is induced or abolished by administration of the inducing or repressing substance, e.g., administration of tetracycline or doxycycline, ecdysone, estrogen, etc., without interfering with the normal profile of gene expression (see, e.g., Bond et al, 2000, Science 289: 1942-46; incorporated herein by reference in its entirety).

5.2.2. MODULATING CONSTRUCT VECTORS The modulating constructs (constructs containing a potential drug target regulated by the key gene protein product) of the invention are preferably introduced into a transgenic animal of the invention (i.e., an animal expressing a key gene under the control of characterizing gene regulatory sequences) in a viral vector. The viral vector can be any viral vector known to be useful to introduce nucleic acid into the species of transgenic animal being used. In a preferred embodiment, the vector is a retroviral vector. They provide high efficiency infection, stable integration and stable expression (Friedmann, 1989, Science 244: 1275-81). Sequences of a gene of interest, e.g., a gene encoding a potential drug target, or portions thereof, can be cloned into a retroviral vector. Delivery of the virus can be accomplished by direct injection or implantation of virus into the desired tissue of the adult animal, a fertilized egg, or an early stage or later stage embryo.

Preferably, the modulating construct is introduced using viral vectors and transduction methods described in Deglon et al. (2000, Human Gene Therapy 11 -.179-190; incorporated herein by reference in its entirety). Deglon et al describe methods for producing and introducing a self-inactivating (non-reproducing) lentiviral vector with enhanced transgene expression into a selected cell population, e.g., neurons in a particular brain region. The self-inactivating vector is used to transduce, and to localize delivery of a potential drug target to, a select population of neurons. The self-inactivating (SIN) lentiviral vector is modified using the methods of Deglon et al by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles are produced with a multiply attenuated packaging system.

The lentiviral vector comprising the modulating construct may also modified so that it has an improved ability to transduce the cells into which it is introduced. The methods of Zennou et al are used to incorporate a central DNA flap into the vector (2000, Cell 101, 173-85; incorporated herein by reference in its entirety). Lentiviruses have the unique property among retroviruses of replicating in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1, reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis, creates a plus strand overlap: the central DNA flap. A key determinant for nuclear import of lentiviral genomes, e.g., HIV-1 genome, is therefore the central DNA flap: the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. A self-inactivating or non-reproducing lentiviral vector comprising the modulating construct is designed using the methods of Zennou et al. The vector comprises a reinsertion of the DNA flap sequence, thereby restoring nuclear import of the vector to wild-type levels. A replication-defective lentiviral vector, such as the one described by Naldini et al

(1996, Proc. Natl. Acad. Sci. USA 93: 11382-88; incorporated herein by reference in its entirety), can also be used for in vivo delivery of a modulating construct. Preferably, the reverse transcription of the vector is promoted inside the vector particles before delivery to enhance the efficiency of gene transfer. The lentiviral vector may be injected into a specific tissue, e.g., the brain.

In another embodiment, a lentivirus-based vector capable of infecting both mitotic and postmitotic cells is used to introduce the modulating construct. Postmitotic cells, in particular postmitotic neurons, are generally refractory to stable infection by retroviral vectors, which require the breakdown of the nuclear membrane during cell division in order to insert the transgene into the host cell genome. Therefore, in a preferred embodiment, a lentivirus vector based on the human immunodeficiency virus (HIV) (Blδmer et al, 1997, J. Virol., Vol. 71(9): 6641-49; incorporated herein by reference in its entirety) is used to infect and stably transduce dividing as well as terminally differentiated cells, preferably neurons, (for a review of lentivirus vectors suitable for infecting non-dividing cells, see Naldini, 1998, Curr. Opin. Biotechnol. 9: 457-63). Retroviral vectors are preferable because they permit stable integration of the transgene into a dividing host cell genome, and because the absence of any viral gene expression reduces the chance of an immune response in the transgenic animal. In addition, retroviruses can be easily pseudo-typed with a variety of envelope proteins to broaden or restrict host cell tropism, thus adding an additional level of cellular targeting for transgene delivery (Welch etal, 1998, Curr. Opin. Biotechnol. 9: 486-96).

Adenoviral vectors can be used to provide efficient transduction, but they do not integrate into the host genome and, consequently, expression is only transient in actively dividing cells. In animals, a further complication arises in that the most commonly used recombinant adenoviral vectors still contain viral late genes that are expressed at low levels and can lead to a host immune response against the transduced cells (Welch et al. , 1998, Curr. Opin. Biotechnol. 9: 486-96). In one embodiment, a 'gutless' adenoviral vector can be used that lacks all viral coding sequences (Parks et al, 1996, Proc. Natl. Acad. Sci. USA 93: 13565-70; incorporated herein by reference in its entirety). Other delivery systems that can be utilized include adeno-associated virus (AAV), lentivirus, alpha virus, vaccinia virus, bovine papilloma virus, members of the herpes virus group such as Epstein-Barr virus, baculovirus, yeast vectors, bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors. Viruses with tropism to central nervous system (CNS) tissue also can be used. Adeno-associated virus (AAV) is attractive because it is a small, non-pathogenic virus that can stably integrate a transgene expression cassette without any viral gene expression (Welch et al, 1998, Curr. Opin. Biotechnol. 9: 486-96). An alpha virus system, using recombinant Semliki Forest virus, provides high transduction efficiencies of mammalian cells along with high cytoplasmic transgene, e.g., ribozyme, expression (Welch et al, 1998, Curr. Opin. Biotechnol. 9: 486-96). Finally, lentiviruses (such as HIV and feline immunodeficiency virus) are attractive as gene delivery vehicles due to their ability to integrate into non-dividing cells (Welch et al, 1998, Curr. Opin. Biotechnol. 9: 486-96).

Site-specific integration of a transgene can be mediated by an adeno-associated virus (AAV) vector derived from a nonpathogenic and defective human parvovirus. In one embodiment, a recombinant adeno-associated virus (rAAV) is used to mediate transgene integration in a population of nondividing cells (Wu et al, 1998, J. Virol. 72(7): 5919-26; incorporated herein by reference in its entirety). In a specific embodiment, the nondividing cells are neurons.

In another embodiment, a recombinant (non-wildtype) AAV (rAAV) is used, such as one of those disclosed by Xiao et al. (1997, Exper. Neurol. 144: 113-24; incorporated herein by reference in its entirety). Such an rAAV vector has biosafety features, a high titer, broad host range, lacks cytotoxicity, does not evoke a cellular immune response in the target tissue, and transduces quiescent or non-dividing cells. It is preferably used to transduce cells in the central nervous system (CNS). In another embodiment, rAAV plasmid DNA is used in a nonviral gene delivery system as disclosed by Xiao et al. (1997, Exper. Neurol. 144: 113-24).

Nondividing cells can be infected by human immunodeficiency virus type 1 (HIV- l)-based vectors, which results in transgene expression that is stable over several months. Preferably, an HIV-1 vector with biosafety features, e.g., a self-inactivating HIV-1 vector is used. In one embodiment, a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3' long terminal repeat (LTR) is used (Zufferey et al, 1998, J. Virol. 72(12): 9873-80^'; incorporated herein by reference in its entirety). The deletion, which includes the TATA box, abolishes the LTR promoter activity but does not affect vector titers or transgene expression in vitro. The self-inactivating vector may be used to transduce neurons in vivo. In another embodiment, a retroviral vector that is rendered replication incompetent, stably integrates into the host cell genome, and does not express any viral proteins, such as a vector based on the Moloney murine leukemia virus (MMLV), is used for gene transfer into the host cell genome (Blδmer et al, 1997, J. Virol., Vol. 71(9): 6641-49).

Pseudorabies virus can also be used as a viral vector for introducing nucleic acid. In a preferred aspect of the invention, a pseudorabies virus is used to introduce the modulating construct. In one embodiment, a strain of pseudorabies virus (PRV 152) is used to introduce a modulating construct, thereby permitting expression of a potential drug target from an inducible or repressible conditional transcription element (see, e.g., Smith et al., 2000, Proc. Natl. Acad. Sci. USA 97(16), 9264-9269). In another embodiment, injection of a pseudorabies vector comprising a transgene into a specific group of neurons transsynaptically infects their postsynaptic targets.

Preferably, the modulating construct is packaged in a viral vector that is used to infect a general type or population of cells (for example, to infect the cells of a mouse in a global fashion) expressing the key protein in a select subpopulation of the general type or population of cells. In a specific embodiment, the viral vector comprising the modulating construct is directly injected into a particular tissue region, e.g., a brain region.

5.3. TRANSGENE VECTORS

In one aspect of the invention, the transgene comprising the characterizing and key gene sequences are inserted into an appropriate vector. A vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked, preferably, the other nucleic acid is incorporated into the vector via a covalent linkage, more preferably via a nucleotide bond such that the other nucleic acid can be replicated along with the vector sequences. One type of vector is a plasmid, which is a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into a viral genome or derivative thereof. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. The invention includes viral vectors, e.g., replication defective retroviruses, adeno viruses and adeno-associated viruses, which serve equivalent functions.

A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene).

Preferably, vectors can replicate (i.e., have a bacterial origin of replication) and be manipulated in bacteria (or yeast) and can then be introduced into mammalian cells. Preferably, the vector comprises a selectable or detectable marker such as Amp^r, tef, LacZ, etc. The recombinant vectors of the invention comprise a transgene of the invention in a form suitable for expression of the nucleic acid in a transformed cell or transgenic animal. Preferably, such vectors can accommodate ( . e. , can be used to introduce into cells and replicate) large pieces of DNA such as genomic sequences, for example, large pieces of DNA consisting of at least 25 kb, 50 kb, 75 kb, 100 kb, 150 kb, 200 kb or 250 kb, such as BACs, YACs, cosmids, etc. Preferably, the vector is a BAC.

The insertion of a DNA fragment into a vector can, for example, be accomplished by ligating the DNA fragment into a vector that has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the vector, the ends of the DNA molecules may be enzymatically modified.

Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and the transgene may be modified by homopolymeric tailing. The vector can be cloned using methods known in the art, e.g., by the methods disclosed in Sambrook et al, 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N.Y.; Ausubel et al, 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., both of which are incorporated herein by reference in their entireties. Vectors have replication origins and other selectable or detectable markers to allow selection of cells with vectors and vector maintenance. Preferably, the vectors contain cloning sites, for example, restriction enzyme sites that are unique in the sequence of the vector and insertion of a sequence at that site would not disrupt an essential vector function, such as replication. In another aspect of the invention, a collection of vectors for making transgenic animals is provided. The collection comprises two or more vectors wherein each vectors comprises a transgene containing a key gene operably linked to regulatory sequences of a characterizing gene corresponding to an endogenous gene or ortholog of an endogenous gene such that said key gene is expressed in said transgenic animal with an expression pattern that is substantially the same as the expression pattern of said endogenous gene in a non-transgenic animal or anatomical region or tissue thereof containing the population of cells of interest.

As discussed above, vectors used in the methods of the invention preferably can accommodate, and in certain embodiments comprise, large pieces of heterologous DNA such as genomic sequences. Such vectors can contain an entire genomic locus, or at least sufficient sequence to confer endogenous regulatory expression pattern and to insulate the expression of coding sequences from the effect of regulatory sequences surrounding the site of integration of the transgene in the genome to mimic better wild type expression. When entire genomic loci or significant portions thereof are used, few, if any, site-specific expression problems of a transgene are encountered, unlike insertions of transgenes into smaller sequences. In a preferred embodiment, the vector into which the transgene comprising the characterizing and key gene sequences is a BAC containing genomic sequences into which key gene coding sequences have been inserted by directed homologous recombination in bacteria, e.g., the methods of Heintz WO 98/59060; Heintz et al, WO 01/05962; Yang et al, 1997, Nature Biotechnol. 15: 859-865; Yang et al, 1999, Nature Genetics 22: 327-35; which are all incorporated herein by reference in their entireties.

Using such methods, a BAC can be modified directly in a recombination-deficient E. coli host strain by homologous recombination. In a preferred embodiment, homologous recombination in bacteria is used for target- directed insertion of the key gene coding sequence into the genomic DNA encoding the characterizing gene and sufficient regulatory sequences to promote expression of the characterizing gene in its endogenous expression pattern, which sequences have been inserted into the BAC. The BAC comprising the key gene coding sequences under the regulation of the characterizing gene sequences is then recovered and introduced into the genome of a potential founder animal for a line of transgenic animals.

In specific embodiments, the key gene is inserted into the 3' UTR of the characterizing gene and, preferably, has its own IRES. In another specific embodiment, the key gene is inserted into the characterizing gene sequences using 5' direct fusion without the use of an IRES, i.e., such that the key gene coding sequences are fused directly in frame to the nucleotide sequence encoding at least the first codon of the characterizing gene coding sequence and even the first two, four, five, six, eight, ten or twelve codons. In yet another specific embodiment, the key gene is inserted into the 5' UTR of the characterizing gene with an IRES controlling the expression of the key gene.

In a preferred aspect of the invention, the key gene sequence is introduced into the BAC containing the characterizing gene by the methods of Heintz et al WO 98/59060 and Heintz et al, WO 01/05962, both of which are incorporated herein by reference in their entireties. The key gene is introduced by performing selective homologous recombination on a particular nucleotide sequence contained in a recombination deficient host cell, . e., a cell that cannot independently support homologous recombination, e.g., Rec A^". The method preferably employs a recombination cassette that contains a nucleic acid containing the key gene coding sequence that selectively integrates into a specific site in the characterizing gene by virtue of sequences homologous to the characterizing gene flanking the key gene coding sequences on the shuttle vector when the recombination deficient host cell is induced to support homologous recombination (for example by providing a functional Rec A gene on the shuttle vector used to introduce the recombination cassette). In a preferred aspect, the particular nucleotide sequence that has been selected to undergo homologous recombination is contained in an independent origin based cloning vector introduced into or contained within the host cell, and neither the independent origin based cloning vector alone, nor the independent origin based cloning vector in combination with the host cell, can independently support homologous recombination (e.g., is RecA^"). Preferably, the independent origin based cloning vector is a BAC or a bacteriophage-derived artificial chromosome (BBPAC) and the host cell is a host bacterium, preferably E. coli. In another preferred aspect, sufficient characterizing gene sequences flank the key gene coding sequences to accomplish homologous recombination and target the insertion of the key gene coding sequences to a particular location in the characterizing gene. The key gene coding sequence and the homologous characterizing gene sequences are preferably present on a shuttle vector containing appropriate selectable markers and the RecA gene, optionally with a temperature sensitive origin of replication (see Heintz et al. WO 98/59060 and Heintz et al, WO 01/05962 such that the shuttle vector only replicates at the permissive temperature and can be diluted out of the host cell population at the non-permissive temperature. When the shuttle vector is introduced into the host cell containing the BAC the RecA gene is expressed and recombination of the homologous shuttle vector and BAC sequences can occur thus targeting the key gene coding sequences (along with the shuttle vector sequences and flanking characterizing gene sequences) to the characterizing gene sequences in the BAC. The BACs can be selected and screened for integration of the key gene coding sequences into the selected site in the characterizing gene sequences using methods well known in the art (e.g., methods described in Section 6, infra, and in Heintz et al. WO 98/59060 and Heintz et al, WO01/05962). Optionally, the shuttle vector sequences not containing the key gene coding sequences (including the RecA gene and any selectable markers) can be removed from the BAC by resolution as described in Section 6 and in Heintz et al. WO 98/59060 and Heintz et al, WO 01/05962. If the shuttle vector contains a negative selectable marker, cells^'can be selected for loss of the shuttle vector sequences. In an alternative embodiment, the functional RecA gene is provided on a second vector and removed after recombination, e.g., by dilution of the vector or by any method known in the art. The exact method used to introduce the key gene coding sequences and to remove (or not) the RecA (or other appropriate recombination enzyme) will depend upon the nature of the BAC library used (for example the selectable markers present on the BAC vectors) and such modifications are within the skill in the art. Once the BAC containing the characterizing gene regulatory sequences and key gene coding sequences in the desired configuration is identified, it can be isolated from the host E. coli cells using routine methods and used to make transgenic animals as described, infra).

BACs to be used in the methods of the invention are selected and/or screened using the methods described in Section 5.3, supra, and Section 6, infra.

Alternatively, the BAC can also be engineered or modified by "Ε-T cloning," as described by Muyrers et al. (1999, Nucleic Acids Res. 27(6): 1555-57, incorporated herein by reference in its entirety). Using these methods, specific DNA may be engineered into a BAC independently of the presence of suitable restriction sites. This method is based on homologous recombination mediated by the recΕ and recT proteins ("ΕT-cloning") (Zhang et al, 1998, Nat. Genet. 20(2): 123-28; incorporated herein by reference in its entirety). Homologous recombination can be performed between a PCR fragment flanked by short homology arms and an endogenous intact recipient such as a BAC. Using this method, homologous recombination is not limited by the disposition of restriction endonuclease cleavage sites or the size of the target DNA. A BAC can be modified in its host strain using a plasmid, e.g., pBAD-αβγ, in which recE and recT have been replaced by their respective functional counterparts of phage lambda (Muyrers et al, 1999, Nucleic Acids Res. 27(6): 1555-57). Preferably, a BAC is modified by recombination with a PCR product containing homology arms ranging from 27-60 bp. In a specific embodiment, homology arms are 50 bp in length.

In another embodiment, a transgene is inserted into a yeast artificial chromosome (YAC) (Burke et al, 1987 Science 236: 806-12; and Peterson et al, 1997, Trends Genet. 13: 61).

In other embodiments, the transgene is inserted into another vector developed for the cloning of large segments of mammalian DNA, such as a cosmid or bacteriophage PI (Sternberg et al, 1990, Proc. Natl. Acad. Sci. USA 87: 103-07). The approximate maximum insert size is 30-35 kb for cosmids and 100 kb for bacteriophage PI.

In another embodiment, the transgene is inserted into a P-l derived artificial chromosome (PAC) (Mejia et al, 1997, Genome Res 7:179-186). The maximum insert size is 300 kb.

