WO2007127254A2 - Compositions and methods for generating transgenic animals - Google Patents

Abstract

The invention provides compositions and methods to screen genomes for cells of a particular phenotype. The methods are used to select and predict the phenotype of a transgenic mammal with selected phenotypic characteristics.

Description

COMPOSITIONS AND METHODS FOR GENERATING TRANSGENIC ANIMAJLS

Background of the Invention

Applications of transgenic mouse models to problems in basic and biomedical research represent a potent new .experimental approach. For example, transgenic mice can be used to study the function of individual genes either by altering the tissue profile of their expression or by producing elevated expression levels. Phenotypic consequences of such alterations can in turn lead to information relating to the function of that particular gene in vivo. The generation of a transgenic mammal, however, is generally a lengthy and time-consuming process. It would be advantageous to develop methods to allow mice to be pre-screened for potential phenotypes in vitro and to facilitate the generation of transgenic mammals for the development of improved therapeutic modalities.

Summary of the Invention

The invention provides compositions and methods to screen genomes for cells of a particular phenotype. Large scale screening of cells containing nucleic acids encoding expressed proteins in an inducible manner allows selection of any phenotypic characteristic. The phenotype of an embryonic stem (ES) cell elucidates in advance of production of a whole animal the phenotype of a transgenic animal, which is produced using that ES cell. This screening strategy, which makes use of a library or bank of transgenic ES cells, is markedly faster than existing transgenic technologies. The bank or library includes a plurality of ES cell clones, each of which comprises a single copy of a transgene integrated into a predetermined genomic site. Alternatively, a target gene is disrupted by integration of a transgenic nucleic acid. The expression of the transgene is inducible, and the plurality of clones is characterized as encompassing a representative fraction of a complete ORFeome. For example, the ORF nucleic acids represent those of a heterologous species relative to the species of the ES cell. An ORFeome is a collection of nucleic acids that define an open reading frame (ORF). A complete ORFeome contains nucleic acids that encode all proteins of a given species. A representative fraction of a full ORFeome is at least 60% of all proteins expressed by the species. For example, the plurality of clones contains at least 85% of the ORFeome. Preferably, the plurality of clones (library) contains at least 90%, 95%, 98%, 99% or 100% of ORFs from a given species.

The predetermined site is one that permits integration of a single copy of the transgene into the target locus; e.g., the site is collagen Al intron 1 (CoIAl) or Rosa26 of a mouse chromosome. Expression of the ORF is preferably inducible, e.g., by contacting the cell with doxicycline (DOX). The cells are typically of murine origin, e.g., mouse ES cells, but any species of cell is used in the production and use of the library. The library or plurality of clones contains 1,000, 5,000, 10,000, 15,000, 20,000, 25,000, or 30,000 clones. Preferably, the plurality of clones encompasses some or all of the mouse expressed genome (ORFeome) or that of another species such as a human.

The plurality of clones is useful in large scale screening endeavors to identity transgenic ES cells that are employed to create a transgenic animal with a desired target phenotype. For example, the method for identifying or predicting a phenotype of a transgenic animal prior to production of the animal is carried out by providing a plurality of ES cell clones as described above. Each cell of the cloned population of cells contains a single copy of a transgene integrated into a predetermined genomic site, the expression of the transgene being inducible. Transgene expression is induced using a compound such as an antibiotic, e.g., DOX or tetracycline (TET) in the case of an antibiotic sensitive expression system. The library of clones is then evaluated to identify an ES cell phenotype, and the ES cell phenotype of the ES cell clone is predictive of the transgenic animal phenotype. Optionally, the cells of the plurality are contacted with a condition or agent (or both) that leads to differentiation of the ES cell into a desired cell type, e.g., neuron, muscle, adipose, heart, liver, pancreas, lung, or skin cells. Exemplary conditions include light, insulin challenge, toxins, DNA damaging agents, cytokines, heat, electricity, or physical forces such as pulsatile flow of tissue culture media. Cells are induced to differentiate as a result of exposure to agents such as insulin, toxins, DNA damaging agents, cytokines, retinoic acid, dexamethasone, and 5-azacytidine. Retinoic acid is useful to induce differentiation of ES cells into neuronal cells. In other examples, agents such as retinoic acid, insulin, T3 thyroid hormone, or Leukemia inhibitory factor (LIF) is used to promote differentiation of ES cells into adipocytes; basic fibroblast growth factor is useful to differentiate them into glial cells; epidermal growth factor, platelet-derived growth factor is used to differentiate them into oligodendrites; bone morphogenic proteins (BMP) such as BMP-2 and BMP-4 are used to differentiate them into chondroyctes; dexamethasone, retinoic acid, ascorbic acid, and beta-glycerophosphate is used to differentiate them into bone cells. Other differentiating agents include basic fibroblast growth factor and nicotinamide for differentiation into pancreatic cells. If the transgenic ES cells are to be differentiated, the plurality of ES cell clones is typically contacted with a differentiating condition or agent, e.g., retinoic acid, prior to the inducing step (e.g., prior to contacting the cells with DOX).