Vectors containing the appropriate characterizing and key gene sequences may be identified by any method well known in the art, for example, by sequencing, restriction mapping, hybridization, PCR amplification, etc.

5.4. INTRODUCTION OF VECTORS INTO HOST CELLS

In one aspect of the invention, a vector containing the transgene comprising the key and/or characterizing gene is introduced into the genome of a host cell, and the host cell is then used to create a transgenic animal. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast or mammalian cells), preferably a mammalian cell, and most preferably a mouse cell. Host cells intended to be part of the invention include ones that comprise a system and/or characterizing gene sequence that has been engineered to be present within the host cell (e.g., as part of a vector), and ones that comprise nucleic acid regulatory sequences that have been engineered to be present in the host cell such that a nucleic acid molecule of the invention is expressed within the host cell. The invention encompasses genetically engineered host cells that contain any of the foregoing system and/or characterizing gene sequences operatively associated with a regulatory element (preferably from a characterizing gene, as described above) that directs the expression of the coding sequences in the host cell. Both cDNA and genomic sequences can be cloned and expressed. In a preferred aspect, the host cell is recombination deficient, i.e., Rec^", and used for BAC recombination.

A vector containing a transgene can be introduced into the desired host cell by methods known in the art, e.g., transfection, transformation, transduction, electroporation, infection, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, liposomes, LIPOFECTIN™ (Bethesda Research Laboratories, Gaithersburg, MD), lysosome fusion, synthetic cationic lipids, use of a gene gun or a DNA vector transporter, such that the transgene is transmitted to offspring in the line. For various techniques for transformation or transfection of mammalian cells, see Keown et al, 1990, Methods Enzymol. 185: 527-37; Sambrook et al, 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N. Y.

Particularly preferred embodiments of the invention encompass methods of introduction of the vector containing the transgene using pronuclear injection of a transgenic construct into the mononucleus of a mouse embryo and infection with a viral vector comprising the construct. Methods of pronuclear injection into mouse embryos are well-known in the art and described in Hogan et al 1986, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, New York, NY and Wagner et al, U.S. Patent No. 4,873,191, issued October 10, 1989, herein incorporate by reference in their entireties.

In preferred embodiments, a vector containing the transgene is introduced into any nucleic genetic material which ultimately forms a part of the nucleus of the zygote of the animal to be made transgenic, including the zygote nucleus. In one embodiment, the transgene can be introduced in the nucleus of a primordial germ cell which is diploid, e.g., a spermatogonium or oogonium. The primordial germ cell is then allowed to mature to a gamete which is then united with another gamete or source of a haploid set of chromosomes to form a zygote. In another embodiment, the vector containing the transgene is introduced in the nucleus of one of the gametes, e.g., a mature sperm, egg or polar body, which forms a part of the zygote. In preferred embodiments, the vector containing the transgene is introduced in either the male or female pronucleus of the zygote. More preferably, it is introduced in either the male or the female pronucleus as soon as possible after the sperm enters the egg. In other words, right after the formation of the male pronucleus when the pronuclei are clearly defined and are well separated, each being located near the zygote membrane.

In a most preferred embodiment, the vector containing the transgene is added to the male DNA complement, or a DNA complement other than the DNA complement of the female pronucleus, of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus. In an alternate embodiment, the vector containing the transgene could be added to the nucleus of the sperm after it has been induced to undergo decondensation. Additionally, the vector containing the transgene may be mixed with sperm and then the mixture injected into the cytoplasm of an unfertilized egg. Perry et al. , 1999, Science 284:1180-1183. Alternatively, the vector maybe injected into the vas deferens of a male mouse and the male mouse mated with normal estrus females. Huguet et al, 2000, Mol. Reprod. Dev. 56:243-247.

Preferably, the transgene is introduced using any technique so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. The transgene is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art. Also known in the art are methods of transplanting the embryo or zygote into a pseudopregnant female where the embryo is developed to term and the transgene is integrated and expressed. See, e.g., Hogan et al. 1986, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, New York, NY. Viral methods of inserting a transgene are known in the art and have been described, supra.

For stable transfection of cultured mammalian cells, only a small fraction of cells may integrate the foreign DNA into their genome. The efficiency of integration depends upon the vector and transfection technique used. In order to identify and select integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene sequence of interest, e.g., the key gene sequence. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). Such methods are particularly useful in methods involving homologous recombination in mammalian cells (e.g., in murine ES cells) prior to introducing the recombinant cells into mouse embryos to generate chimeras.

A number of selection systems may be used to select transformed host cells. In particular, the vector may contain certain detectable or selectable markers. Other methods of selection include but are not limited to selecting for another marker such as: the herpes simplex virus thymidine kinase (Wigler et al, 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adenine phosphoribosyltransferase (Lowy et al, 1980, Cell 22: 817) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, 1980, Natl. Acad. Sci. USA 77: 3567; O'Hare et al, 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al, 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistance to hygromycin (Santerre et al, 1984, Gene 30: 147). The transgene may integrate into the genome of the founder animal (or an oocyte or embryo that gives rise to the founder animal), preferably by random integration. In other embodiments the transgene may integrate by a directed method, e.g., by directed homologous recombination ("knock-in"), Chappel, U.S. Patent No. 5,272,071; and PCT publication No. WO 91/06667, published May 16, 1991; U.S. Patent 5,464,764; Capecchi et al, issued November 7, 1995; U.S. Patent 5,627,059, Capecchi et al. issued, May 6, 1997; U.S. Patent 5,487,992, Capecchi et al, issued January 30, 1996). Preferably, when homologous recombination is used, it does not knock out or replace the host's endogenous copy of the characterizing gene (or characterizing gene ortholog). Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. The construct will comprise at least a portion of the characterizing gene with a desired genetic modification, e.g., insertion of the key gene coding sequences and will include regions of homology to the target locus, i.e., the endogenous copy of the characterizing gene in the host's genome. DNA constructs for random integration need not include regions of homology to mediate recombination.

Markers can be included for performing positive and negative selection for insertion of the transgene.

To create a homologous recombinant animal, a homologous recombination vector is prepared in which the key gene is flanked at its 5' and 3' ends by characterizing gene sequences to allow for homologous recombination to occur between the exogenous gene carried by the vector and the endogenous characterizing gene in an embryonic stem cell. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous characterizing gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Thomas and Capecchi, 1987, Cell 51: 503; Bradley, 1991, Curr. Opin. Bio/Technol. 2: 823-29; and PCT Publication Nos. WO 90/11354, WO 91/01140, and WO 93/04169.

5.4.1. INTRODUCTION OF MODULATING CONSTRUCTS

COMPRISING GENES ENCODING POTENTIAL DRUG TARGETS

The drug validation method of the invention does not involve the production of transgenic lines for each potential drug target to be validated but, rather, involves introduction of the potential drug target (or an inhibitor thereof) into existing transgenic animal lines such that the potential drug target is either expressed or inhibited only in a particular subset of cells (i.e., expression is spatially or temporally restricted).

Preferably, a coding region for a potential drug target is operably linked to an inducible or repressible conditional transcription element. The modulating construct is cloned into a viral vector that is used to infect a general type or population of cells (for example, the cells of a mouse in a global fashion) expressing the key protein in a select subpopulation of the general type or population of cells using any method know in the art. In a specific embodiment, the viral vector comprising the modulating construct is directly injected into a particular tissue region, e.g., a brain region. In one embodiment, the invention provides a method of expressing a potential drug target protein (or inhibitor thereof) in a specific subset of cells in a non-human animal. The method comprises introducing into cells of the transgenic non-human animal a vector comprising a first nucleotide sequence encoding the potential drug target protein (or inhibitor thereof), the expression of the potential drug target protein or inhibitor thereof being under the control of a conditional expression element. The transgenic non-human animal comprises a transgene containing a key gene that encodes an inducer or suppressor of the conditional expression element. The key gene is operably linked to regulatory sequences of a characterizing gene corresponding to an endogenous gene or homolog of an endogenous gene such that the key gene is expressed in the transgenic non-human animal with an expression pattern that is substantially the same as the expression pattern of the endogenous gene in a non-transgenic animal of the same species as the transgenic non- human animal. Preferably, the transgene is located at a site in the mouse genome other than the site of the endogenous characterizing gene. The potential drug target protein (or inhibitor thereof) is thereby selectively expressed in the cells expressing the key gene. In a preferred aspect, the invention provides a method of determining whether the modulation of expression of a potential target gene in a particular cell type is causally linked to a desired effect, for example, expression of the potential target causes the expression of a certain cell or tissue phenotype associated with a particular disease or disorder or with the treatment, prevention or amelioration of that disease or disorder. In one embodiment, homogeneous populations of cells expressing a particular key gene or group of key genes are isolated and purified from a transgenic animal line of the collection. A modulating construct comprising a gene encoding a selected potential drug target is introduced into the genomes of the homogeneous cell populations. The expression of the potential target gene is then modulated to determine whether expression of the potential target causes the expression of a certain cell or tissue phenotype associated with a particular disease or disorder or with the treatment, prevention or amelioration of that disease or disorder. In another embodiment, the modulating construct is introduced into the genomes of cells in vivo.

The drug validation system of the invention is more flexible, convenient and efficient than other existing drug validation systems because it uses one of a limited set of transgenic mouse lines instead of requiring the production of a transgenic mouse line for each target to be validated. The subject methods are advantageous because they enable the validation of drug targets to proceed rapidly and efficiently, limited only by the rate at which modulating constructs and viral vectors containing those modulating constructs can be produced, and not by the rate at which a transgenic animal line can be produced. A collection of transgenic animal lines expressing key proteins can be used repeatedly to validate many potential drug targets introduced via modulating constructs.

5.5. METHODS OF PRODUCING TRANSGENIC ANIMALS A transgenic animal is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene, i.e., has a non-endogenous (i.e., heterologous) nucleic acid sequence present as an extrachromosomal element in a portion of its cell or stably integrated into its germline DNA (i.e., in the genomic sequence of most or all of its cells). Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. Unless otherwise indicated, it will be assumed that a transgenic animal comprises stable changes to the germline sequence. Heterologous nucleic acid is introduced into the germ line of such a transgenic animal by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal. Methods for producing transgenic animal lines and collection of transgenic animal lines are described in Serafmi, U.S. Patent Application Serial No. (to be assigned) (Attorney Docket Number 10239-010-999) entitled "Collections of Transgenic Animal Lines (Living Library)" filed February 14, 2001, which is incorporated herein by reference in its entirety. As discussed above, the transgenic animals of the invention are preferably generated by random integration of a vector containing a transgene of the invention into the genome of the animal, for example, by pronuclear injection in the animal zygote, or injection of sperm mixed with vector DNA as described above. Other methods involve introducing the vector into cultured embryonic cells, for example ES cells, and then introducing the transformed cells into animal blastocysts, thereby generating a "chimeras" or "chimeric animals", in which only a subset of cells have the altered genome. Chimeras are primarily used for breeding purposes in order to generate the desired transgenic animal. Animals having a heterozygous alteration are generated by breeding of chimeras. Male and female heterozygotes are typically bred to generate homozygous animals.

A homologous recombinant animal is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

In a preferred embodiment, a transgenic animal of the invention is created by introducing a transgene of the invention, encoding the characterizing gene regulatory sequences operably linked to the key gene sequence, into the male pronuclei of a fertilized oocyte, e.g., by microinjection or retroviral infection, and allowing the egg to develop in a pseudopregnant female foster animal. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent No. 4,873,191, in Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986) and in Wakayama et al, 1999, Proc. Natl. Acad. Sci. USA, 96:14984-89; see also infra. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene as described supra. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes, animals of the same species that are disease models, etc. In another embodiment, the transgene encoding the characterizing gene regulatory sequences operably linked to the key gene sequence, is inserted into the genome of an embryonic stem (ES) cell, followed by injection of the modified ES cell into a blastocyst- stage embryo that subsequently develops to maturity and serves as the founder animal for a line of transgenic animals. In another embodiment, a vector bearing a transgene encoding the characterizing gene regulatory sequences operably linked to the key gene sequence is introduced into ES cells (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected. See, e.g., Li et al, 1992, Cell 69:915. For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc.

After transformation, ES cells are grown on an appropriate feeder layer, e.g., a fibroblast-feeder layer, in an appropriate medium and in the presence of appropriate growth factors, such as leukemia inhibiting factory (LIF). Cells that contain the construct of interest may be detected by employing a selective medium. Transformed ES cells may then be used to produce transgenic animals via embryo manipulation and blastocyst injection. (See, e.g., U.S. Pat. Nos. 5,387,742, 4,736,866 and 5,565,186 for methods of making transgenic animals.)

Stable expression of the construct is preferred. For example, ES cells that stably express a key gene product may be engineered. Rather than using vectors that contain viral origins of replication, ES host cells can be transformed with DNA, e.g., a plasmid, controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered ES cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.- "The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and expanded into cell lines. This method may advantageously be used to engineer ES cell lines that express the key gene product.

The selected ES cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL, Oxford, 113-52. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are implanted into the uterine horns of suitable pseudopregnant female foster animal. Alternatively, the ES cells may be incorporated into a morula to form a morula aggregate which is then implanted into a suitable pseudopregnant female foster animal. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct encoding the characterizing gene regulatory sequences operably linked to the key gene sequence, . The chimeric animals are screened for the presence of the characterizing gene regulatory sequences operably linked to the key gene sequence. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected. Males and female chimeras having the modification are mated to produce homozygous progeny. Only chimeras with transformed germline cells will generate homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allergenic or congenic grafts or transplants, or in in vitro culture.

Progeny harboring homologously recombined or integrated DNA in their germline cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA or randomly integrated transgene by germline transmission of the transgene.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al, 1997, Nature 385: 810-13 and PCT Publication NOS. WO 97/07668 and WO 97/07669. Once the transgenic mice are generated they may be bred and maintained using methods well known in the art. By way of example, the mice may be housed in an environmentally controlled facility maintained on a 10 hour dark: 14 hour light cycle or other appropriate light cycle. Mice are mated when they are sexually mature (6 to 8 weeks old). In certain embodiments, the transgenic founders or chimeras are mated to an unmodified animal (i.e., an animal having no cells containing the transgene). In a preferred embodiment, the transgenic founder or chimera is mated to C57BL/6 mice (Jackson Laboratories). In a specific embodiment in which the transgene encoding the characterizing gene regulatory sequences operably linked to the key gene sequence is introduced into ES cells and a chimeric mouse is generated, the chimera is mated to 129/Sv mice, which have the same genotype as the embryonic stem cells. Protocols for successful breeding are known in the art (see also Section 6). Commercial breeding services, e.g., Tosk, Inc. (Santa Cruz, CA) are also well known in the art and may be used to breed transgenic animals. Preferably, a founder male is mated with two females and a founder female is mated with one male. Preferably two females are rotated through a male's cage every 1-2 weeks. Pregnant females are generally housed 1 or 2 per cage. Preferably, pups are ear tagged, genotyped, and weaned at approximately 21 days. Males and females are housed separately. Preferably log sheets are kept for any mated animal, by example and not limitation, information should include pedigree, birth date, sex, ear tag number, source of mother and father, genotype, dates mated and generation.

More specifically, founder animals heterozygous for the transgene encoding the characterizing gene regulatory sequences operably linked to the key gene sequence may be mated to generate a homozygous line as follows: A heterozygous founder animal, designated as the P_j generation, is mated with an offspring designated as the F, generation from a mating of a non-transgenic mouse with a transgenic mouse heterozygous for the transgene (backcross). Based on classical genetics, one fourth of the results of this backcross are homozygous for the transgene. In a preferred embodiment, transgenic founders are individually backcrossed to an inbred or outbred strain of choice. Different founders should not be intercrossed, since different expression patterns may result from separate transgene integration events.

The determination of whether a transgenic mouse is homozygous or heterozygous for the transgene is as follows:

An offspring of the above described breeding cross is mated to a normal control non-transgenic animal. The offspring of this second mating are analyzed for the presence of the transgene by the methods described below. If all offspring of this cross test positive for the transgene, the mouse in question is homozygous for the transgene. If, on the other hand, some of the offspring test positive for the transgene and others test negative, the mouse in question is heterozygous for the transgene.

An alternative method for distinguishing between a transgenic animal which is heterozygous and one which is homozygous for the transgene is to measure the intensity with radioactive probes following Southern blot analysis of the DNA of the animal. Animals homozygous for the transgene would be expected to produce higher intensity signals from probes specific for the transgene than would heterozygote transgenic animals. In a preferred embodiment, the transgenic mice are so highly inbred to be genetically identical except for sexual differences. The homozygotes are tested using backcross and intercross analysis to ensure homozygosity. Homozygous lines for each integration site in founders with multiple integrations are also established. Brother/sister matings for 20 or more generations define an inbred strain. In another preferred embodiment, the transgenic lines are maintained as hemizygotes. In an alternative embodiment, individual genetically altered mouse strains are also cryopreserved rather than propagated. Methods for freezing embryos for maintenance of founder animals and transgenic lines are known in the art. Gestational day 2.5 embryos are isolated and cryopreserved in straws and stored in liquid nitrogen. The first and last straws are subsequently thawed and transferred to foster females to demonstrate viability of the line with the assumption that all embryos frozen between the first and last straws will behave similarly. If viable progeny are not observed a second embryo transfer will be performed. Methods for reconstituting frozen embryos and bringing the embryos to term are known in the art.

5.6. METHODS OF SCREENING FOREXPRESSION OFTRANSGENES

Potential founder animals for a line of transgenic animals can be screened for expression of the key gene sequence in the population of cells characterized by expression of the endogenous characterizing gene. Transgenic animals that exhibit appropriate expression (e.g., detectable expression having substantially the same expression pattern as the endogenous characterizing gene in a corresponding non-transgenic animal or anatomical region thereof, i.e., detectable expression in at least 80%, 90%, 95% or, preferably, 100% of the cells shown to express the endogenous gene by in situ hybridization) are selected as transgenic animal lines. Additionally, in situ hybridization using probes specific for the key gene coding sequences may also be used to detect expression of the key gene product. In a preferred embodiment, immunohistochemistry using an antibody specific for the key gene product or associated marker is used to detect expression of the key gene product. Alternatively, expression of the key gene may be detected by in situ hybridization to detect the key gene mRNA.

In another aspect of the invention, key and/or marker gene expression is visualized in single living mammalian cells. In one embodiment where the expression of a marker gene is linked to the key gene expression, the method of Zlokarnik et al, (1998, Science 279: 84-88; incorporated herein by reference in its entirety) is used to visualize marker gene expression. The marker gene encodes an enzyme, e.g., β-lactamase. To image single living cells, an enzyme assay is performed in which β-lactamase hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester. Each molecule of β-lactamase changes the fluorescence of many substrate molecules from green to blue by disrupting resonance energy transfer. This wavelength shift can be detected by eye or photographically (either on film or digitally) in individual cells containing less than 100 β-lactamase molecules.

In another embodiment, the non-invasive method of Contag et al. is used to detect and localize light originating from a mammal in vivo (Contag et al. , U.S. Patent No. 5,650,135, issued July 22, 1997; incorporated herein by reference in its entirety) . Light- emitting conjugates are used that contain a biocompatible entity and a light-generating moiety. Biocompatible entities include, but are not limited to, small molecules such as cyclic organic molecules; macromolecules such as proteins; microorganisms such as viruses, bacteria, yeast and fungi; eukaryotic cells; all types of pathogens and pathogenic substances; and particles such as beads and liposomes. In another aspect, biocompatible entities may be all or some of the cells that constitute the mammalian subject being imaged.

Light-emitting capability is conferred on the entities by the conjugation of a light- generating moiety. Such moieties include fluorescent molecules, fluorescent proteins, enzymatic reactions giving off photons and luminescent substances, such as bioluminescent proteins. The conjugation may involve a chemical coupling step, genetic engineering of a fusion protein, or the transformation of a cell, microorganism or animal to express a bioluminescent protein. For example, in the case where the entities are the cells constituting the mammalian subject being imaged, the light-generating moiety may be a bioluminescent or fluorescent protein "conjugated" to the cells through localized, promoter-controlled expression from a vector construct introduced into the cells by having made a transgenic or chimeric animal.

Light-emitting conjugates are typically administered to a subject by any of a variety of methods, allowed to localize within the subject, and imaged. Since the imaging, or measuring photon emission from the subject, may last up to tens of minutes, the subject is usually, but not always, immobilized during the imaging process.

Imaging of the light-emitting entities involves the use of a photodetector capable of detecting extremely low levels of light (typically single photon events) and integrating photon emission until an image can be constructed. Examples of such sensitive photodetectors include devices that intensify the single photon events before the events are detected by a camera, and cameras (cooled, for example, with liquid nitrogen) that are capable of detecting single photons over the background noise inherent in a detection system.

Once a photon emission image is generated, it is typically superimposed on a "normal" reflected light image of the subject to provide a frame of reference for the source of the emitted photons (i.e. localize the light-emitting conjugates with respect to the subject). Such a "composite" image is then analyzed to determine the location and/or amount of a target in the subject.

5.7. ISOLATION AND PURIFICATION OF CELLS FROM THE

TRANSGENIC ANIMALS

Homogeneous populations of cells that express a particular key gene can be isolated and purified from transgenic animals of the invention. Methods for cell isolation include, but are not limited to, surgical excision or dissection, dissociation, fluorescence-activated cell sorting (FACS), panning, and laser capture microdissection (LCM). Methods for the isolation and purification of cells from transgenic animals of a collection are described in Serafmi, U.S. Patent Application Serial No. (to be assigned) (Attorney Docket Number 10239-010-999) entitled "Collections of Transgenic Animal Lines (Living Library)" filed February 14, 2001, which is incorporated herein by reference in its entirety. In certain embodiments, cells expressing a particular key gene are isolated using surgical excision or dissection. Before dissection, the transgenic animal may be perfused. Perfusion is preferably accomplished using a perfusion solution that contains α-amanitin or other transcriptional blockers to prevent changes in gene expression from occurring during cell isolation. In other embodiments, cells expressing a particular key gene are isolated from adult rodent brain tissue which is dissected and dissociated. Methods for such dissection and dissociation are well-known in the art. See, e.g., Brewer, 1997, J. Neurosci. Methods 71(2):143-55; Nakajima et al, 1996, Neurosci. Res. 26(2):195-203; Masuko et al, 1992, Neuroscience 49(2):347-64; Baranes et al, 1996, Proc. Natl. Acad. Sci. USA 93(10):4706-11; Emerling et al, 1994, Development 120(10):2811-22; Martinou (1989, J. Neurosci. 9(10):3645-56; Ninomiya, 1994, Int. J. Dev. Neurosci. 12(2): 99-106; Delree, 1989, J. Neurosci. Res. 23(2): 198-206; Gilbert, 1997, J. Neurosci. Methods 71(2):191-98; Huber, 2000, J. Neurosci. Res. 59(3):372-78; which are all incorporated herein by reference in their entireties. .. , _ In other embodiments cells expressing a particular key gene are dissected from tissue slices based on their morphology as seen by transmittance light direct visualization and cultured, using, e.g., the methods of Nakajima et al, 1996, Neurosci. Res. 26(2):195-203; Masuko et al, 1992, Neuroscience 49(2):347-64; which are incorporated herein by reference in their entireties. Tissue slices are made of a particular tissue region and a particular subregion, e.g., a brain nucleus, is isolated under direct visualization using a dissecting microscope.

In yet other embodiments, cells expressing a particular key gene can be dissociated using a protease such as papain (Brewer, 1997, J. Neurosci. Methods 71(2):143-55; Nakajima et al, 1996, Neurosci. Res. 26(2):195-203;) or trypsin (Baranes, 1996, Proc. Natl. Acad. Sci. USA 93(10):4706-11; Emerling et al, 1994, Development 120(10):2811-22; Gilbert, 1997, J. Neurosci. Methods 71(2):191-98; Ninomiya, 1994, Int. J. Dev. Neurosci. 12(2): 99-106; Huber, 2000, J. Neurosci. Res. 59(3):372-78; which are incorporated herein by reference in their entireties). Cells can also be dissociated using collagenase (Delree, 1989, J. Neurosci. Res. 23(2):198-206; incorporated herein by reference in its entirety). The dissociated cells are then grown in cultures over a feeder layer. In one embodiment, the dissociated cells are neurons that are grown over a glial feeder layer.

In another embodiment, tissue that is labeled with a fluorescent marker, e.g., a marker gene protein, can be microdissected and dissociated using the methods of Martinou (1989, J. Neurosci. 9(10):3645-56; incorporated herein by reference in its entirety). Microdissection of the labeled cells is followed by density-gradient centrifugation. The cells are then purified by fluorescence-activated cell sorting (FACS) (see infra). In other embodiments, cells can be purified by a cell-sorting procedure that only uses light-scatter parameters and does not necessitate labeling (Martinou, 1989, J. Neurosci. 9(10):3645-56). In one aspect of the invention, a subset of cells within a heterogeneous cell population derived from a transgenic animal in the collection of transgenic animals lines is recognized by expression of a key gene and/or marker gene. The regulatory sequences of the characterizing gene are used to express a key gene and/or a marker gene protein in transgenic cells, and the targeted population of cells is isolated based on expression of the key gene and/or marker gene. Selection and/or separation of the target subpopulation of cells may be effected by any convenient method. For example, where the marker is an externally accessible, cell-surface associated protein or other epitope-containing molecule, immuno-adsorption panning techniques or fluorescent immuno-labeling coupled with fluorescence activated cell sorting (FACS) are conveniently applied.

Cells that express a marker gene product, e.g., an enzyme, can be detected using flow cytometric methods such as the one described by Mouawad et al, 1997, J. Immunol. Methods, 204(1), 51-56; incorporated herein by reference in its entirety). The method is based on an indirect immunofluorescence staining procedure using a monoclonal antibody that binds specifically to the marker enzyme encoded by the marker gene sequence, e.g. , β- galactosidase or a β-galactosidase fusion protein. The method can be used for both quantification in vitro and in vivo of enzyme expression in mammalian cells. The method is preferably used with a construct containing a lacZ selectable marker. Using such a method, cells expressing a key gene and/or marker gene can be quantified and gene regulation, including transfection modality, promoter efficacy, enhancer activity, and other regulatory factors studied (Mouawad et al, 1997, J. Immunol. Methods 204(1): 51-56).

In another embodiment, a FACS-enzyme assay, e.g., a FACS-Gal assay, is used (see, e.g., Fiering et al, 1991, Cytometry 12(4): 291-301; Nolan et al, 1988, Proc. Natl. Acad. Sci. USA 85(8): 2603-07; which are incorporated herein by reference in their entireties). The FACS-Gal assay measures E. coli lacZ-encoded β-galactosidase activity in individual cells. Enzyme activity is measured by flow cytometry, using a fluorogenic substrate that is hydrolyzed and retained intracellularly. In the system described by Fiering et al , lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence-activated cell sorting based on their lacZ expression level. Preferably, phenylethyl-beta-D-thiogalactoside (PETG), is used as a competitive inhibitor in the reaction, to inhibit β-galactosidase activity and slow reaction with the substrate. Also preferably, interfering endogenous host (e.g., mammalian) β-galactosidases are inhibited by the weak base chloroquine. Further, false positives may be minimized by performing two- color measurements (false-positive cells tend to fluoresce more in the yellow wavelengths. In another specific embodiment, a fluorescence-activated cell sorter (FACS) is used to detect the activity of a marker gene encoding E. coli β-glucuronidase (gus) (Lorincz et al, 1996, Cytometry 24(4): 321-9). When loaded with the Gus substrate fluorescein-di-beta- D- glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis by flow cytometry. This assay can be used to FACS-sort viable cells based on Gus enzymatic activity, and the efficacy of the assay can be measured independently by using a fluorometric lysate assay. In another specific embodiment, the intracellular fluorescence generated by the activity of both beta-glucuronidase and E. coli β-galactosidase enzymes are detected by FACS independently. Because each enzyme has high specificity for its cognate substrate, each reporter gene can be measured by FACS independently.

The invention provides methods for isolating individual cells harboring a fluorescent protein reporter from tissues of transgenic mice by FACS. See Hadjaantonakis and Naki, 2000, Genesis, 27(3):95-8, which is incorporated herein by reference it its entirety. In certain embodiments of the invention, the reporter is a autofluorescent (AFP) reporter such as, but not limited to, wild type Green Fluorescent Protein (wtGFP) and its variants, including enhanced green fluorescent protein (EGFP) and enhanced yellow fluorescent protein (EYFP).

In one embodiment of the invention, cells are isolated by FACS using fluorescent antibody staining of cell surface proteins. The cells are isolated using methods known in the art as described by Barrett et al. , 1998, Neuroscience, 85(4): 1321 -8, incorporated herein in its entirety. In another embodiment, cells are isolated by FACS using fluorogenic substrates of an enzyme transgenically expressed in a particular cell-type. The cells are isolated using methods known in the art as described by Blass-Kampmann et al, 1994, J. Neurosci. Res., 37(3):359-73, which is incorporated herein by reference in its entirety. The invention also provides methods for isolating cells from primary culture cells.

Using methods known in the art, whole animal sorting (WACS) is accomplished whereby live cells derived from animals harboring a lacZ transgene are purified according to their level of beta-galactosidase expression with a fluorogenic beta-galactosidase substrate and FACS. See Krasnow et al, 1991, Science 251:81-5, which is incorporated herein by reference in its entirety.

In other embodiments of the invention, cells are isolated by FACS using fluorescent, vital dyes to retrograde label cells with fluorescent tracers. Cells are isolated using the methods described by St. John and Stephens, 1992, Dev. Biol. 151(l):154-65, Martinou et al, 1992, Neuron 8(4):737-44. Clendening and Hume, 1990, J Neurosci. 10(12):3992-4005 and Martinou et al, 1989, J Neurosci, 9(10):3645-56, which are all incorporated herein by reference in their entireties.

In yet other embodiments of the invention, cells are isolated by FACS using fluorescent-conjugated lectins in retrograde labeled cells. The cells are isolated using the methods described in Schaffner et al, 1987, J Neurosci, 7(10):3088-104 and Armson and Bennett, 1983, Neurosci. Eett., 38(2):181-6, which are all incorporated herein by reference in their entireties.

In certain embodiments of the invention, cells are isolated by panning on antibodies against cell surface markers. In preferred embodiments, the antibody is a monoclonal antibody. Cells are isolated and characterized using methods known in the art described by Camu and Henderson, 1992, J Neurosci. Methods 44(l):59-79, Kashiwagi et al, 2000, 41(l):2373-7, Brocco and Panzetta, 1997, 75(l):15-20, Tanaka et al, 1997, Dev. Neurosci. 19(1):106-11, and Barres et al, 1988, Neuron l(9):791-803, which are all incorporated herein by reference in their entireties.

In another embodiment, cells are isolated using laser capture microdissection (LCM). Methods for laser capture microdissection of the nervous system are well known in the art. See, e.g., Emmert-Buck et α ., 1996, Science 274, 998-1001 ; Luo, et al, 1999, Nature Med. 5(1), 117-122; Ohyama et al, 2000, Biotechniques 29(3):530-36; Murakami et al, 2000, Kidney Int. 58(3),1346-53; Goldsworthy et al, 1999, Mol. Carcinog. 25(2): 86-91; Fend et al, 1999, Am. J. Pathol. 154(l):61-66); Schutze et al, 1998, Nat. Biotechnol. Aug;16(8):737-42, which are all incorporated herein by reference in their entireties.

In a specific embodiment, a collection of transgenic mouse lines of the invention is used to isolate neurons expressing the key gene that are located in the arcuate nucleus of the hypothalamus and that regulate feeding behavior.

5.8. METHODS OF TARGET VALIDATION

The transgenic animal lines of the invention and cells isolated from the transgenic animal lines of the invention may be used for target validation, drug discovery, pharmacological, behavioral, electrophysiological, and gene expression assays, etc. but, preferably target validation.

In certain embodiments, cells expressing the key gene and/or marker gene coding sequences are detected in vivo in the transgenic animal, or in explanted tissue or tissue slices from the transgenic animal, to analyze the population of cells marked by the expression of the key gene and/or marker gene coding sequences. In particular, the population of cells can be examined in transgenic animals in which a modulating construct comprising a particular drug target has or has not been introduced.

The cells are detected by methods known in the art depending upon the marker gene . used (see Sections 5.1.3 and 5.6, above). In a particular embodiment, the marker gene coding sequences encode or promote the production of an agent that enhances the contrast of the cells expressing the key gene coding sequences and such cells are detected by MRI. Additionally, the transgenic animals may be bred to existing disease model animals or treated pharmacologically or surgically, or by any other means, to create a disease state in the transgenic animal. The animals can then be compared to such animals in which a modulating construct comprising a particular drug target has been introduced, e.g. for phenotypic changes, particularly changes in symptoms, indicators of the particular disease or disorder.

Additionally, treatments for the disease may be evaluated by administering a treatment (e.g. a candidate compound) to the transgenic mice of the invention expressing the target protein and, preferably that have been bred to a disease state or a disease model otherwise induced in the transgenic mice. The mice are then evaluated for morphological, physiological or electrophysiological changes, changes in gene expression, protein-protein interactions, protein profile in response to the treatment is an indication of efficacy or toxicity, etc. of the treatment.

In other preferred embodiments, cells expressing the key gene and a potential drug target are isolated from the transgenic animal using methods known in the art, preferably, for analysis or for culture of the cells and subsequent analysis. In certain embodiments, the transgenic animal expressing the key gene and a potential drug target in a select population of cells may be subjected to a treatment (for example a surgical treatment or administered a candidate compound of interest) prior to isolation of the cells. In other embodiments, the transgenic animal may be bred to a disease model or a disease state induced in the transgenic animal, for example by surgical or pharmacological manipulation, prior to isolation of the cells.

Once isolated, the populations of cells expressing a key gene and a potential drug target of interest can be analyzed by any method known in the art. In one aspect of the invention, the gene expression profile of the cells is analyzed using any number of methods known in the art, for example but not by way of limitation, by isolating the mRNA from the isolated cells and then hybridizing the mRNA to a microarray to identify the genes which are or are not expressed in the isolated cells. Gene expression in cells treated and not treated with a compound of interest or in cells from animals treated or untreated with a particular treatment, e.g., surgical treatment, may be compared. In addition, mRNA from the isolated cells may also be analyzed, for example by northern blot analysis, PCR, RNase protection, etc., for the presence of mRNAs encoding certain protein products and for changes in the presence or levels of these mRNAs depending on the treatment of the cells. In another aspect, mRNA from the isolated cells may be used to produce a cDNA library and, in fact, a collection of such cell type specific cDNA libraries may be generated from different populations of isolated cells. Such cDNA libraries are useful to analyze gene expression, isolate and identify cell type-specific genes, splice variants and non-coding RNAs. In another aspect, such cell type specific libraries prepared from cells isolated from treated and untreated transgenic animals of the invention or from transgenic animals of the invention having and not having a disease state can be used, for example in subtractive hybridization procedures, to identify genes expressed at higher or lower levels in response to a particular treatment or in a disease state as compared to untreated transgenic animals. Data from such analyses may be used to generate a database of gene expression analysis for different populations of cells in the animal or in particular tissues or anatomical regions, for example, in the brain. Using such a database together with bioinformatics tools, such as

10: hierarchical and non-hierarchical clustering analysis and principal components analysis, cells are "fingerprinted" for particular indications from healthy and disease-model animals or tissues.

In yet another embodiment, specific cells or cell populations that express a potential drug target are isolated from the collection and analyzed for specific protein-protein interactions or an entire protein profile using proteomics methods known in the art, for example, chromatography, mass spectroscopy, 2D gel analysis, etc.