The transgenic ES cells (differentiated or not) are screened for a chosen phenotype, and the phenotype foretells or indicates the phenotype of a transgenic animal made with the chosen transgenic ES cell. A target transgenic animal phenotypes are selected from a variety of characteristics or conditions such as increased longevity compared to a wild type animal, or a symptom or cluster of symptoms of a pathologic condition. For example, the ES cell phenotype is impaired neurotransmission and the transgenic animal phenotype is characterized by a symptom of a neurodegenerative disease. In another example, the ES or differentiated cell phenotype is resistance to a toxin and the transgenic animal phenotype is increased stress resistance. In another example, the differentiated adipocyte or muscle cell phenotype is increased sensitivity to insulin or fat moblilization, and the transgenic animal phenotype is improved insulin sensitivity or leanness. In another example, the ES cell phenotype is reduced superoxide dismutase (SOD) activity and the transgenic animal phenotype is characterized by a symptom of amyotrophic lateral sclerosis (ALS). In yet other example, an ES cell phenotype characterized by increased p25 expression indicates that the transgenic animal phenotype is characterized by a symptom of Alzheimer's Disease, and an ES cell phenotype characterized by increased sirtuin activity indicates a transgenic animal phenotype is increased resistance to neurodegeneration, diabetes, cancer and other diseases of aging.

Also within the invention are kits and assemblies of reagents for making and using the libraries and screening methods described herein. For example, the invention encompasses a surface containing a plurality of wells (e.g., a 96-well plate) in which each of the wells contains an ES cell clone that contains a transgene. Preferably the collection of clones represents ORFs from a representative fraction of the expressed proteins in the genome of a given species. The surface comprises at least 10, 50, 110, 500 or more different ES cell clones, each of which comprises a different transgene.

The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. AU patents and publications cited in this specification are incorporated herein by reference.

Other embodiments are described in the figures and the description below.

Brief Description of the figures

FIGURE 1 is a diagram showing exemplary vectors that are used in generating a transgenic mouse.

FIGURE 2 is a diagram showing the overall strategy for generating SIRT transgenic mice.

FIGURE 3A is a diagram showing the DNA construct used to generate transgenic mice that over- express SIRTl.

FIGURE 3B is a series of photographs showing confirmation of the integration of the vector carrying the SIRTl transgene into the ES cell genome using PCR and the resulting protein levels when the STOP cassette is excised in ES cells. FIGURE 3C is a series of PCR gel photographs.

FIGURE 4 is a diagram showing DNA contructs that encode Cre recombinase under the control of a rat nestin promoter or a CamKII promoter.

FIGURE 5 is a diagram illustrating the recombination event that results from breeding a SIRTl mouse and a Cre recombinase mouse.

FIGURES 6A-6C are diagrams showing tissue-specific inducible expression of SIRTl -7 using targeted transgenes. FIGURE 6 A illustrates the generation of an ES cell line for targeting to a well- behaved locus, namely the Collagenl A or Rosa26 locus. FIGURE 6B shows the recombination event that results in the integration of SIRTs into the Collagen IA or Rosa26 locus. FIGURE 6C illustrates the tissue-specific inducible expression of SIRT 1-7 using a DOX-Cre driver.