Other types of assays may be used to analyze the cell population expressing the potential drug target, either in vivo, in explanted or sectioned tissue or in the isolated cells, for example, to monitor the response of the cells to a certain treatment or candidate compound or to compare the response of the animals, tissue or cells to expression of the target or inhibitor thereof, with animals, tissue or cells from animals not expressing the target or inhibitor thereof. The cells may be monitored, for example, but not by way of limitation, for changes in electrophysiology, physiology (for example, changes in physiological parameters of cells, such as intracellular or extracellular calcium or other ion concentration, change in pH, change in the presence or amount of second messengers, cell morphology, cell viability, indicators of apoptosis, secretion of secreted factors, cell replication, contact inhibition, etc.), morphology, etc.

In a particular embodiment, a subpopulation of cells in the isolated cells is identified and/or gene expression analyzed using the methods of Serafmi et al. (PCT Publication WO 99/29877, entitled Methods for Defining Cell Types, published June 17, 1999) which is hereby incorporated by reference in its entirety.

6. EXAMPLE 1: METHODS USED FOR CREATION OF TRANSGENIC ANIMAL LINE

This example describes the methods used for creation of a transgenic animal line for use in the drug validation methods of the invention.

6.1. ISOLATION AND INITIAL MAPPING OF BACS A BAC clone is isolated with either a unique cDNA or genomic DNA probe from

BAC libraries for various species, (in the form of high density BAC colony DNA membrane) The BAC library is screened and positive clones are obtained, and the BACs for specific genes of interest are confirmed and mapped, as described in detail below.

Probes Overlapping oligonucleotide ("overgo") probes are highly useful for large-scale physical mapping and whenever sequence is available from which to design a probe for hybridization purposes. In particular, the short length of the overgo probe is advantageous when there is limited available sequence known from which to design the probe. In addition, overgo probes obviate the need to clone and characterize cDNA fragments, which traditionally have been used as hybridization probes. Overgo probes can be used for identifying homologous sequences on DNA macroarrays printed on nylon membranes (i.e., BAC DNA macroarrays) or for Southern blot analysis. This technique can be extended to any hybridization-based gene screening approach. The following protocol describes a method for generating hybridization probes of high specific activity and specificity when sequence data is available. The method is used for identifying homologous DNA sequences in arrays of BAC library clones.

Design of Overgo Probes Overgo probes are designed through a multistep process designed to ensure several important qualities:

(1) Overgos are gene-specific so that they do not hybridize to each other (when probes are pooled) or to sequences in the genome other than those that belong to the gene of interest. (2) Probes are designed with similar GC contents. This allows probes to be labeled to ^■ similar specific activities and to hybridize with similar efficiencies, thus enabling a probe pooling strategy that is essential for high throughput screening of BAC library macroarrays.

The starting point for overgo design is to obtain sequence information for the gene of interest. The software packages required for overgo design require this sequence to be in FASTA format. A sequence in FASTA format begins with a single-line description, followed by lines of sequence data. The description line is distinguished from the sequence data by a greater-than (">") symbol in the first column. It is recommended that all lines of text be shorter than 80 characters in length. Sequences are expected to be represented in the standard IUB/IUPAC amino acid and nucleic acid codes, with these exceptions: lower-case letters are accepted and are mapped into upper-case; a single hyphen or dash can be used to represent a gap of indeterminate length; and in amino acid sequences, U and * are acceptable letters (see below). Before submitting a request, any numerical digits in the query sequence should either be removed or replaced by appropriate letter codes (e.g., N for unknown nucleic acid residue or X for unknown amino acid residue). The nucleic acid codes supported are: A --> adenosine M — > A C (amino)

C --> cytidine S --> G C (strong)

G --> guanine W — > A T (weak)

T --> thymidine B --> G T C U --> uridine D --> G A T

R -> G A (purine) H --> A C T

Y --> T C (pyrimidine) V --> G C A

K -> G T (keto) N --> A G C T (any) gap of indeterminate length The sequence used for overgo design is preferably genomic, but cDNA sequences have been used successfully. For overgo design, programs known in the art such as OvergoMaker (John D. McPherson, Ph.D., Genome Sequencing Center/Department of Genetics, Washington University School of Medicine, Box 8501,4444 Forest Park Blvd., St. Louis, MO 63108) may be used. To design a probe, a region of approximately 500bp is selected. The 500bp region should flank the gene's start codon (ATG) for probe design. This strategy gives a high probability of identifying BACs containing the 5' end of the gene (and presumably many or all of the relevant transcriptional control elements. Selected sequences are screened for the presence of known murine DNA repeat sequences using the RepeatMasker program (Bioinformatics Applications Note 16 (11)(2000): 1040-1041). Oligonucleotides or "overgos" are then designed using OvergoMaker (John D. McPherson, Ph.D., Genome Sequencing Center/Department of Genetics, Washington University School of Medicine, Box 8501,4444 Forest Park Blvd., St. Louis, MO 63108). The overgo design program scans sequences and identifies two overlapping 24mers that have a balanced GC content, and an overall GC content between 40-60%. Once gene specific overgos have been designed, they are checked for uniqueness by using the BLAST program (NCBI) to compare them to the nr nucleic acid database (NCBI). Overgos that have significant BLAST scores for genes other than the gene of interest, i.e., could hybridize to genes other than the gene of interest, are redesigned.

Creation of Overgo Probes

To create an overgo probe, a pair of 24mer oligonucleotides overlapping at the 3' ends by 8 base pairs are annealed to create double stranded DNA with 16 base pair overhangs. The resulting overhangs are filled in using Klenow fragment. Radionucleotides are incorporated during the fill-in process to label the resulting 40mer as it is synthesized. The overgo probe is then hybridized to immobilized BAC DNA. Following hybridization, the filter is washed to remove nonspecifically bound probe. Hybridization of specifically bound probe is visualized through autoradiography or phosphoimaging.

Materials 1. Target BAC clone DNA immobilized on nylon filters, for example, a macroarray of a BAC library, e.g., the CITB BAC library (Research Genetics) or the RPCI-23 library (BACPAC Resources, Children's Hospital Oakland Research Institute, Oakland, CA). 2. 10 μCi/μl [³²P]dATP (-3000 Ci/mmol, 1 OmCi/ml) 3. 10 μCi/μl [³²P]dCTP (-3000 Ci/mmol, lOmCi/ml)

4. Sephadex G-50 Microspin Column (e.g. ProbeQuant Spin Columns; Amersham Pharmacia Biotech)

5. 60 ° C hybridization oven

6. SSC (sodium chloride/sodium citrate) 20x: 701.2 g NaCI

352 g NaCitrate Add ddH₂O to make 4 L. ^• - pH to 7.0 with 6M HCI

7. 10% SDS (sodium dodecyl sulfate) : 100 g SDS/l L dd H₂O

8. Church's hybridization buffer:

1 mM EDTA

7% SDS (use 99.9% pure SDS) 0.5 M Sodium phosphate IM Sodium phosphate, pH 7.2:

268 g Na2HPO4; 7H₂O in 1700 ml ddH₂O

Add 8 ml 85% H₃PO₄ and ddH₂O to make 2000 ml.

9. 0.5M EDTA, pH 8.0:

To make 500 ml: 93 g EDTA (disodium dihydrate) in 400 ml ddH₂O. pH to 8.0 with 6M NaOH and add ddH₂O to make 500 ml. To make 4000 ml:

To 2000 ml IM sodium phosphate, add 1200 ml ddH₂O, 8 ml 0.5M EDTA and 280g SDS. Heat and stir until SDS is dissolved (approximately 1 hr.). Add ddH₂O to bring volume to 4000 ml. Warm to 60 °C before using.

10. Wash Buffer B: 1% SDS, 40 mM NaPO₄ , ImM EDTA, pH 8.0

4x: 48 ml 0.5M EDTA 240 g SDS

960mllMNaHPO₄,pH7.2 Add ddH₂O to make 6 L.

11. Wash Buffer 2: 1.5x SSC, 0.1% SDS

1125 ml 20x SSC 150 ml 10% SDS

AddddH₂Otomakel5L.

12. Wash Buffer 3: 0.5x SSC, 0.1% SDS

375 ml 20x SSC 150 ml 10% SDS Add ddH₂O to make 15 L.

13. 2%BSA:200mgBSA10mlddH₂O

14. Stripping Buffer: O.lx SSC, 0.1% SDS

10ml20xSSC 20 ml 10% SDS Add ddH₂O to make 2 L.

15. Overgo Labeling Buffer (OLB)

Solution O:

125mMMgCl₂ 1.25 M Tris-HCl, pH 8.0 15.1 gTris-base

2.54 g MgCl₂.6H₂O Add ddH₂O to make 100 ml. Solution A:

1 ml Solution O 18 μl 2-mercaptoethanol

5μl0.1MdGTP 5μl0.1MdTTP Store up to 1 year at -80 °C. Solution B: 2 M HEPES-NaOH, pH 6.6 2.6 g HEPES to 5 ml ddH₂O pH to 6.6 with approximately 2 drops 6M NaOH

Store up to 1 year at room temperature Solution C: 3 mM Tris-HCl pH 7.4 / 0.2 mM Na₂EDTA

36 mg Tris-base

7 mg EDTA

Add ddH₂O to make 100 ml. pH to 7.4 with IM NaOH Store up to 1 year at room temperature.

OLB:

A:B:C, in a 2:5:3 ratio

1 ml Solution A

2.5 ml Solution B 1.5 ml Solution C

Store in 0.5 ml aliquots at -20 °C for up to 3 months.

Methods

Annealing oligonucleotides to generate a overhang Step 1: combine 1.0 μl of partially complementary 10 μM oligos (1.0 μl forward primer + 1.0 μl reverse primer) with 3.5 μl ddH₂O (10 pmol each oligo/reaction) to either a tube or microtiter plate well.

Step 2: Cap each tube or microtiter well and heat the paired oligonucleotides for 5 min at 80 °C to denature the oligonucleotides. Step 3: Incubate the labeling reactions for 10 min at 37°C to form overhangs.

Step 4: Store the annealed oligonucleotides on ice until they are labeled. If the labeling step is not done within 1 hour of annealing the oligonucleotides, repeat steps 2 and 3 before proceeding.

A thermocycler can be programmed to perform steps 2 through 4.

Overgo Labeling

Overgo probes can be labeled and hybridized using methods well-known in the art, for example, using the protocols described in Ross et al, 1999, Screening Large-Insert Libraries by Hybridization, In Current Protocols in Human Genetics, eds. N.C. Dracopoli, J.L. Haines, B.R. Korf, D.T. Moir, C.C. Morton, C.E. Seidman, J.G. Seidman, D.R. Smith. pp. 5.6.1-5.6.52 John Wiley and Sons, New York; incorporated herein by reference in its entirety.

The following protocol is modified after Ross et al, supra. Prepare a master mix containing the following reagents for each overgo probe to be labeled: 0.5 μl 2% BSA

2.0 μl overgo labeling buffer

0.5 μl [³²P]dATP

0.5 μl [³²P]dCTP

1.0 μl 2U/μl Klenow fragment

When making a master mix to label a number of overgo probes, prepare more than needed to ensure that there will be sufficient mix to account for small losses when transferring. An extra 10% is usually sufficient.

This protocol uses both [³²P]dATP and [³²P]dCTP for labeling. This is recommended; however, the composition of the dNTP mix in the overgo labeling buffer can be altered to allow different labeled deoxynucleotides to be used.

Pipet 4.5 μl of overgo labeling master mix to each of the annealed oligonucleotide pairs from step 4.

Incubate labeling reactions at room temperature for 1 hour.

Removal of unincorporated nucleotides

Remove unincorporated nucleotides using a Sephadex G-50 microspin column following the manufacturers protocol. If probes will be pooled, multiple labeling reactions can be combined and processed simultaneously as long as the total volume specified by the manufacturer is not exceeded.

Checking incorporation

The following method can be used as a quick measure of the success of the labeling reaction. Dilute the probes 1 : 100 (1 μl probe + 99 μl H₂O), and use 1 μl of diluted probe for scintillation counting. For optimal hybridization, the probe specific activity should be approximately 5 x 10⁵ cpm/ml. 6.1.1. BAC SCREENING

BACs containing specific characterizing genes of interest are identified by using ³²P labeled overgo probes, as described above, to probe nylon membranes onto which BAC- containing bacterial colonies have been spotted. Traditionally, BAC screening is accomplished by hybridizing a single probe to BAC library filters, and identifying positive clones for that single characterizing gene of interest. The use of overgo probes makes it possible to adopt a probe pooling strategy that permits higher throughput while using fewer library filters. In this strategy, probes are arrayed into a two- dimensional matrix (i.e., 5x5 or 6x6). Then probes are combined into row and column pools (e.g., 10 pools total for a 5x5 array). Each probe pool is hybridized to a single copy of the BAC library filters (10 separate hybridizations) e.g., the CITB or RPCI-23 BAC library filters.

Following hybridization and autoradiography or phosphoimaging, clones hybridizing to each probe pool ( 4-5 probes) are manually identified. Assignment of positive clones to individual probes is done by pairwise comparisons between each row and each column. The intersection of each row pool and column pool defines a single probe within the probe array. Thus, all positive clones that are shared in common by a specific row pool and a specific column pool are known to hybridize to the probe defined by the unique intersection between the row and column. Deconvolution of hybridization data to assign positive clones to specific probes in the probe array is done manually, or by using an MICROSOFT EXCEL™-based Visual Basic program.

Using this strategy increases screening efficiency, and throughput, while decreasing the number of library filters required. For example, without probe pooling, hybridizing 25 probes would require 25 sets of library filters. In contrast, a 5x5 probe array requires only 10 probe pools, thus 10 hybridizations and 10 filter sets. This approach can also be extended using 3 dimensional probe arrays. For example, a 3x3x3 array allows for identification of 27 genes and only requires 9 hybridization experiments.

Hybridization of overgo probe to nylon filter

The nylon filters are prehybridized by wetting with 60 °C Church's hybridization buffer and rolling the filters into a hybridization bottle filled halfway or approximately 150 ml of 60 °C Church's hybridization buffer. All of the filters are rolled in the same direction (DNA and writing side up), with a nylon mesh spacer in between each and on top, and the bottle is placed in the oven to keep them rolled. The rotation speed is set to 8-9 speed. The filter is incubated at 60 °C for at least 4 hours the first time (1-2 hours for subsequent prehybridizations of the same filters).

Following prehybridization of the filters, labeled probes are denatured by heating to 100°C for 10 min and then placed on slushy ice for 2 min or longer. 5 The Church's hybridization buffer is replaced before adding probes if the filter is used for the first time. Filters are incubated with the probe at 60°C overnight. The rotation speed is set to 8-9 speed.

The next day, the Church's hybridization buffer is drained from the bottle and 100 ml Washing Buffer B pre-heated to 60 °C is added. The hybridization bottle is returned to 10 the incubation oven for 30 min. The rotation speed is set to 8-9 speed. Church's hybridization buffer and Washing Buffer B are radioactive and must be disposed of in a liquid radioactive waste container.

Washing Buffer B is drained from the bottle and 80 ml Washing Buffer 2 pre-heated to 60 °C is added. The hybridization bottle is returned to the incubation oven for 20 min. 15 The rotation speed is set to 8-9 speed.

Washing Buffer 2 is drained from the bottle and 80 ml Washing Buffer 2 pre-heated to 60 °C is added. The hybridization bottle is returned to the incubation oven for 20 min. The rotation speed is set to 8-9 speed.

Filters are removed from the hybridization bottles and washed in a shaking bath for 20 5 min. at 60°C with 2.5 L Washing Buffer 3, shaking slowly, without overwashing.

Filters are soaked in Church's hybridization buffer.

Filters are removed from the bath, spacers are set aside, and placed in individual Kapak, 10" x 12," Sealpak pouches. All air bubbles are removed by rolling with a glass pipette. The pouches are sealed and checked for leaks. A damp tissue removes any 25 remaining solution on the outside of the bag.

Each filter is placed in an autoradiograph cassette at room temperature with an intensifying screen. An overnight exposure at room temperature is usually adequate. Alternatively, the data can be collected using a phosphorimager if available.

Probes may be stripped from the filters (not routinely done) by washing in 1.5 L 30 70 °C Stripping Buffer for 30 min. Counts are checked with a survey meter to verify the efficacy of stripping procedure. This is repeated for an additional 10 minutes, if necessary. Filters should not be overstripped. Overstripping removes BAC DNA and reduces the life of the filters.

Stripping may be incomplete, so it is preferable to autoradiograph the stripped filter 35 if residual probe may confuse subsequent hybridization results. Identification and confirmation of clones

The CTIB and RPCI-23 BAC library filters come as sets of 5-10 filters that have 30- 50,000 clones spotted in duplicate on each filter. Following autoradiography, positive clones appear as small dark spots. Because clones are spotted in duplicate, true positives always appear as twin spots within a subdivision of the macroarray. Using templates and positioning aids provided by the filter manufacturer, unique clone identities are obtained for each positive clone. Once the identities of clones for each probe have been identified, they are ordered from BACPAC Resources (Children's Hospital Oakland - Bacpac Resources 747 52nd St., Oakland, CA 94609) or Research Genetics (ResGen, an Invitrogen Corporation, 2130 Memorial Parkway, Huntsville, AL 35801). To confirm that clones have been correctly identified, each clones is rescreened by PCR using gene specific primers that amplify a portion of the 5' or the 3' end of the gene. In some cases, clones are tested for the presence of both 5' and 3' end amplicons. Other BAC libraries, including those from noncommercial sources may be used. Clones may be identified using the hybridization method described above to filters with arrayed clones having an identifiable location on the filter so that the corresponding BAC of any positive spots can be obtained.

6.1.2. BAC QUALITY CONTROL BY COLONYPCR

1. Perform positive control PCRs with mouse genomic DNA template and primers to be used. 500ng genomic DNA per reaction generally produces a clean and strong band after 25 cycles. The PCR reaction can be optimized by varying the annealing temperatures.

2. Streak out BAC clones on LB-Chloramphenicol plates.

3. Set up a PCR as follows:

Make a master reaction mixture of dNTP, buffer, MgCl₂, water, Taq and primers.

Master Reaction Mixture:

5' primer (10 μM) 0.8μl 3* primer (10 μM) 0.8μl

GibcoBRL 1 OX PCR buffer 2μl

MgCl₂ (50 mM) 0.8μl

DNTP mix (10 mM) 0.4μl

Platinum Taq (GibcoBRL) 0.1 μl Nuclease free H₂0 13.1 μl Total volume = 20 μl

Dispense 20 μl of reaction mix to PCR tubes. Use a 20μl thin tip to transfer a colony from plate to the PCR tube. Pipet up and down a couple of times to dispense the colony into the PCR mixture. Include positive control (genomic DNA) and negative control (no DNA template).

4. Run PCR using the following cycles:

1. 95°C 10 min 2. 94°C 30 sec

3. 55-60°C 30 sec

4. 72 °C 45 sec

5. go back to step 2 for 25 cycles. 6. 72°C 10 min 7. 4°C hold

5. Load all (or 20μl) of the reaction on a gel.

6.1.3. TPF (TIGR PROCIPITATE™ FILTER METHOD BAC ISOLATION PROTOCOL

Materials

1. 96 deep well blocks

2. 96 well micro-titer plates

3. Qiagen Turbofilter 4. Qiagen solutions R1, R2 & R3, with RNAse A

5. Ambion RNAse TI

LB

Appropriate antibiotic

Ice-cold Isopropanol 70% Ethanol

New Brunswick C25 Incubator Shaker w/Lab Line microtiter plate clamps

Troemner tube vortexer

Sorvall RT7 centrifuge w/micro plate carriers Methods

1. Start deep well cultures from fresh cultures. Inoculate (with pipette tips or

5 toothpicks) the wells of a 96 well micro-titer plate with 150 μl of LB with the appropriate antibiotic . Grow overnight at 37 ° C .

2. Using a V&P 96-pin replicator, inoculate 3 x 96 deep well blocks with 1.3 ml LB with the appropriate antibiotic. Seal with Qiagen Air-Pore tape sheets. Grow 18-20 hours at 37 °C and 325 rpm.

10 3. Combine 3 blocks and pellet cells in a centrifuge (Sorvall RT7), by spinning at 3500 rpm for 5 min each block. Dump media into a waste bucket by quick inversion, and then tap on a paper towel, and let media drain for 5 min. 4. Resuspend completely in 300 μl RI with 14 U/ml RNAse A & 100 U/ml RNAse TI , using a tube vortexes (Troemner).

15 5. Add 300 μl R2. Gently invert 5 times, and incubate at room temperature for 5 min.

6. Add 300 μl R3. Gently invert 5 times, and incubate on ice for 5 min.

7. Add 100 μl ProCipitate™ (Ligochem, Inc.). Gently invert a few times over a 5 -minute period at room temperature. Let stand 1 min.

8. Transfer to a Turbofilter in Qiagen vacuum manifold, with a collection deep well 20 block underneath it. Vacuum at 250-350 mm Hg until lysates are completely transferred, about 5-10 min.

9. Add 0.62 ml ice-cold isopropanol and mix gently by inverting 3 times.

10. Incubate on ice or at -20 ° C for 30 min.

11. Pellet DNA by spinning at 3365 rpm and 4°C in the Sorvall RT7 centrifuge for 20 25 min. Decant supernatant gently.

12. Add 0.5 ml 70% ethanol.

13. Pellet DNA by spinning at 3365 rpm and room temperature in the Sorvall RT7 centrifuge for 15 min. Decant supernatant gently, and blot dry.

14. Air-dry completely.

30 15. Resuspend pellet in 30 μl of 1 mM Tris, pH 8.0.

6.1.4. ALKALINE LYSIS MINIPREP

For a 3 ml BAC Miniprep: 1. Centrifuge overnight cultures: 35 a) Decant 1.5 ml culture to microfuge tube and spin 10,000 rpm for 2 min at 4°C. b) Decant supernatant and add 1.5 ml more of remaining culture. c) Spin 10,000 rpm for 2 min at 4°C.

2. Resuspend by adding 250λ PI buffer (Qiagen) and vortexing until no visible pellet pieces remain. 3. Add 250λ P2 buffer (Qiagen), mix by inverting gently 6 times; let stand 4 min (and preferably should not exceed 5 min).

4. Add 350λ N3 buffer (Qiagen), mix by inverting gently 6 times.

5. Immediately spin at top speed (14,000 rpm) for 10 min at 4°C.

6. Decant supernatant to fresh tube; if some precipitate transfers, re-spin at top speed for 10 10 min at 4°C, and re-decant to fresh tube.

7. Add 1ml 100% EtOH, mix gently by inverting a few times.

8. Spin at top speed for 15 min at 4°C.

9. Wash with 500λ 70%EtOH; decant wash.

10. Wash again with 500λ 70% EtOH and centrifuge at top speed for 5 min at 4°C. 15 11. Aspirate as much wash as possible and let stand uncapped to air dry for approximatelylO min.

12. Resuspend in 50λ EB buffer (Qiagen).

6.1.5. MAPPING OF BACS

20 Once BACs for a characterizing gene of interest have been identified, the position of the gene within the BAC must be determined. To design reporter systems that faithfully reproduce the normal expression pattern of the gene of interest, it is preferable that the BAC contain the transcriptional control elements required for wild-type expression. As a first approximation, it can be hypothesized that if the characterizing gene lies near the center of a 5 BAC that is 150-200 kb in length, then the BAC will likely contain the control elements required to reproduce the wild type expression pattern. Thus, it is preferable to use methods for approximating the position of the gene of interest within the BAC.

Fingerprinting of BACs

30 Fingerprinting methods rely on genome mapping technology to assemble BACs containing the characterizing gene of interest into a contig, i.e., a continuous set of overlapping clones. Once a contig has been assembled, it is straightforward to identify 1 or 2 center clones in the contig. Since all clones in the contig hybridize to the 5' end of the gene (because the probe sequence is designed to hybridize at or near the start codon of the

35 gene's coding sequence), the center clones of the contig should have the gene in the central- most position.

A mouse BAC library, e.g., a RPCI-23 BAC library, can be fingerprinted using the methods of Soderlund et al. (2000, Genome Res. 10(11): 1772-87; incorporated herein by reference in its entirety). BACs are fingerprinted using Hindlll digestion digests. Digests are run out on 1% agarose gels, stained with sybr green (Molecular Probes) and then visualized on a Typhoon fluoroimager (Amersham Pharmacia). Gel image data is acquired using the "IMAGE" program (Sanger Center; UK; Sulston et al., 1989, Image analysis of restriction enzyme fingerprint autoradiograms, CABIOS 5(2): 101-106; Sulston et al., 1988, Software for genome mapping by fingerprinting techniques, CABIOS 4 (1): 125-132). Data from "IMAGE" is then passed along to the analysis program "FPC" (fingerprinting contig)(Sanger Center, UK; Soderlund et al., 1997, FPC: a system for building contigs from restriction fingerprinted clones. CABIOS, 13: 523-535; Soderlund et al.,2000, Contigs built with fingerprints, markers and FPC V4.7, Genome Research 10:1772-1787). Using FPC, the data from a publicly available genome database can be queried to determine if the insert of a particular BAC has been fingerprinted and contigged. BAC fingerprint information has been generated by the University of British Columbia Genome Mapping Project (Genome Sequence Centre, BC Cancer Agency, 600 West 10th Avenue, Vancouver, British Columbia, V5Z 4E6) and can be used for assembling BAC contigs. Preferably, contig information from publicly available databases is used to select clones for BAC modification as described above.

If an existing contig cannot be identified from publicly available data, three alternative strategies are used to determine which BAC is the best candidate for recombination:

1) Restriction mapping

In the first step of the BAC recombination process, the shuttle vector (containing the homology region and the key gene coding sequences) integrates into the BAC to form the cointegrate. This process introduces a unique Asc-1 restriction site into the BAC at the site of cointegration. It is possible to map the position of this site, by first cutting the cointegrate with Not-1, which releases the BAC insert (approx 150-200 kb) from the BAC vector. Subsequent digestion with Asc-1 (which cuts very rarely in mammalian genomes), should cleave the BAC insert once, yielding two fragments. The fragment sizes can be accurately resolved using the CHEF gel mapping system (Bio-Rad). If the Asc-1 site is centrally located, then the insert should be cleaved into 2 nearly equal fragments of large size (-75-100 kb each). If the Asc-1 site is located asymmetrically, then the homology region is not centered in the BAC, and thus is not a good candidate for transgenesis. Alternatively, if the size of the smaller fragment falls below a predetermined size (for example 50 kb), then that BAC should be ruled out as a candidate.

2) Fingerprinting

The fingerprinting method described above can also be used to generate additional fingerprint data. This data is used to generate contigs of currently uncontigged BACs from which center clones can be selected. In addition, this data can be combined with data from publicly available databases to generate novel contig information.

3) Alternative mapping method

If neither of the above methods is successful, then the following alternative mapping method is used to roughly localize a gene within a BAC clone. This method takes advantage of the fact that one end of the BAC genomic insert is linked to the SP6 promoter while the other end is linked to the T7 promoter. The alternative mapping method involves the following steps: a) digestion with notl to release the BAC insert b) digestion with another enzyme that cuts no more than 4-7 times in the BAC (in practice, we usually use several different enzymes). Digests are run out on a 0.7% agarose gel. c) The gel is transferred to nylon, hybridized to alkaline phosphatase conjugated T7 oligo probe-develop and the blot is exposed according to the alternative mapping protocol described below. This step identifies that fragment containing the T7 end of the BAC insert. d) Hybridization to alkaline phosphatase conjugated SP6 oligo probe. The blot is developed and exposed according to the alternative mapping protocol described below. This identifies fragment containing the SP6 end of the BAC insert. e) Finally, the blot is hybridized to a gene specific probe. This identifies which fragment contains the gene.

If the gene-hybridizing fragment is different from the T7-or SP6- hybridizing fragments, and the latter two fragments are >30-50 kb, then these data show that the gene must be at least 30-50 kb away from the ends of the BAC, and thus is a likely candidate for transgenesis. Alternative mapping protocol

1. Double digest each BAC DNA with four different rare cutters, together with Notl .

Four lOμl BAC DNA (out of 50μl of alkaline lysis miniprep with 3ml starting culture, roughly lOng pure BAC DNA) per digest are used.

DNA 4μl

10xB(NEB₄) lμl Clal 0.3μl

Notl 0.3 μl ddH₂0 4.4μl lOμl

1. A similar double digest is performed with SacII/Notl (with NEB buffer4), Sall/Notl (Sal buffer), and Xhol/Notl (buffer3). The digests are incubated for '2 hours at 37°C.

2. Loading dye is added (orange dye preferred for Typhoon fluoroimager) to the above entire reaction, and the reactions are loaded into a 0.7% agarose gel. The gel is run . at 80V (for a 7x11 inch large gel) overnight.

3. The gel is stained with Vista green (1 :10,000 dilution in TAE buffer) for 10-20 min and imaged on a Typhoon fluoroimager (Amersham Pharmacia) using the

Fluorescence mode, 526 SP/Green (532nm) setting. The gain and sensitivity are varied until the bands look dark but not saturated. Alternatively, bands can usually be visualized using standard ethidium bromide stain and visualized on a UV lightbox. 4. The gel is transferred into a large container and depurinated with 0.125M HCI for 10 min, rinsed with ddH₂0 once, then neutralized with 1.5M NaCI and 0.5M Tris-HCl (pH 7.5) for 30 min, and denatured with 0.5M NaOH and 1.5M NaCI for 30 min. 5. A capillary wet transfer in 0.5M NaOH and 1.5M NaCI is set up, following the instructions that come with the H+ nylon membrane, and the transfer runs overnight. 6. Next day, the well and lane positions are marked as well as the upper-right corner of the membrane (to keep track of which side is up and the location of the left and right lanes). The membrane is UV crosslinked. Hybridization with alkaline phosphatase (APVconjugated T7 and SP6 probes

T7 and SP7 hybridizations and exposures are done sequentially and are not to be performed together.

5 7. Wash buffer #1 and wash buffer #2 are prewarmed at 37 °C.

8. The membrane is prewet with ddH₂O. The membrane is prehybridized in hybridization buffer at 37 °C for 10 min. For the prehybridization and hybridization steps, exactly 50 μl of buffer is used per 1.0 cm² of membrane.

9. During the prehybridization step, the probe is diluted to a 2 nM final concentration 10 in hybridization buffer. The volume is calculated as done in step 8. The correct probe concentration is crucial. The tubes containing these solutions are incubated at 37 °C during the prehybridization step.

10. After 10 min, all of the prehybridization buffer is removed and the hyb buffer containing probe is added. A hybridization step is done at 37° C for 60 min.

15

The membrane should not dry out during the following wash, detection and film exposure.

11. 100 ml of prewarmed wash buffer 1 is poured into a container. The membrane is 20 transferred into the container, swirled gently for 1 min. The buffer solution is poured out and 150-200 ml of wash buffer 1 is added and the membrane is washed for 10 min with gentle agitation.

12. Buffer 1 is removed and prewarmed buffer 2 is added. Washes are done as in step 11 for another 10 min.

25 13. Washes with 2xSSC are done for 10 min at RT. The CSPD chemiluminescent substrate is removed from refrigeration and allowed to warm up to room temperature (RT).

14. The substrate buffer is prepared and 50μl is used per 1.0 cm² of membrane.

15. The membrane is rinsed 2 times for 5 min. each in assay buffer. The membrane is 30 incubated in substrate buffer inside heat-sealable bags at RT for 10 min. while manually agitating the bag to ensure that the membranes are covered with substrate buffer.

16. The membrane is removed from the substrate buffer and placed into a seal bag and exposed to KODAK® film (Eastman Kodak Co.) immediately.

35 Southern hybridization with gene specific probes

17. Probes are labeled using purified PCR product as a template with the Ready-Prime kit. The prehybridization and hybridization steps are carried out as in standard Southern blot hybridization. The membranes are exposed at room temperature or at 37°C. Alternatively, one can probe with a gene-specific overgo probe using the

BAC screening protocol as described above.

Band identification

18. The two blots are aligned with the original DNA gel. Positive bands are identified for T7/SP6 and the gene-specific probe.

1. Wash buffer 1: 2x SSC 1% (w/v) SDS 2. Wash buffer 2:

2xSSC l% Triton-X-100

3. Substrate buffer:

5 ml of assay buffer 30 μl of CSPD chemiluminescent substrate

4. Hybridization buffer lxSSC 1%SDS 0.5% BSA 0.5% PVP

0.01% NaN₃

5. Assay buffer

0.96 ml ofDEA 0.1 ml of lM MgCl₂ 0.21 ml of 2M NaN₃ add ddH₂0 to 80ml adjust to pH 10.0 with dilute HCI add ddH₂0 to make final 100 ml 6.2. CLONING HOMOLOGY BOXES

Methods for introducing the key gene coding sequences into the characterizing gene sequences on the BAC through homologous recombination in bacteria are described below. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. The construct comprises at least a portion of the characterizing gene with a desired genetic modification, e.g., insertion of the key gene coding sequences and will include regions of homology to the target locus, i.e., the endogenous copy of the characterizing gene in the host's genome.

A homologous recombination shuttle vector is prepared in which the key gene is positioned next to characterizing gene sequences to allow for homologous recombination to occur between the exogenous gene carried by the shuttle vector and the characterizing gene sequences on the BAC. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the characterizing gene on the BAC. Homology boxes are these regions of DNA and are used to direct site specific recombination between a shuttle vector and a BAC of interest. In one embodiment, the homologous regions comprise the 3' portion of the characterizing gene. In preferred embodiments, the homologous regions comprise the 5' portion of the characterizing gene, more preferably to target integration of the key gene coding sequences in frame with the ATC of the characterizing gene sequences. PCR is used for cloning a homology box from genomic DNA or BAC DNA. The homology box is cloned into the shuttle vector that is used for BAC recombination, as described below.

Design of PCR primers

Using Primer3 program (Massachusetts Institute of Technology Cambridge, MA; Steve Rozen, Helen J. Skaletsky, 1998, Primer3), a Ascl site is added in the 5' forward primer and a Smal site is added in the 3' reverse primer.

Using the Primer3 default temperature calculations, primers are designed so that they have T_ms of 57-60°C and so that the amplicons are between 300 and 500 bp in length. If a 5' UTR sequence of the characterizing gene sequence is available, amplicons are designed against this sequence. If the 5' UTR sequence is not available, then homology boxes are designed to include the 3' UTR or the 3' stop codon, or any other desired region of the characterizing gene.

PCR reactions PCR reactions are performed with the following reagents: 1.0 μl Mouse genomic DNA or BAC having characterizing gene insert (500ng/μl)

1.0 μl Forward primer 10 pmol/μl

1.0 μl Reverse primer 10 pmol/μl

0.5 μl 10 mM dNTP mix 2.5 μl 10XPCR buffer without MgCl₂

2.0 μl 25mM MgCl₂

0.125μl Taq AmpliGold (Perkin Elmer)

15.875μl H₂O

DNA template for PCR should be from the BAC to be modified, or genomic DNA from the same strain of mouse from which the BAC library was constructed. The homology boxes must be cloned from the same mouse strain as the BACs to be modified.

Preferably, Pfu DNA polymerase (Stratagene) is used. This reduces errors introduced into the amplified sequence via PCR with Taq polymerase. Total volume is 25 μl.

1 drop (approximately 25 μl) of mineral oil is added to the PCR tubes before running the PCR reactions. PCR reactions are run on a thermal cycler using the following program:

1. 95°C 10 min

2. 94 °C 30 sec

3. 55-60°C 30 sec (annealing temperature is determined based on the Tm of the primers used)

4. 72 °C 45 min 5. go back to step 2 for 40 cycles.