FIGURE 7 is a photograph of a Southern blot analysis and a diagram illustrating the recombination events leading to the generation of SIRTl transgenic cell.

FIGURE 8 is a series of photographs showing the integration of the transgene into the cell genome by PCR and Southern blot analysis.

FIGURE 9 is a series of immunoblots showing the level of SIRTl and phospho-SIRTl-T530 in SIRTl transgenic and knock-out mice.

FIGURE 10 is a series of immunoblots showing the induction of SIRTl expression in various tissues of LC-l-Cre/SIRTl(CollagenAl) transgenic mice following the addition of DOX into the drinking water.

FIGURE 11 is a series of photographs showing the genotyping of COLAl /ES cells transfected with pCAGGS-SIRT2, SIRT4, and SIRT5 by PCR and southern blot analysis.

FIGURE 12 is a diagram and a series of photographs showing germline transmission of the SIRT3 transgene in mice by PCR and southern blot analysis.

FIGURE 13 is a diagram and a series of photographs showing germline transmission of the SIRT2 transgene in mice by PCR and southern blot analysis.

FIGURE 14 is a series of photographs showing immunofluorescent stains of the hippocampus of Camk2a-Cre/ColAl -SIRTl transgenic mice.

FIGURE 15 is a series of photographs showing immufluorescent stains in Camk2a-Cre/ColAl- SIRTl transgenic mice.

FIGURE 16 is a series of immunohistochemistry stains of the cortex of non-transgenic and nestin- Cre/SIRTl mice.

FIGURE 17 is a series of immunohistochemistry stains of the hippocampus of non-transgenic and nestin-Cre/SIRTl mice. FIGURE 18 is a series of immunohistochemistry stains of the dentate gyrus of non-transgenic and nestin-Cre/SIRTl mice.

FIGURE 19 is a series of immunohistochemistry stains of the cerebellum of non-transgenic and nestin-Cre/SIRTl mice.

FIGURE 20 is a series of immunohistochemistry stains of the striatum of non-transgenic and nestin-Cre/SIRTl mice.

FIGURE 21 is a series of immunohistochemistry stains showing the pancreatic expression of SIRTl in non-transgenic mice and LC 1-Cre/SIRTl (CoIlA) transgenic mice.

FIGURE 22 is a series of immunohistochemistry stains showing the liver expression of SIRTl in non-transgenic mice and LCl-Cre/SIRTl(CollA) transgenic mice.

FIGURE 23 is a series of photographs showing immunofluorescent stains of the hypothalamus of wildtype and PomC-Cre/SIRTl (SIRTl^⁰¹⁵) transgenic mice.

FIGURE 24 is a photograph of a Western blot analysis illustrating the level of SIRT2 in the cerebellum, cortex and hippocampus of wildtype and Nestin-Cre/SIRT2 transgenic mice.

FIGURE 25 is a photograph of a Western blot analysis showing the level of SIRT5 in the pancreas of wildtype and transgenic mice using the doxycycline inducible Cre driver.

FIGURE 26 is a diagram showing the recombination event that results from breeding a SIRTl^stop mouse and a Villin-Cre mouse.

FIGURE 27 is a series of photographs showing immunofluorescent stains of the intestine of _SIRT1 ^sto _{P aπd} siRTl^stop/Villin-Cre transgenic mice (SIRTl^⁰P).

FIGURE 28 is an immunoblot showing the level of SIRTl in SIRTl^stop and SIRTl^⁰" transgenic mice.

FIGURE 29 is a series of photographs illustrating the color of the paws of SIRTl^3top and SIRTl^^10P transgenic mice.

FIGURE 30 is a series of photographs illustrating adenomas in the duodenum and ileum of _SIRT1sto_{P and srR}τiA«°^p transgenic mice. Detailed Description

Transgenic and gene-targeted mutant cells and mammals provide powerful tools for analysis of the cellular processes involved in early development and in the pathogenesis of many diseases. The invention provides libraries of embryonic stem (ES) cells clones. These ES cell clones are genetically altered so that a transgene is integrated in their genome while others have a gene-targeted disruption in their genome. Furthermore, each cell clone is physically segregated from other ES cell clones. For example, each ES cell clone of an ES library expresses a specific cDNA from a mammalian genome (e.g., murine or human) and collectively, these ES cell clones express a portion or the entire mammalian expressed genome (ORFeome). A transgene integration strategy mediated by site-specific recombination allows establishment of multiple embryonic stem (ES) cell lines carrying drug-inducible (e.g., tetracycline- inducible) genes targeted to a specific locus to assure predictable temporal and spatial expression in ES cells and mice (Beard et al., Genesis 44:22-28, 2006). Using homologous recombination, an fit homing site allows for drug-inducible transgenes to be integrated efficiently into the genome in the presence of a recombinase (e.g., Cre recombinase or FLPe recombinase). This strategy and the vectors described herein are generally applicable to any locus in ES cells and allow for the rapid production of transgenic mammals with transgenes efficiently targeted to a defined site.

As a specific example, a library of ES cell clones is provided in a 96-well format and includes independent clonal lines, each of which expresses a specific tagged mouse ORF that is expressed after treatment of the ES cells with DOX. There is little or no (0%) leakage of expression in the absence of DOX. The addition of DOX to the cell media induces the expression of the Cre recombinase protein causing the excision of a transcriptional STOP cassette upstream of the cDNA and in turn, resulting in the expression of the cDNA or transgene.

If desired, ES cell clones are differentiated into various cell types within the 96-well format and screened for specific phenotypes including, for example, the expression of cell-specific proteins. ES cell clones may be differentiated into tissue-specific cells such as adipocytes, astrocytes, neurons, smooth muscle cells, cardiomyocytes, pancreatic cells, skeletal cells, fibroblasts, and endothelial cells. ES cell- derived cardiomyocytes express cardiac gene products in a developmentally controlled manner. As in early myocardial development, mRNAs encoding GAT A-4 and Nkx2.5 transcription factors are detected before mRNAs encoding atrial natriuretic factor (ANF), myosin light chain (MLC)-2v, alpha-myosin heavy chain -MHC), β-myosin heavy chain (β-MHC), Na⁺-Ca²⁺ exchanger, and phospholamban. Sarcomeric proteins of ES cell-derived cardiomyocytes are also established developmentally in the following order: titin (Z disk), alpha-actinin, myomesin, titin (M band), MHC, alpha-actin, cardiac troponin T, and M protein. Cardiomyocytes with characteristics of fetal/neonatal rodent cardiomyocytes express slow skeletal muscle troponin I isoforms and a greater proportion of β-MHC versus alpha-MHC, whereas cardiomyocytes that more rapidly contract preferentially express cardiac troponin I and alpha- MHC. Thus, the appearance of cardiac-associated gene products is a function of differentiation time, similar to that seen in normal myocardial development.

ES cell clones are induced to differentiate into different cell types by using specific culture conditions (e.g., presence of particular growth factors, basement membrane substrates, or pharmaceutical agents). Addition of dimethyl sulfoxide into ES cell medium, for example, induces the differentiation of ES cells into cardiomyocyytes. Alternatively, the ES cell clone contains a transgene that upon expression, commits the ES cell to a particular cell lineage. Cells that are committed to a certain cell lineage may also be isolated by using a selectable marker such as EGFP that is placed under the control of a tissue-specific promoter. Cells that express the EGEP are identified as cells that are committed to differentiate into the cell type that form the tissue to which the promoter is specific. The hybrid line of 129 x C57BL/6 Fl is readily differentiated into muscle, adipocytes or neurons. Optionally, the ES cells are used to make transgenic mammals, in which the expression of the gene of interest (transgene) is titratable or is regulated temporally or spatially by having the expression of the recombinase protein be regulated by a constitutional promoter, inducible and/or tissue-specific promoter. For example, the transgene is specifically expressed in endothelial cells by placing the recombinase gene under the control of an endothelial cell specific promoter (e.g., Tek/Tie2). Alternatively, the transgene is expressed by feeding the transgenic mammal with DOX for a period of time (e.g., 1, 2, 5, 10, 12, 15 or more days). Typically, the animal is administrated DOX for 5-10 days.