6. 72°C 10 min

7. 4°C hold

Analysis of PCR product 5μl of the PCR reaction is run on 0.8% agarose gel. The bands are visualized with

EtBr staining. Good PCR reactions produce a single product at the expected size. The yield of one PCR reaction is between 50 to 200 ng. Cloning of PCR product

A TOPO-TA cloning kit (Invitrogen) may be used to clone the PCR product. Ligation reactions are carried out at room temperature for 3 min with the following reagents:

lμl TOPO vector

2-4 μl PCR reaction aliquot (depending on the yield of the reaction, no purification is needed if only a single band is produced) 0-2 μl ddH₂O Optional: 1 μl salt solution (provided in the TOPO kit)

2μl of the ligation reaction is transformed into Top 10 cells (Invitrogen) following the manufacturer's protocol.

A blue-white selection is used (spreading IPTG and X-gal solutions on the LB-Amp plates prior to plating the transformation mixture).

Analysis of TOPO-PCR clones

Four white colonies are picked to start overnight 2ml LB-Amp cultures. The DNA is extracted using a Qiagen miniprep kit. 2μl (1/25) of the miniprep DNA is digested with EcoRI, which excises the inserts from the TOPO vectors. The identity of the clones is confirmed by sequence analysis using either T3 or T7 primers.

6.3. HOMOLOGOUS RECOMBINATION BETWEEN A SHUTTLE VECTOR AND THE BAC

6.3.1. PREPARATION OF COINTEGRATES OFABAC AND A SHUTTLEVECTOR(ALTERNATIVE 1)

Preparation of cointegrates of the BAC and a shuttle vector may be prepared as follows. A shuttle vector containing IRES, GFP and the homology box (FIGS. 12 and 13; see PCT publication WO 01/05962), containing the key gene of interest is transformed into competent cells containing the BAC of interest by electroporation using the following protocol. A 40-μl aliquot of the BAC-containing competent cells is thawed on ice, the aliquot is mixed with 2 μl of DNA(0.5μg /μl), and the mixture is placed on ice for 1 minute. Each sample is transferred to a cold 0.1 cm cuvette. A Gene Pulser apparatus (Bio-Rad) is used to carry out the electroporation. The Gene Pulser apparatus is set to 25 μf, the voltage to 1.8KV and pulse controller to 200Ω. lml SOC is added to each cuvette immediately after conducting the electroporation. The cells are resuspended. The cell suspension is transferred to a 17x100mm polypropylene tube and incubated at 37° C for one hour with shaking at 225 RPM.

The 1 ml culture is spun off and plated onto one chloramphenicol (Chi) (12.5μg/ml) and ampicillin (Amp) (50μg/ml) plate and incubated at 37 °C for 16-20 hours.

The colonies are picked and inoculated with 5ml LB supplemented with Chi (12.5μg/ml) and Amp (50 μg/ml), and incubated at 37°C overnight. Miniprep DNA from 3 ml of culture by alkaline lysis method described supra. Cointegrates for each clone are identified by Southern blot. Using a homology box as a probe in Southern blot analysis, the cointegrate can be identified by the appearance of an additional homology box that is introduced via the recombination process.

The resolved clones (i.e., clones in which the shuttle vector sequences have been removed, leaving the key gene sequences) from the modified BACs are screened and each colony of cointegrate from the Chi/ Amp plates is picked and used to innoculate 5ml of LB + Chi (12.5μg/ml) and 6% sucrose, and incubated at 37 °C for 8 hours.

The culture is diluted 1 :5000 and plated on the agar plate with Chi (12.5 μg/ml) and 6% sucrose and incubated at 37 °C overnight. Five colonies per plate are picked and inoculated with 5ml of LB + Chl(12.5μg/ml) only and incubated at 37 °C overnight. DNA from those cultures are miniprepped by alkaline lysis method known in the art. The resolved BACs are screened by Southern blot.

6.3.2. PREPARATION OF COINTEGRATES OF A BAC AND A SHUTTLE VECTOR (ALTERNATIVE 2

Alternatively, preparation of cointegrates of the BAC and a shuttle vector may be prepared as follows.

Clone the shuttle vectors for each BAC:

1. Transform pLD53PA shuttle vector (e.g., FIGS. 12 and 13) into pir2 cells (Invitrogen), and amplify DNA through a Qiagen column.

2. Prepare 100 μg (enough for 1000 litigation reactions) of Ascl/Smal digested shuttle vector by incubation overnight in appropriate amounts of the enzymes. Purify digested vector, test its aliquot in ligation to determine background of undigested or single digested shuttle vector, redigest it until the disappearance of the background. Aliquot and store this stock of predigested vector for use in "A box" cloning.

3. PCR amplify (using an enzyme that does not leave an overhang, such as Pfu DNA polymerase) a 300-500 bp "A box" homology regions from C57bl/6J genomic DNA using primers to the gene of interest (see Section 6.2, cloning homology boxes). Use of the 5' primer results in incorporation of an Ascl site. Digest products overnight with Ascl, purify digested fragments by gel electroelution.

4. Ligate the digested shuttle vector (100 ng) with each individual fragment (25 ng), transform into pir2 cells (Invitrogen) and plate the transformed cells in LB Amp (30 μg/ml) plates.

5. Pick a few colonies individually and test for correct insertion by PCR. Prepare DNA for each positive shuttle vector and confirm these clones with restriction enzymes by comparing the digestion pattern with the vector.

During this step, the A box should not contain an internal Asc I site. If the A box contains an Ascl site, then incorporate an Mlul site using the 5' primer and use that enzyme for cloning.

Since this shuttle vector contains a R6kr DNA replication origin, which can only replicate in bacteria expressing the pir replication protein, use of pir2 cells (Invitrogen) is preferable.

Prepare competent cells for electroporation:

1. Inoculate 200 ml of LB with 1/1000 volume of a fresh overnight culture.

2. Grow cells at 37°C with vigorous shaking to OD600=0.5-0.8 (To reach an OD600 of 0.7 usually takes about 5-6 hours).

3. Harvest cells by centrifugation in a cold rotor at 3000 rpm for 10 min (in a Beckmann J6-MI centrifuge) at -5 °C.

4. Resuspend pellets in equal volume of 10% cold glycerol. Centrifuge as in step 3.

5. Repeat ltime. 6. Decant the supernatant as much as possible. 7. Gently resuspend cells to a final volume of 400 μl with 10% cold glycerol.

8. Dispense 40 μl aliquots into sterile tubes and freeze.

Prepare the cointegrates for BACs

1. Transform pLD 53 -modified shuttle vector (PLD53PA) containing the gene of interest into BAC competent cells by electroporation: Thaw 40 μl of the BAC containing competent cells on ice, mix it with 2 μl of DNA (0.5 μg/μl), and place the mixture on ice for 1 minute. Transfer each sample to a cold 0.1cm cuvette. Use a Gene Pulser apparatus to carry out the electroporation. Set the Gene Pulser apparatus at 25 μF, the voltage to 1.8KV and pulse controller to 200Ω.

2. Add 1ml of SOC to each cuvette immediately following the electroporation. Resuspend the cells, transfer the cell suspension to a 17x100mm polypropylene tube, and incubate at 37° C for one hour with shaking at 225 rpm.

3. Select those transformed cells using 5 ml of LB supplemented with chloramphenicol (12.5 μg/ml) and ampicillin (30 μg/ml), and incubate at 37 °C overnight.

4. Dilute the overnight culture 1 to 1000 and grow in 5 ml of LB with chloramphenicol (12.5μg/ml) and ampicillin (50μg/ml) at 37 °C for about 14 hours. Dilute this culture 1 to 5000 and grow in the same media at 37° C for 8 hours. Make a series of dilution, and place them on chloramphenicol(12.5μg/ml) and ampicillin (lOOμg/ml) plates, incubate at 37 °C overnight.

5. Pick up four colonies per plate and inoculate each colony with 5 ml of LB supplemented with chloramphenicol (12.5μg/ml) and ampicillin (lOOμg/ml), streak the same colony onto chloramphenicol/ampicillin (chl/amp) master plates, grow overnight at 37 °C. Miniprep DNA from 3 ml of cultures by the alkaline lysis method. Identify proper cointegrates for each clone by PCR or by Southern blot.

Screen the resolved clones from the modified BACs.

1. Pick up each colony of cointegrate from the (chl/amp) master plates, inoculate each colony with 5 ml of LB supplemented with chloramphenicol(12.5μg/ml) and 6% sucrose, and incubate at 37 °C for eight hours. 2. Dilute the culture 1 to 5000 and plate them on the agar plate with chloramphenicol (12.5μg/ml) and 6% sucrose, incubate at 37 °C overnight.

3. Pick up colonies and plate them on two agar plates, incubate the master plate at 37 °C directly. Expose the second plate with UV light for 30 seconds and incubate at 37 °C overnight to check the deletion of RecA gene (second recombination). After the resolution, colonies that have lost the excised recombination vector including SacB and RecA genes become UV light sensitive. Therefore, the UV light experiment helps to screen out the false positive clones.

4. Following the protocol for UV screening of resolvant BACs disclosed hereinbelow, pick up the colonies that are sensitive to UV light and inoculate each colony with 3ml of LB supplemented with chloramphenicol(12.5μg/ml) only. Streak the same colony onto a Chi master plate, incubate a 37 °C overnight. Miniprep DNA from those cultures by the alkaline lysis method. Screen the resolved BACs by PCR or by

Southern blot.

UV Screening of Resolvant BACS

Materials: 96-well block of resolvant cultures 96-pin replicator

4 LB-agar-12.5 μg/ml chloramphenicol plates STRATALINKER® UV Crosslinker (Stratagene) Troemner Tube Vortexer

Protocol:

I. Stamping Replica Plates

1. Dry the plates by placing them in the 37° C incubator upside down and slightly ajar. Plates should be dried until there is no moisture on the LB-agar or lid. Moisture could cause culture spots to run together and must be removed.

2. Because of culture precipitation, the 96-well culture block must be vortexed before being replicated. Place block in Troemner Vortexer and vortex briefly. Observe culture for uniform appearance. Repeat if necessary.

3. Because of variability in culture densities, a series of UV-exposed plates must be prepared. Label plates: Control, 10 mJ, 15 mJ, and 20 mJ. Also, place a spot on the back of each plate to orient it: pin Al will be placed on this spot (i.e., the colony on this spot will correspond to the culture in well Al).

4. Flame sterilize the 96-pin replicator.

5. Insert replicator into the 96-well culture block and remove carefully. There will be small volumes of culture on the tips of the replicator pins. Carefully position the replicator over the large LB plate to be stamped. Align pin Al over its orienting spot. Gently rest the replicator on the LB-agar surface. Try not to break the surface with the pins. Remove the replicator by pulling it straight off the plate, being careful not to smear the spots together.

6. Cover the plate and allow it to sit on the bench for a few minutes until spots appear dry.

II. UV-Expose Plates

Using an appro jpprriiaattee UUVV ccrroosssslliinnkkeerr ssuucchh aass tthhee SSTTRRAATALINKER® UV Crosslinker, expose the plates without covers to 0 mJ, 10 mJ, 15 mmJJ aanndd 2200 mmJJ rreespectively. I Innccuubbaattee n pllaatteess a att 3 ^"377°°C C n ovveerrnniiVghhtt..

III. Optimum Killing Curve

Select the plate with the lowest effective dose. The colonies on this plate will have grown well or not at all. A plate in which some colonies look "sick" (i.e., lack an even, round morphology) has been overdosed and will have false positives. Choosing the plate with the lowest effective dose will select against false positives and will insure that cells that did not grow are recA-.

Construct verification In summary, to ensure that a cointegrate is formed properly, PCR or Southern blotting is performed to ensure that the first step of recombination has occurred properly. In addition, this step may be verified to determine that the key gene sequences have been juxtaposed adjacent to the characterizing gene sequences.

After the shuttle vector is recombined into the BAC to form a cointegrate, the vector sequences are removed in a resolution step, as described in WO 01/05962, herein incorporated by reference in its entirety. After cointegrates are resolved, Southern blotting and PCR are used to confirm that resolution products are correct, i.e., the only modification to the BAC is that the reporter has been inserted at the homology box. 6.4. CHEF MAPPING

The following protocol describes the CHEF gel mapping system (Bio-Rad). The protocol is run according to the manufacturer's instructions in the Bio-Rad CHEF gel mapping system reference manual. Restriction mapping is described in general in Section 6.1.5.

Parameters to be used in CHEF Mapping:

0.5XTBE

14°C 1% pulse field agarose

6 V/cm angle=120 degrees, int. sw. time = 0.4 sec fin. sw. time = 40 sec ramping factor a = linear, run time = 16 hrs calibration factor = no change

1 :10,000 dilution of Vistra Green in the gel (or alternatively, post-stain the gel with Vistra

Green)

DNA used:

Unmodified BAC (from 3ml prep total 50ul): 3ul in three digests (Notl, Ascl, Notl/ Ascl double)

Col BAC (from 96 prep total 30ul): 5ul in three digests (Notl, Ascl, Notl/ Ascl double) NEB low range PFG marker: small piece of agar to put into the well

If a Southern blot is performed: transfer: 1.5 hr in 0.25 M HCI

1 hr in 0.5 M NaOH/ 1.5 M NaCI

Set up wet transfer overnight in the above NaOH/NaCl buffer. The next day, mark the orientation of the membrane and UV crosslink.

Hybridization with AP-T7 or AP-SP6 probe: Prehybridization: in small roller bottle, at 37°C for lhr, 50 ul of buffer/ 1 cm² of membrane. Hybridization buffer: 1 X SSC, 1% SDS, 0.5% BSA, 0.5% PVP, 0.01% NaN3 Hybridization: add fresh, warmed hybridization buffer (50 ul of buffer/1 cm² of membrane), and add in the probe at 2 nM final concentration. Run the hybridization at 37 °C overnight. Wash in: 2XSSC/1% SDS, 37 °C, 30 min

2XSSC/1% triton X-100, 37°C, 30 min 2XSSC, room temperature, 10 min

CSPD substrate buffer (see below), room temperature, 5 min (minimal buffer is enough) 0.96 ml of DEA, 0.1 ml of IM MgCl₂, 0.2 ml of 2 M NaN₃, adjust pH to 10.0 with diluted HCI, and final vol. = 100 ml. AP reaction: prepare CSPD substrate (Roche) in substrate buffer (50 ul of buffer/ 1 cm₂ of membrane). Dilute it 1 :100 to use.

Incubate the membrane with the substrate inside heat-sealable bag at RT for 10 min. Manually agitate the bag to ensure contact with the buffer.

Remove the substrate buffer, and expose to film, at room temperature for preferably 1-2 hr.

6.5. ISOLATION AND PREPARATION OF BAC DNA FOR INJECTION

BAC DNA is preferably purified using one of the two following alternative methods and is then used for pronuclear injection or other methods known in the art to create transgenic mice. The injection concentration is preferably 1 ng/μl.

6.5.1. MAXIPREP BY ALKALINE LYSIS FOR BACS (ALTERNATIVE 1 1. 250 ml cultures are centrifuged to pellet bacteria.

2. The pellet is resuspended in PI buffer (RNase-free, Qiagen), 20 ml, by pipetting.

3. Cells are lysed for 4-5 min in P2 buffer (Qiagen), 40 ml, by inversion or swirling.

4. 20 ml cold P3 buffer is added, mixed briefly, and incubated on ice for 10 min.

5. The pellet is spun down on a swing bucket rotor at maximum speed for 20 min. 6. The supernatant is filtered through four layers of cheesecloth into clean 250 ml tubes.

7. 2x volume of 95% EtOH is added and the suspension is spun on a swing bucket rotor at maximum speed for 20 min.

8. The pellet is resuspended. 9. DNA is precipitated with 5ml 5M LiCI (final cone. 2.5M), on ice for 10 min. 10. Precipitate is spun at 4000 rpm for 20 min by a Sorval tabletop centrifuge.

11. The supernatant is transferred to fresh 50 ml Falcon tubes.

12. lx volume isopropanol is added.

13. The precipitate is spun at 4000 rpm for 20 min on Sorval tabletop centrifuge.

14. The pellet is washed with 1 ml 70% EtOH.

15. The DNA is resuspended in 500λ TE.

16. 5λ RNase, DNAse-free. (Roche) is added to the DNA.

17. RNase A is added to a final concentration of 25μg/ml. (Qiagen).

18. The DNA is incubated for 1 hr at 37°C.

10 19. The DNA is phenol extracted 10 min on ADAMS™ Nutator Mixer (BD Diagnostic Systems).

20. 250 μl NH₄OAc +750 μl isopropanol is added.

21. Precipitate is spun for 10 min at maximum speed on Eppendorf at 4°C

22. The pellet is resuspended in 50 μl TE.

15

The DNA is purified for injection by either treatment with plasmid safe endonuclease (Epicenter Technologies) or by gel filtration using Sephacryl S-500 column or CL4b Sepharose column (both from Amersham Pharmacia Biotech).

20

6.5.2. PURIFICATION OF BAC DNA BY CESIUM

CHLORIDE/ETHIDIUM BROMIDE EQUILIBRIUM CENTRIFUGATION (ALTERNATIVE 2~)

I. Grow and Concentrate Cells

25

1. Inoculate 5 ml LB medium containing 12.5 ug/ml chloramphenicol with an isolated colony of E. coli containing the desired BAC. Grow at 37°C with vigorous shaking overnight.

2. Inoculate 1 L LB medium containing 12.5 ug/ml chloramphenicol with 1 ml of overnight culture. Grow at 37 °C with vigorous shaking until culture is saturated

30 (16-20 hours).

3. Harvest cells by centrifuging 10 minutes at 6000 x g at 4°C.

4. Resuspend pellet in 8ml glucose/Tris/EDTA solution and transfer to 250 ml centrifuge bottle.

35 II. Lyse the Cells

5. Add 2 ml of 25 mg/ml hen egg white lysozyme in glucose/Tris/EDTA solution (Ausubel et al, 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y.). Mix with pipette and allow it to stand 10

5 minutes at room temperature.

6. Add 40 ml freshly prepared 0.2 M NaOH/1% SDS and mix by stirring gently with a pipette until solution becomes homogeneous and clears. Let stand 10 minutes on ice. Solution will become viscous.

7. Add 30 ml of 3 M potassium acetate solution and again stir gently with a pipette 10 until viscosity is reduced and a large precipitate forms. Let stand 10 minutes on ice.