The technology described herein provides various advantages over existing transgenic technologies. DNA constructs are generated and stable ES cell clones are established within a much shorter period of time. The ES cell library can either be pooled for screening or kept as individual cell lines in 96-well format. Furthermore, the ES cells can be differentiated into other cell types prior to the generation of transgenic mice.

The methods described herein have been reliably and successfully used to generate panels of ES cells and to generate transgenic mice. For example, transgenic mice that contain members, e.g., SIRT 1-7, of the SIRT family of longevity genes (FIGURES 1-6C) were produced. Proper integration of the transgene and genotyping was confirmed by PCR and Southern blot analysis (FIGURES. 7, 8 and 11-13), while expression of SIRT proteins in various tissues was confirmed by Western blot (FIGURES 9 and 10). Further confirmation of SIRT protein production in situ was shown by immunohistochemical analysis of tissue sections (FIGURES 14-22). Example 1: Production of Transgenic ES cells by Site-Directed Gene Targeting

One strategy for performing site-directed gene targeting involves the following steps (see, for example, Beard et al. supra, Novak et al., Genesis 28: 147-155, 2000, and Gertsenstein et al., Methods MoI. Biol. 185: 285-307, 2002, as well as U.S. Serial No. 11/191,157, all of which are hereby incorporated by reference in their entirety). A first resistance gene (e.g., antibiotic resistance gene such as neomycin) which is flanked by recombination sites (e.g., loxP or FRT sites) and a second resistance gene (e.g., antibiotic resistance gene such as hygromycin) that lacks a promoter and an ATG initiation codon and that has a recombination site embedded in the 5' coding region are introduced into targeting arms that will be used to mediate homologous recombination at a specific site in the genome (see FIGURE 1). Next, the modified locus is targeted using a plasmid that contains a promoter (e.g., PGK promoter), an ATG initiation codon, a recombination site, and a drug-inducible minimal promoter (e.g., CMV promoter) that drives expression of the gene of interest. Co-electroporation of this plasmid with a plasmid expressing the recombinase protein (e.g., Cre recombinase or FLPe) is performed and is followed by exposure to selection conditions (e.g., hygromycin treatment). The cells that survive in such selection conditions are those in which recombination has occurred between recombination sites such that the entire plasmid in which recombination has occurred is inserted with the promoter (e.g., PGK promoter) and the ATG initiation codon upstream and into frame with the second (e.g., hygromycin) resistance gene.

The site-specific recombinases catalyze the recombination between two consensus sequences. The sites are designed into a transgene or a gene-targeting vector such that the site-specific recombination event triggers the expression of the transgene or the loss-of-function allele of the targeted gene. Exemplary recombinases include the Cre recombinase which recombines DNA between two loxP recognition sites and the FIp recombinase (or the enhanced Flpe recombinase) which recognizes the consensus recombination site FRT. The integrase from Streptomyces phage ΦC31, which also functions in human cells, carries out a site-specific recombination between attP and attB sites. Other recombinases include the Gin recombinase of phage Mu, the Pin recombinase of E. coli, and the R/RS system of the pSRl plasmid.

Optionally, the recombinase protein used in recombination events are modified or bonded in an operating manner (in particular by fusion) to a sequence which provides them with a property of induction by an exogenous agent. For example, a recombinase is fused to the domain for fixing of the receptor for estrogens (ER) which has been mutated so that it no longer fixes the endogenous estrogens but is now activated by tamoxifen or by one of its analogues. As another example, a recombinase is fused to other domains such as the domain for fixing of the Iigand of the receptor for progesterone (PR) or the domain for fixing of the Iigand of the receptor for glucocorticoids (GR). These domains are mutated so that they are no longer activated by their ligand and are activated by synthetic molecules such as dexamethasone, tamoxifen (an estrogen antagonist), or RU486 (a synthetic steroid). These resulting inducible systems use the nuclear localization capability of estrogen or a progesterone receptor ligand-binding domain in the presence of a ligand. Accordingly, the recombinase which is fused to a mutant ligand-binding domain (which no longer binds endogenous estrogen or progesterone but binds tamoxifen or RU-486, respectively) translocates to the nucleus in the presence of the synthetic ligand and executes its function.