8. Centrifuge 10 minutes at 20,000 x g at 4C.

III. Precipitate BAC DNA

9. Decant the supernatant into a clean 250 ml centrifuge bottle. If supernatant is 15 cloudy or contains floating material, repeat centrifugation (Step 8) before proceeding.

10. Add 0.6 volume isopropanol, mix by inversion, and let stand 10 minutes at room temperature.

11. Recover nucleic acids by centrifuging 10 minutes at 15,000 x g at room temperature. 20 12. Wash pellet with 2 ml of 70% ethanol. Centrifuge 5 minutes at 15,000 x g at room temperature to collect pellet. Aspirate ethanol and dry pellet under vacuum.

IV. Purify BAC DNA by Cesium Chloride/Ethidium Bromide Equilibrium Centrifugation

25 13. Resuspend pellet in 4 ml TE buffer. Transfer to test tube. Add 4.4 g CsCl, dissolve, and add 0.4 ml of 10 mg/ml ethidium bromide.

14. Ethidium bromide will form a complex with the remaining protein to form a deep red flocculent precipitate. Centrifuge 5 minutes at 2000 x g. This will cause to the complex to form a disc at the top of the solution. Carefully transfer the- solution

30 beneath the disc to a fresh tube.

15. Transfer the solution to a 6 ml Sorvall Ultracrimp tube. Fill any remaining volume in the tube with a 1 g/ml cesium chloride in TE. Seal tube.

16. Band BAC DNA by overnight centrifugation in a Sorvall ultracentrifuge. Centrifuge parameters at Renovis: Rotor=Sorvall 70V6, temp=25, speed=60,500, acc=5, dec=7

35 17. Carefully remove the tube from the centrifuge. Visualize the BAC DNA band by side illumination with a low-intensity shortwave UV light. Insert a 20-G needle into the top of the tube. Recover the BAC DNA band: insert a 3 ml syringe with a 20-G needle bevel side up into the side of the tube just below the BAC DNA band.

5 Carefully direct the needle to the bottom of the BAC DNA band and remove it by gently pulling the syringe plunger out.

18. Extract the band with an equal volume of water-saturated isobutanol. Ethidium will partition to the organic phase. Let phases separate by waiting a minute or so. Repeat until there is no more pink in aqueous phase.

10 19. Add 2 volumes TE + 6 volumes EtOH (Usually the band is' 1 ml, so add 2 ml TE+6 ml EtOH) Spin 10 x kg 15'. Wash Pellet with 70%EtOH.

20. Resuspend pellet in 200 λ TE.

21. Add RNase to a final concentration of 1 Omg/ml (e.g. , 2λ of a 1 : 100 dilution of Qiagen RNase lOOmg/ml). Incubate at 37°C for 1 hour.

15 22. Phenol/chloroform extract (no vortex, gentle agitation)

23. Precipitate supernatant (20λ 3M NaAc+400λ EtOH). Wash pellet with 70% EtOH

24. Resuspend in lOOλ injection buffer-overnight at 4 degrees

25. The next day, make sure pellet is resuspended, filter through a 0.45 micron filter.

26. Quantitate by OD

20 27. Quantitate by running H3 digests of BAC on CHEF gel comparing to known amounts of lambda H3 run on the same gel.

6.6. PRODUCTION OF TRANSGENIC MICE

The following protocol discloses transgenic production in FVB strain mice: 25

1. 4 week old FVB female mice are superovulated using 5 IU PMSG (11 :00AM) followed 47 hours later by 7.5 IU HCG (10:00AM) and mated to FVB male studs after, the HCG injection.

30 2. The next morning, the FVB female egg donors are checked for copulation plugs (8:00AM), sacrificed via cervical dislocation, the oviducts harvested and the embryos are isolated from the oviducts for subsequent microinjection. Microinjection generally takes place between 10:00 AM and 2:00PM. The injection concentration is preferably lng/μl.

35 3. Injected embryos are transferred into the oviducts of ICR outbred strain pseudopregnant female mice. 20-25 eggs are transferred unilaterally into an oviduct. 19 days later the pups are bom.

4. Birthed pups are tail clipped (approximately 0.5cm of tail is obtained) at 7-10 days of age.

5. DNA is extracted from the tail biopsy (see tail biopsy protocol disclosed hereinbelow in Section 6.7).

6. PCR is performed as disclosed hereinbelow (Section 6.8).

6.7. TAIL DNA ISOLATION FOR PCR ANALYSIS

1. Digest tail clippings overnight at 55°C in 490λ sterile filtered lysis buffer + 1 Oλ proteinase K ( 1 OOmg) .

Lysis buffer: 100 mM Tris HCl pH 8.5 5 mM EDTA 0.2% SDS 200 mM NaCI

2. Spin samples down for 10 minutes at 14K rpm to remove hair.

3. Transfer contents to a newly labeled tube (approximately 500λ) by pouring. No pipette manipulation is necessary.

4. Add an equal volume (500λ) of 100% isopropanol. Gently shake tubes until DNA precipitates. Do not vortex.

5. Spin samples down for 5 minutes at 14K rpm.

6. Pour off isopropanol, being careful not to lose the pellet.

7. Wash 1 time in 1 ml 70% EtOH at room temperature. 8. Dab tubes dry using a tissue and allow tubes to air-dry for 5-10 min. An overnight dry is not necessary.

9. Resuspend pellets in 300λ Lo TE. Briefly vortex and place in a 65°C incubator with agitation to aid in resuspension. The length of time needed to completely resuspend pellets may vary but usually falls within the range of 20 min - 1.5 hrs. Periodically check the samples until the desired suspension is attained.

10. Randomly O.D. 10% of the samples to check for concentration uniformity (i.e., 5 of 50 samples). The samples are now ready to be analyzed by PCR.

6.8. PCR ANALYSIS PROCEDURE

1. Use a Perkin-Elmer 0.5ml PCR tube for each sample.

2. Using a cellulose acetate plugged pipet tip, add 400ng of template DNA to each tube in a volume of lul (always change tips). Set these samples aside and make up the PCR premix.

3. Use Template Free Pipets to make up this premix. Make up a PCR premix and add 49 ul of premix to each sample tube. Listed below is an example of what a typical PCR reaction contains; amounts of each component may vary from experiment to experiment:

PCR Mix per tube

1 OX PCR Buffer 5. Oul/reaction

1.25mM dNTP's 5. Oul/reaction OR

25mM dNTP's 0.3ul/reaction

3' primer (20uM) 0.5ul/reaction

(Approximately lOOng) 5' primer (20uM) 0.5ul/reaction

(Approximately lOOng)

Taq Polymerase (5U/ul) 0.25ul/reaction

Sterile H₂O Amount will vary

Total volume 49ul/tube *

*Total reaction volume is 50ul in the above example. If the total volume of the DNA required for the reaction is not lul then adjust the amount of H₂O accordingly.

4. Run samples on the appropriate file in the PCR machine (Applied Biosystems

GeneAmp PCR System 9700).

GFP primers: egfpl32F CCTGAAGTTCATCTGCACCA (SEQ ID NO:2)

egfp 61 Or TGCTCAGGTAGTGGTTGTCG (SEQ ID NO:3)

Reaction Volume: 25 μl Amount of each primer per reaction: 5' primer: 5-10 pmol 3' primer: 5-10 pmol

Amount of source DNA: 100 ng

Amount of fragment used in one copy control: 0.7 pg

PCR Reaction Kit: Invitrogen Thermal Ace Kit E0200

PCR Cycles:

Step 1 = 3 min at 95° C (hot start) Denaturing Temperature: 95 °C Denaturing Time: 30 sec Annealing Temperature: 58 °C Annealing Time: 30 sec Extension Temperature: 74 °C Extension Time: 45 sec Number of Cycles: 30

The following precautions are preferably taken when doing PCR experiments according to the methods described herein:

Always use plugged tips.

Change gloves frequently.

Use PCR pipets when making PCR premix. Avoid having any DNA template near when making PCR premix.

Analysis of GFP-PCR results

The presence of positive GFP PCR product indicates that the transgenic mouse test carries the gene of interest.

6.9. CREATION OF TRANSGENIC MOUSE LINE EXPRESSING A 5HT6 RECEPTOR BAC

This is an example of making a transgenic mouse line, expressing the 5HT6 receptor BAC, according to the methods of the invention disclosed hereinabove. A transgenic mouse line expressing the 5HT6 receptor BAC was constructed as follows.

An overgo probe was made for the 5HT6 gene as described in Section 6.1 using the following oligos.

5HT6 overgo sequences:

5HT6-Ova

TGCGCAACACGTCTAACTTCTTCC (SEQ ID NO:4)

5HT6-Ovb GTGAAGAGCGACACCAGGAAGAAG (SEQ ID NO:5)

Four BAC clones were identified using the overgo probe in a screen of CITB filters (see Section 6.1). PCR (Section 6.8) was used to verify BACs as containing the 5HT6 gene.

The following oligos were used to obtain the A box: "A" box primers used to amplify 5HT6 A box fragment:

AF134158/5HT6.AscI.fl

GTCTGGCGCGCCAATGGCTGGGATACTGTAATAGCA (SEQ ID NO:6)

AF134158/5HT6.SmaI.rl GTCTCCCGGGAATCTTGACCTGGTCAGTTCATG (SEQ ID NO:7)

The sequence of this A box for the 5HT6 gene was determined to be:

"A" box sequence:

TGGCTGGGATACTGTAATAGCACCATGAACCCTATCATCTATCCCCTCTTCATG

CGGGACTTCAAGAGGGCCCTGGGCAGGTTCGTGCCGTGTGTCCACTGTCCCCCG

GAGCACCGGGCCAGCCCCGCCTCCCCCTCCATGTGGACCTCTCACAGTGGTGCC AGGCCAGGCCTCAGCCTGCAGCAGGTGCTGCCCCTGCCTCTGCCACCCAACTC A

GATTCAGACTCAGCTTCAGGGGGCACCTCGGGCCTGCAGCTCACAGCCCAGCTT

TTGCTGCCTGGAGAGGCGACCCGGGACCCCCCGCCACCCACCAGGGCCCCTAC

TGTGGTCAACTTCTTCGTCACAGACTCTGTGGAGCCTGAGATACGGCAGCATCC

ACTTGGTTCCCCCATGAACTGACCAGGTCAAGA (SEQ ID NO:8)

The A box was cloned into a shuttle vector such that recombination with the 5HT6 gene in a BAC would place an IRES-EGFP sequence downstream of the stop codon in the

5HT6 gene coding sequence.

Three different BACs were used to make cointegrates (see Section 6.3.2). DNA from pμtative cointegrates was prepared using the methods disclosed in hereinabove (see

Sections 6.1 and 6.4).

A DNA fingerprint (performed as disclosed in Section 6.1.5) is shown in FIG. 1A.

A corresponding Southern blot, shown in FIG. IB, was used to verify duplication of A boxes in cointegrate clones. CHEF mapping (see Section 6.4) was used to determine that one of the BACs was constructed such that one of the BAC clones had a sufficiently large DNA fragment upstream of the 5HT6 start site (FIG. 2).

Resolution of this cointegrate was performed as described hereinabove (see Section

6.3); the DNA fingeφrint and corresponding Southern blot are shown in FIG. 3. Two of the four putatives tested contained only one copy of EGFP, verifying resolution. After preparing large amounts of the BAC DNA for injection (Section 6.5), transgenic animals were constructed (Section 6.6), and genotyped for the presence of GFP sequences genotyped for the presence of GFP sequences (Sections 6.7 and 6.8). Founders were bred in order to obtain progeny containing the transgene (and verify that a line had indeed been established). Again, PCR (Section 6.8) was used to genotype FI animals.

Sections of brain tissue showed that the transgene was indeed expressed in subsets of neurons in the transgenic animals (FIGS. 4 and 5).

6.10. CREATION OF TRANSGENIC MOUSE LINE EXPRESSING A 5HT2A RECEPTOR BAC

This is an example of making a transgenic mouse line expressing a 5HT2A receptor BAC, according to the methods of the invention disclosed hereinabove.

A transgenic mouse line expressing the 5HT2A receptor BAC was constructed as follows. An overgo probe was made for the 5HT6 gene as described in Section 6.1 using the following oligos.

5HT2A overgo sequences:

5HT2A-Ova GTCTCTCCACACTTCATCTGCTAC (SEQ ID NO:9)

5HT2A-Ovb

GTCTAAGCCGGAAGTTGTAGCAGA (SEQ ID NO: 10)

Seven BAC clones were identified using the overgo probe in a screen of CITB filters

(see Section 6.1). PCR (Section 6.8) was used to verify BACs as containing the 5HT2A gene.

The following oligos were used to obtain the A box:

"A" box primers used to amplify 5HT2A A box fragment:

5HT2A-5'AscFl:

GTCTGGCGCGCCAACTCGTTTGGATCTCATGCTG (SEQ ID NO: 11)

5HT2A-5'SmaRl : GTCTCCCGGGAAAAGCCGGAAGTTGTAGCAGA (SEQ ID NO: 12)

The sequence of this A box for the 5HT2A gene was determined to be:

"A box" sequence:

CTCGTTTTGGATCTCATGCTGTTTTAACTTTGTGATGGCTGAACTCTTGAAAGCA GCATATCCAACCCGAGAATTGGCTGAAAGATTCTCACCGGATACAAAACTTTTC TTCCTTAACCAGGAACACGTTTGTGTCTCCAAATGCTCCACACTGCTTTTTTTGC CTTTGCTTCCGTGAGAACTTACCTGCCGCCGTGACTCTCCCTAGCACTGTGAAG CGAGGCATAATCAAGAGCCATCACACTTCTGTAACTCTTACTATGGAAGAGGA GAAAGCAGCCAGAGGAGCCACACAGGTCTCCGCTTCAGCATGCCCTAGCTCCA GGACGTAAAGATGAATGGTGACCCCGGCTATGACTCGCTAGTCTCTCCACACTT CATCTGCTACAACTTCCGGCT (SEQ ID NO: 13)

The A box was cloned into a shuttle vector such that recombination with the 5HT2A gene in a BAC would place an Emerald sequence at the 5' end of the 5HT2A gene such that expression of the gene would result in only Emerald production, and not 5HT2A production.

Seven different BACs were used to make cointegrates (see Section 6.3). DNA from putative cointegrates was prepared using the methods disclosed in Sections 6.1 and 6.4. ■

A DNA fingerprint (performed as disclosed in Section 6.1.5) is shown in FIG. 6. A corresponding Southern blot, shown in FIG. 7, was used to verify duplication of A boxes in cointegrate clones. CHEF mapping (see Sections 6.1.5 and 6.4) was used to determine that one of the

BACs was constructed such that one of the BAC clones had a sufficiently large DNA fragment upstream of the 5HT6 start site (FIG. 8).

Resolution of this cointegrate was performed (see Section 6.3); the DNA fingeφrint and corresponding Southern blot are shown in FIGS. 9 and 10. Two of the four putatives tested contained only one copy of EGFP, verifying resolution.

After preparing large amounts of the BAC DNA for injection (Section 6.5), transgenic animals were constructed (Section 6.6), and genotyped for the presence of GFP sequences (Sections 6.7 and 6.8). Founders were bred in order to obtain progeny containing the transgene (and verify that a line had indeed been established). Again, PCR (Section 6.8) was used to genotype FI animals. Sections of brain tissue showed that the transgene was indeed expressed in subsets of neurons in the transgenic animals (FIG. 11, arrows point to two fluorescent cells).

Using the methods described hereinabove, the inventors have obtained useable BACs comprising a gene of interest in approximately 96% of cases. Of these useable BACs, typically all can be can be converted to recombinant BACs and used to create transgenic founder animals according to the methods of the invention. Approximately 83% of founders tested by the inventors passed the transgene to progeny to create a transgenic line of the invention.

6.11. CONSTRUCTION AND INTRODUCTION OF A MODULATING

CONSTRUCT

6.11.1. CONSTRUCTION OF A MODULATING CONSTRUCT

Using the methods of Utomo et al (1999, Nat. Biotechnol. 17, 1091-96; incoφorated herein by reference in its entirety)), a transgenic construct is designed that contains two expression modules: (1) a Cre recombinase (key protein)-encoding sequence under the regulation of the rtTA-responsive hybrid promoter consisting of a tetO heptad repeat and a characterizing gene regulatory element (e.g., a hCMV minimal promoter), and (2) a rtTA cassette containing rtTA encoding sequence and SV40 polyadenylation site. Using the methods of Utomo et al. (1999), conditional Cre-loxP-mediated recombination is as follows. Without doxycycline, rtTA is inert and unable to activate transcription of the key protein, Cre recombinase. In the presence of doxycycline, rtTA binds to the tetO-characterizing gene promoter leading to Cre expression. Cre-mediated DNA recombination is assayed as follows. In the absence of Cre, expression of the potential drug target gene from the modulating construct is prevented by the intervening transcriptional STOP sequence flanked by loxP sites. Cre-mediated DNA recombination results in removal of the STOP sequence followed by potential drug target expression. PCR amplification using primers that recognize sequences of chicken β-actin (or any other promoter used in the modulating construct), promoter and the potential drug target gene is used to assay for recombination (see Fig. 2 and accompanying text of Utomo et al. (1999)). In the absence of recombination, the intervening STOP sequence between the loxP sites is amplified inefficiently, whereas Cre-mediated recombination permits amplification of a PCR product comprising the chicken β-actin promoter and the potential drug target gene. 6.11.2. INTRODUCTION OF A MODULATING CONSTRUCT

The modulating construct is introduced using transduction methods described in Deglon et al. (2000, Human Gene Therapy 11 :179-190; incoφorated herein by reference in its entirety). Deglon et al. describe methods for producing and introducing a self-inactivating (non-reproducing) lentiviral vector with enhanced transgene expression into a selected cell population, e.g., neurons in a particular brain region. The self-inactivating vector is used to transduce, and localize delivery of a potential drug target to, a select population of neurons. The self-inactivating (SIN) lentiviral vector is modified using the methods of Deglon et al. by insertion of the posttranscriptional regulatory element

10 of the woodchuck hepatitis virus, and particles are produced with a multiply attenuated packaging system.