Recombinase expression is regulated in various ways. The tetracycline-inducible gene expression system uses the DNA-binding domain of the bacterial tet-repressor protein and a strong transcriptional activator domain (e.g., VPl 6), which are fused together. Such a heterologous protein can bind to the tetracycline operator element and activate transcription depending on the presence of tetracycline. The combination of Cre/loxP and the doxycycline system may also be used. Optionally, in such a system, the Cre recombinase and a reverse tetracycline-dependent transactivator (rtTA) under the control of the same bi-directional DOX responsive promoter are used. Transgene expression occurs following the excision of the STOP codon 5' to the transgene by the recombinase protein. Following such excision, the transgene is now under the control of the promoter.

The transgenic animal used in the methods of the invention is any mammal including rodents; ruminants; ungulates; domesticated mammals; and dairy animals. Commonly used animals include: rodents, goats, sheep, camels, cows, pigs, horses, oxen, llamas, chickens, geese, and turkeys. Desirably, the mammal is a mouse.

As a specific example, a flp-in vector carrying the gene encoding EGFP is constructed. In addition, this flp-in vector contains a splice acceptor (SA) and a polyadenylation sequence upstream of the minimal tetracycline-responsive promoter that should reduce tetracycline-independent transcription by blocking read-through from potential upstream promoters. This vector and a vector that expresses the FLPe recombinase from the highly expressed CAGGS promoter are co-electroporated into KH2 ES cells. Integration of this vector into the frt site linked to an ATG initiation codon and a Pgklpromoter restores the hygr gene. Hygromycin-resistant colonies are selected and screened by Southern blot analysis. Very few hygromycin colonies survive selection, but 80% of the clones show correct targeting to the frt site upstream of the hygromycin cassette. To confirm that EGFP is tetracycline-inducible, ES cells are cultured in the presence of increasing amounts of DOX for 2 days and then analyzed for intensity of GFP fluorescence by FACS. The percentage of cells expressing high levels of GFP increase with increasing concentrations of DOX. Without DOX all cells express a very low level of GFP that is detected by FACS analysis but not by fluorescence microscopy. Thus, tight and titratable regulation of this reporter gene is possible in ES cells. To test the tetracycline-inducible system in vivo, mice are derived by tetraploid embryo complementation from ES cells carrying both the R26-M2rtTA allele and the flp-in tetO-EGFP allele. The production of "ES mice" from ES cells that have been subjected to multiple rounds of in vitro selection is highly efficient. Since mice produced by this method are derived entirely from the ES cells, they can be used directly without further breeding. Mice are treated with DOX administered in drinking water for 5 days, and tissues are harvested and analyzed by immunocytochemistry.

Example 2: ES Clonal Cell Libraries

The ES cell clone of the present ES cell library expresses any desired gene. The ES cell library is provided on a surface having a plurality of wells, each of which contains a single ES cell clone and media suitable for the growth of ES cells. Preferably, at least one cell of 1, 5, 10, 20, 50, 100, or more of the ES cell clones have been tested for and known to express a particular transgene or have a disrupted gene. Desirably, a layer of feeder cells coats the bottom of each well. ES cell libraries may include between 5 and 5000 genes, between 5 and 1000 genes, between 5 and 500 genes, between 5 and 100 genes, or between 5 and 50 genes. If desired, the library is designed to contain organ- or tissue-specific genes. For example, the library may contain ES clonal cells expressing genes that are specifically expressed in cardiac, lung, thymus, testis, kidney, skeletal, neuronal, or liver tissue. Alternatively, the library may contain genes that have similar functions. Accordingly, the ES cell library contains ES clonal cells with ami- and pro-apoptotic genes. As an example, an ES cell library is provided as a 96 well plate (24 well plate or 6 well plate) containing a plurality of ES cell clones. Each cell clone is segregated from other cell clones by being placed in a separate well in the plate and each cell clone has an inserted transgene that encodes for a muscle cell specific protein.

Example 3: Construction of an ES library

Commercially available normalized mouse cDNA libraries of ~8 x 10⁵ clones are available from brain, muscle and adipose tissue, in the Gateway® (INVITROGEN, Carlsbad, CA) or similar cloning system. The ORF may be transferred using the "flp-In" approach in vitro from the donor vector to an ES targeting clone between the recombination sites, attLl and attL2. In another example, the ORF may be transferred using lambda-mediated recombination known as Red®/ET® (Gene Bridges, GMBG, Dreseden, Germany). Alternatively, the cDNA is obtained from existing libraries and ligated into the vector. Selecting for Amp^R eliminates the starting vector and the by-product. The complexity of library is validated then shot-gun targeted to the collagen (CoIAl) locus of B6/C57 embryonic stem (ES) cell line, using "Flp-in" technology (Beard et al., supra and Hochedlinger et al., Genes Dev. 18: 1875-1885, 2004). Hundreds of hygromycin-resistant (Hy g¹¹) colonies are generated per plate, and approximately 500 colonies are picked per day into deep-well 96-well microtitre trays, amplified, and quadruplicated and duplicate aliquots are frozen down for storage. The vast majority of Hyg^R clones are correctly integrated as a single copy at the target locus, CoIAl (Collagen Al intron 1) such that the use of Southern blot analysis for each clone is not required. Each clonal ES line is induced by the addition of DOX and expression is discernable by detecting fluorescence as a result of the incorporation of the green fluorescent protein (GFP) in an EMCV internal ribosome entry site (IRES) 3' to the cDNA (see Lindeberg and Ebendal, Nucleic acids Res. 27: 1552-1554, 1999). The B6/C57 ES cell line is engineered such that the expression of the Cre recombinase protein is inducible upon the addition of DOX. In turn, the Cre recombinase excises the transcriptional STOP cassette 5' to the transgene and results in the expression of the inserted transgene. Each cell line is sequenced for ID and verification purposes. Optionally, the DOX- inducible rtTA transactivator driving Cre recombinase expression from the Rosa26 locus is bred out of the eventual transgenic mouse at a later stage if a tissue-specific Cre driver should be bred in.

Example 4: Transgenic mice

The methods described herein have been reliably and successfully used to predict and control tissue-specific target gene expression in transgenic mice. For example, transgenic mice that contain members, e.g. SIRT 1-7, of the SIRT family of longevity genes were produced. Figures 1-7 illustrate the methods used herein for selection and control of tissue specific inducible expression of desired transgenes. Immunohistochemical staining demonstrated tissue-specific overexpression of SIRTl in the hypothalamus of PomC-Cre/SIRTl transgenic mice as compared to wildtype mice (Figure 23). Moreover, western blot analysis illustrated increased expression of SIRT2 in three different regions of the brain (cerebellum, cortex, and hippocampus) of Nestin-Cre/SIRT2 transgenic mice as compared to wildtype mice (Figure 24). Additionally, SIRT5 was specifically overexpressed in the pancreas of transgenic mice as compared to wildtype mice (Figure 25).

The APC^min mouse model of colorectal cancer provides a quantitative measurement of tumor multiplicity, allowing for quantitative trait locus analysis. Immunohistochemical staining of the intestinal villi of SIRTl ^Stop/Villin-Cre transgenic mice in the APC^mm background revealed increased expression of SIRTl in the villi of the intestine as compared to control mice, demonstrating that tissue-specific expression of a target gene was controlled using the methods described herein (Figure 27). The expression of SIRTl increased 3 to 5 fold in SIRTl^⁰" mice in the APC^min background as compared to SIRTl^stop mice (Figure 28). No structural abnormalities were detected in the intestines of the transgenic mice. Without the transgene, mice become anemic due to bleeding in the gut. In contrast, after 14 weeks, the SIRTl ^¹⁰¹* transgenic mice (expressing the transgene) showed no signs of anemia (Figure 29) and 5 fold fewer adenomas in the intestine as compared to SIRTl^stop mice (Figure 30).