The lentiviral vector comprising the modulating construct is also modified so that it has an improved ability to transduce the cells into which it is introduced. The methods of Zennou et al. are used to incoφorate a central DNA flap into the vector (2000, Cell 101,

15 173-85; incoφorated herein by reference in its entirety). Lentiviruses have the unique property among retroviruses of replicating in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1 , reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis, creates a plus

20 strand overlap: the central DNA flap. A key determinant for nuclear import of lentiviral genomes, e.g., HIV-1 genome, is therefore the central DNA flap: the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. A self-inactivating or non-reproducing lentiviral vector comprising the modulating construct is designed using the methods of Zennou et al. The vector comprises a reinsertion of the DNA flap sequence, thereby

25 restoring nuclear import of the vector to wild-type levels.

Using the methods of Deglon et al. (2000, Human Gene Therapy 11:179-190), 2 ml of the modified, potential drug target-expressing lentiviral vector is injected into the cell population or region of interest, e.g., a select population of neurons.

30 All references cited herein are incoφorated herein by reference in their entirety and for all puφoses to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incoφorated by reference in its entirety for all puφoses.

35 The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims along with the full scope of equivalents to which such claims are entitled.

Claims

I CLAIM:

1. A method of expressing a potential drug target protein or inhibitor thereof in a subset of cells in a transgenic non-human animal, said method comprising introducing into cells of said transgenic non-human animal a modulating construct comprising a first nucleotide sequence encoding said potential drug target protein or inhibitor thereof, the expression of said potential drug target protein or inhibitor thereof being under the control of a conditional expression element, said transgenic non-human animal comprising a transgene containing a key gene which encodes an inducer or suppressor of said conditional expression element, said key gene being operably linked to regulatory sequences of a characterizing gene corresponding to an endogenous gene or homolog of an endogenous gene such that said key gene is expressed in said transgenic non-human animal with an expression pattern that is substantially the same as the expression pattern of said endogenous gene in a comparable non-transgenic animal or anatomical region thereof, whereby said potential drug target protein or inhibitor thereof is expressed in the cells expressing said key gene, and wherein said transgene is present in the genome at a site other than where the endogenous gene is located.

The method of claim 1 wherein said modulating construct is a viral vector.

The method of claim 2 wherein said viral vector is a lentiviral vector.

The method of claim 2 wherein said viral vector is an adenovirus vector.

5. The method of claim 2 wherein said viral vector is an adeno-associated virus

(AAV) vector.

6. The method of claim 1 wherein said conditional expression element is a promoter.

7. The method of claim 1 wherein said conditional expression element is target sites for recombination positioned such that in the presence of an appropriate recombinase, the orientation of the key gene is reversed thereby operably linking the first nucleotide sequence to a promoter such that the first nucleotide sequence is expressed.

8. The method of claim 7 wherein said target sites for recombination are lox sites.

9. The method of claim 1 wherein said conditional expression element is a tet operator and said key gene encodes a tet repressor.

10. The method of claim 1 wherein said conditional expression element is a UAS site and said key gene encodes a GAL4 protein.

11. The method of claim 1 wherein said key gene is expressed selectively in neural cells.

12. The method of claim 1 wherein said modulating construct is introduced into cells that have been isolated from said transgenic non-human animal.

13. The method of claim 12 wherein substantially all of said cells express said key gene.

14. The method of claim 1 wherein said modulating construct is introduced into cells in said transgenic non-human animal.

15. The method of claim 1 wherein substantially all of said cells expressing said key gene are marked by expression of a marker gene.

16. The method of claim 1 wherein said transgenic non-human animal is a transgenic mouse.

17. The method of claim 1 which further comprises assaying said potential drug target protein by detecting a difference in said transgenic non-human animal or said cells from said transgenic non-human animal expressing said first nucleotide sequence in comparison with a comparable non-transgenic animal or anatomical region thereof, or cells isolated therefrom corresponding to said cells from said transgenic non-human animal.

18. A method of expressing two or more potential drug target proteins or inhibitors thereof in a subset of cells in a transgenic non-human animal, said method comprising

(a) introducing into cells of said transgenic non-human animal a modulating construct comprising a first nucleotide sequence encoding one of said potential drug target proteins or inhibitors thereof, the expression of said potential drug target protein or inhibitor thereof being under the control of a conditional expression element, said transgenic non-human animal comprising a transgene containing a key gene which encodes an inducer or suppressor of said conditional expression element, said key gene being operably linked to regulatory sequences of a characterizing gene corresponding to an endogenous gene or homolog of an endogenous gene such that said key gene is expressed in said transgenic non-human animal with an expression pattern that is substantially the same as the expression pattern of said endogenous gene in a comparable non-transgenic animal or anatomical region thereof, whereby said two or more potential drug target proteins or inhibitors thereof are expressed in the cells expressing said key gene, and wherein said transgene is present in the genome at a site other than where the endogenous gene is located; and (b) repeating step (a) at least one more time with the same transgenic non-human animal and a different potential drug target protein or inhibitor thereof.

19. The method of claim 18 which comprises expressing five or more potential drug target proteins or inhibitors thereof.

20. The method of claim 18 which comprises expressing ten or more potential drug target proteins or inhibitors thereof.

21. The method of claim 18 wherein said modulating construct is a viral vector.

22. The method of claim 21 wherein said viral vector is a lentiviral vector.

23. The method of claim 21 wherein said viral vector is an adenovirus vector.

24. The method of claim 21 wherein said viral vector is an adeno-associated virus (AAV) vector.

25. The method of claim 18 wherein said conditional expression element is a promoter.

26. The method of claim 18 wherein said conditional expression element is target sites for recombination positioned such that in the presence of an appropriate recombinase the orientation of the key gene is reversed thereby operably linking the first nucleotide sequence to a promoter such that the first nucleotide sequence is expressed.

27. The method of claim 26 wherein said target sites for recombination are lox sites.

28. The method of claim 18 wherein said conditional expression element is a tet operator and said key gene encodes a tet repressor.

29. The method of claim 18 wherein said conditional expression element is a UAS site and said key gene encodes a GAL4 protein.

30. The method of claim 18 wherein said key gene is expressed selectively in neural cells.

31. The method of claim 18 wherein said modulating construct is introduced into cells that have been isolated from said transgenic non-human animal.

32. The method of claim 31 wherein substantially all of said cells express said key gene

33. The method of claim 18 wherein said modulating construct is introduced into cells in said transgenic non-human animal.

34. The method of claim 18 wherein substantially all of said cells expressing said key gene are marked by expression of a marker gene.

35. The method of claim 18 wherein said transgenic non-human animal is a transgenic mouse.

36. The method of claim 18 which further comprises assaying said potential drug target proteins by detecting a difference in said transgenic non-human animal or said cells from said transgenic non-human animal expressing said first nucleotide sequence in comparison with a comparable non-transgenic animal or anatomical region thereof, or cells isolated therefrom corresponding to said cells from said transgenic non-human animal.

37. A transgenic non-human animal expressing a potential drug target protein or inhibitor thereof in a subset of cells, said transgenic non-human animal comprising

(a) at least one modulating construct comprising a first nucleotide sequence encoding a potential drug target protein or inhibitor thereof, the expression of said potential drug target protein or inhibitor thereof being under the control of a conditional expression element; and

(b) a transgene containing a key gene which encodes an inducer or suppressor of said conditional expression element, said key gene being operably linked to regulatory sequences of a characterizing gene corresponding to an endogenous gene or homolog of an endogenous gene such that said key gene is expressed in said transgenic non-human animal with an expression pattern that is substantially the same as the expression pattern of said endogenous gene in a comparable non-transgenic animal or anatomical region thereof, such that said potential drug target protein or inhibitor thereof is expressed in the cells expressing said key gene, and wherein said transgene is present in the genome at a site other than where the endogenous gene is located.

38. The transgenic non-human animal of claim 37 wherein said modulating construct is a viral vector.

39. The transgenic non-human animal of claim 38 wherein said viral vector is a lentiviral vector.

40. The transgenic non-human animal of claim 38 wherein said viral vector is an adenovirus vector.

41. The transgenic non-human animal of claim 38 wherein said viral vector is an adeno-associated virus (AAV) vector.

42. The transgenic non-human animal of claim 37 wherein said conditional expression element is a promoter.

43. The transgenic non-human animal of claim 37 wherein said conditional expression element is target sites for recombination positioned such that in the presence of an appropriate recombinase the orientation of the key gene is reversed thereby operably linking the first nucleotide sequence to a promoter such that the first nucleotide sequence is expressed.

44. The transgenic non-human animal of claim 43 wherein said target sites for recombination are lox sites.

45. The transgenic non-human animal of claim 37 wherein said conditional expression element is a tet operator and said key gene encodes a tet repressor.

46. The transgenic non-human animal of claim 37 wherein said conditional expression element is a UAS site and said key gene encodes a GAL4 protein.

47. The transgenic non-human animal of claim 37 wherein said key gene is expressed selectively in neural cells.

48. The transgenic non-human animal of claim 37 which is a transgenic mouse.

49. A transgenic non-human animal expressing two or more potential drug target proteins or inhibitors thereof in a subset of cells, said transgenic non-human animal comprising (a) at least two modulating constructs each comprising a first nucleotide sequence encoding a potential drug target protein or inhibitor thereof, the expression of said potential drug target protein or inhibitor thereof being under the control of a conditional expression element, wherein each of said modulating constructs encodes a different potential drug target protein or inhibitor thereof; and (b) a transgene containing a key gene which encodes an inducer or suppressor of said conditional expression element, said key gene being operably linked to regulatory sequences of a characterizing gene corresponding to an endogenous gene or homolog of an endogenous gene such that said key gene is expressed in said transgenic non-human animal with an expression pattern that is substantially the same as the expression pattern of said endogenous gene in a comparable non-transgenic animal or anatomical region thereof, such that said two or more potential drug target proteins or inhibitors thereof are expressed in the cells expressing said key gene, and wherein said transgene is present in the genome at a site other than where the endogenous gene is located.

50. The transgenic non-human animal of claim 49 wherein said modulating construct is a viral vector.

51. The transgenic non-human animal of claim 50 wherein said viral vector is a lentiviral vector.

52. The transgenic non-human animal of claim 50 wherein said viral vector is an adenovirus vector.

53. The transgenic non-human animal of claim 50 wherein said viral vector is an adeno-associated virus (AAV) vector.

54. The transgenic non-human animal of claim 49 wherein said conditional expression element is a promoter.

55. The transgenic non-human animal of claim 49 wherein said conditional expression element is target sites for recombination positioned such that in the presence of an appropriate recombinase the orientation of the key gene is reversed thereby operably linking the first nucleotide sequence to a promoter such that the first nucleotide sequence is expressed.

56. The transgenic non-human animal of claim 55 wherein said target sites for recombination are lox sites.

57. The transgenic non-human animal of claim 49 wherein said conditional expression element is a tet operator and said key gene encodes a tet repressor.

58. The transgenic non-human animal of claim 49 wherein said conditional expression element is a UAS site and said key gene encodes a GAL4 protein.

59. The transgenic non-human animal of claim 49 wherein said key gene is expressed selectively in neural cells.

60. The transgenic non-human animal of claim 49 which is a transgenic mouse.

61. A collection of transgenic non-human animals for drug target validation, said collection comprising at least five transgenic non-human animals each expressing a potential drug target protein or inhibitor thereof, each potential drug target protein or inhibitor thereof being different from the other potential drug target proteins or inhibitors thereof expressed in the transgenic non-human animals of the collection and expressed in a subset of cells in said transgenic non-human animal, each of said transgenic non-human animals of the collection comprising

(a) at least one modulating construct comprising a first nucleotide sequence encoding said potential drug target protein or inhibitor thereof, the expression of said potential drug target protein or inhibitor thereof being under the control of a conditional expression element; and

(b) a transgene containing a key gene which encodes an inducer or suppressor of said conditional expression element, said key gene being operably linked to regulatory sequences of a characterizing gene corresponding to an endogenous gene or homolog of an endogenous gene such that said key gene is expressed in said transgenic non-human animal with an expression pattern that is substantially the same as the expression pattern of said endogenous gene in a comparable non-transgenic animal or anatomical region thereof, such that said two or more potential drug target proteins or inhibitors thereof are expressed in the cells expressing said key gene, and wherein said transgene is present in the genome at a site other than where the endogenous gene is located.

62. The collection of claim 61 wherein said modulating construct is a viral vector.

63. The collection of claim 62 wherein said viral vector is a lentiviral vector.

64. The collection of claim 62 wherein said viral vector is an adenovirus vector.

65. The collection of claim 62 wherein said viral vector is an adeno-associated virus (AAV) vector.

66. The collection of claim 61 wherein said conditional expression element is a promoter.

67. The collection of claim 61 wherein said conditional expression element is target sites for recombination positioned such that in the presence of an appropriate recombinase the orientation of the key gene is reversed thereby operably linking the first nucleotide sequence to a promoter such that the first nucleotide sequence is expressed.

68. The collection of claim 67 wherein said target sites for recombination are lox sites.

69. The collection of claim 61 wherein said conditional expression element is a tet operator and said key gene encodes a tet repressor.

70. The collection of claim 61 wherein said conditional expression element is a

UAS site and said key gene encodes a GAL4 protein.

71. The collection of claim 61 wherein said key gene is expressed selectively in neural cells.

72. The collection of claim 61 wherein each transgenic non-human animal is a transgenic mouse.

73. A method of making a collection of transgenic non-human animals for drug target validation, said collection comprising at least five transgenic non-human animals each expressing a potential drug target protein or inhibitor thereof in a subset of cells in said transgenic non-human animal, said method comprising

(a) introducing into a transgenic non-human animal at least one modulating construct comprising a first nucleotide sequence encoding a potential drug target protein or inhibitor thereof, the expression of said potential drug target protein or inhibitor thereof being under the control of a conditional expression element, said transgenic non-human animal comprising a transgene containing a key gene which encodes an inducer or suppressor of said conditional expression element, said key gene being operably linked to regulatory sequences of a characterizing gene corresponding to an endogenous gene or homolog of an endogenous gene such that said key gene is expressed in said transgenic non-human animal with an expression pattern that is substantially the same as the expression pattern of said endogenous gene in a comparable non-transgenic animal or anatomical region thereof, such that said potential drug target protein or inhibitor thereof is expressed in the cells expressing said key gene, and wherein said transgene is present in the genome at a site other than where the endogenous gene is located; and

(b) repeating step (a) at least four more times, wherein each time said first nucleotide sequence encodes a different potential drug target or inhibitor thereof, thereby making said collection of at least five transgenic non-human animals.

74. The method of claim 73 wherein said modulating construct is a viral vector.

75. The method of claim 74 wherein said viral vector is a lentiviral vector.

76. The method of claim 74 wherein said viral vector is an adenovirus vector.

77. The method of claim 74 wherein said viral vector is an adeno-associated virus (AAV) vector.

78. The method of claim 73 wherein said conditional expression element is a promoter.

79. The method of claim 73 wherein said conditional expression element is target sites for recombination positioned such that in the presence of an appropriate recombinase the orientation of the key gene is reversed thereby operably linking the first nucleotide sequence to a promoter such that the first nucleotide sequence is expressed.

80. The method of claim 79 wherein said target sites for recombination are lox sites.

81. The method of claim 73 wherein said conditional expression element is a tet operator and said key gene encodes a tet repressor.

82. The method of claim 73 wherein said conditional expression element is a UAS site and said key gene encodes a GAL4 protein.

83. The method of claim 73 wherein said key gene is expressed selectively in neural cells.

84. The method of claim 73 wherein each transgenic non-human animal is a transgenic mouse.

85. A method of validating a potential drug target, said method comprising (a) introducing into a transgenic non-human animal at least one modulating construct comprising a first nucleotide sequence encoding a potential drug target protein or inhibitor thereof, the expression of said potential drug target protein or inhibitor thereof being under the control of a conditional expression element, said transgenic non-human animal comprising a transgene containing a key gene which encodes an inducer or suppressor of said conditional expression element, said key gene being operably linked to regulatory sequences of a characterizing gene corresponding to an endogenous gene or homolog of an endogenous gene such that said key gene is expressed in said transgenic non-human animal with an expression pattern that is substantially the same as the expression pattern of said endogenous gene in a comparable non-transgenic animal or anatomical region thereof, such that said potential drug target protein or inhibitor thereof is expressed in the cells expressing said key gene, and wherein said transgene is present in the genome at a site other than where the endogenous gene is located;

(b) introducing into the transgenic non-human animal a test compound; and

(c) detecting a change in a phenotype or activity associated with the potential drug target after introducing said test compound, wherein detection of a change in said phenotype or activity validates said potential drug target.

86. The transgenic non-human animal of claim 85 wherein said modulating construct is a viral vector.

87. The transgenic non-human animal of claim 86 wherein said viral vector is a lentiviral vector.

88. The transgenic non-human animal of claim 86 wherein said viral vector is an adenovirus vector.

89. The transgenic non-human animal of claim 86 wherein said viral vector is an adeno-associated virus (AAV) vector.

90. The transgenic non-human animal of claim 85 wherein said conditional expression element is a promoter.

91. The transgenic non-human animal of claim 85 wherein said conditional expression element is target sites for recombination positioned such that in the presence of an appropriate recombinase the orientation of the key gene is reversed thereby operably linking the first nucleotide sequence to a promoter such that the first nucleotide sequence is expressed.

92. The transgenic non-human animal of claim 91 wherein said target sites for recombination are lox sites.

93. The transgenic non-human animal of claim 85 wherein said conditional expression element is a tet operator and said key gene encodes a tet repressor.

94. The transgenic non-human animal of claim 85 wherein said conditional expression element is a UAS site and said key gene encodes a GAL4 protein.

95. The transgenic non-human animal of claim 85 wherein said key gene is expressed selectively in neural cells.

96. The transgenic non-human animal of claim 85 which is a transgenic mouse.