These data demonstrate the specificity of gene expression in transgenic mice produced from ES cells selcted by screening an ES cell library for a desired phenotype. The data also show that the transgene is regulated, e.g., switch on in the desired target tissue, e.g. neurons. The methods are applicable to any gene in any target tissue, i.e., any tissue or organ of interest. The libraries and methods described herein are useful to reliably select and predict the phenotype of a transgenic mammal, e.g., a mouse, with selected phenotypic characteristics. Transgenic animal models for human conditions and pathologies have been successfully and predictably produced in this manner.

Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A plurality of embryonic stem (ES) cell clones, each of said clones comprising a single copy of a transgene integrated into a predetermined genomic site, the expression of said transgene being inducible, wherein said plurality comprises a representative fraction of an ORFeome of an species.

2. The plurality of claim 1, wherein said species is a heterologous species relative to the species of said ES cell.

3. The plurality of claim 1, wherein said plurality comprises at least 85% of said ORFeome.

4. The plurality of claim 1, wherein said plurality comprises at least 90% of said ORFeome.

5. The plurality of claim 1, wherein said plurality comprises at least 95% of said ORFeome.

6. The plurality of claim 1, wherein said predetermined site is CoIAl.

7. The plurality of claim 1, wherein said expression is induced by contacting said cell with DOX.

8. The plurality of claim 1, wherein said ES cell is of murine origin.

9. The plurality of claim 1, wherein said plurality comprises 5,000 clones.

10. The plurality of claim 1, wherein said plurality comprises 15,000 clones

11. The plurality of claim 1, wherein said plurality comprises 25,000 clones

12. The plurality of claim 1, wherein said plurality comprises 30,000 clones

13. A method for predicting a phenotype of a transgenic animal prior to production of said animal comprising, providing a plurality of embryonic stem (ES) cell clones, each of said clones comprising a single copy of a transgene integrated into a predetermined genomic site, the expression of said transgene being inducible, said plurality comprising at least 85 % of an ORFeome of a heterologous species relative to the species of said ES cell; inducing transgene expression; and evaluating said plurality of clones to identify an ES cell phenotype, wherein said ES cell phenotype of said ES cell clone is predictive of said transgenic animal phenotype.

14. A method for predicting a transgenic animal phenotype prior to production of said animal, comprising contacting a plurality of ES cell clones with a differentiating agent, each of said clones comprising a single copy of a transgene integrated into a predetermined genomic site, the expression of said transgene being inducible; inducing transgene expression; and evaluating said plurality of clones to identify an ES cell phenotype, wherein said ES cell phenotype of said ES cell clone is predictive of said transgenic animal phenotype.

15. The method of claim 14, wherein said differentiating agent is selected from the group consisting of retinoic acid, dexamethasone, and 5-azacytidine.

16. The method of claim 14, wherein said transgenic animal phenotype is increased longevity compared to a wild type animal.

17. The method of claim 14, wherein said transgenic animal phenotype is characterized by a symptom of a pathologic condition.

18. The method of claim 14, wherein said ES cell phenotype is impaired neurotransmission and wherein said transgenic animal phenotype is characterized by a symptom of a neurodegenerative disease.

19. The method of claim 14, wherein said ES cell phenotype is reduced superoxide dismutase (SOD) activity and wherein said transgenic animal phenotype is characterized by a symptom of amyotrophic lateral sclerosis (ALS).

20. The method of claim 14, wherein said ES cell phenotype is increased p25 expression and wherein said transgenic animal phenotype is characterized by a symptom of Alzheimer's Disease.

21. The method of claim 14, wherein ES cell phenotype is increased sirtuin activity and wherein said transgenic animal phenotype is increased longevity.

22 A surface comprising a plurality of wells, wherein each of said wells contains an ES cell clone, said ES cell clone comprising a transgene and wherein each ES cell clone comprises at least one ES cell that has been identified as expressing or not expressing a transgene.