WO2008137031A2 - Panels of genetically diverse samples and methods of use thereof - Google Patents

Abstract

Provided herein are panels of genetically diverse biological samples and methods of use thereof. Exemplary biological samples are those that include cells or tissue. Panels comprise at least 5 genetically diverse samples. The samples may be used for a variety of purposes including examination of the toxicity, effectiveness and/or selectivity of a compound on a genetically diverse population.

Description

PANELS OF GENETICALLY DIVERSE SAMPLES AND METHODS

OF USE THEREOF

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/927,639, filed May 4, 2007, which application is hereby incorporated by reference in its entirety.

BACKGROUND

In recent years it has become accepted that drug efficacy and safety are highly dependent upon the genetic makeup of the individual. This led to the development of the field of pharmaco genomics/pharmacogenetics which involves the study of the correlation between genetic variation and differential drug responses. With the realization of the influence of genetics on drug efficacy and toxicity, it has become apparent that a specific inbred mouse strain or a single individual will give only limited information regarding a drug's activity and metabolism. Furthermore, many assays that utilize cell lines are of limited value. Usually cell lines are established transformed cell lines that are used for in vitro analysis of the efficacy or mechanism of action of a compound. The advantage of such cell lines is that they are easily available, widely used and well characterized. However, a downside is that due to in vitro culturing and transformation these cell lines have highly abnormal karyotypes, genotypes and hence phenotypes, and therefore are biased regarding the results obtained and are not representative of real populations of individuals. Such cell lines are not useful for understanding the influence of genetics on compound efficacy. Therefore new methods are needed to evaluate and address pharmacogenomic effects during drug development.

SUMMARY

In one aspect, the invention provides a panel comprising at least five genetically diverse biological samples from a given species. The biological samples may comprise cells or tissue (including, for example, tissue slices). In certain embodiments, the panel may comprise at least 5, 10, 20, 30, 40, 50, or 100 genetically diverse biological samples. In an exemplary embodiment, the biological samples are provided in a standard multi-well plate format, e.g., 4, 6, 12, 24, 48, 72, 96, 384, or 1536 well plate format.

In certain embodiments, the biological samples of a panel may comprise cells from the same tissue type or types, or cells that are same cell type or types. hi certain embodiments, at least one of the biological samples in a panel is from an inbred population or an outbred population. hi certain embodiments, the biological samples are vertebrate samples (such as, for example, fish, mouse, rat or human samples), plant samples, or algae samples, hi an exemplary embodiment, the biological samples are mammalian samples, hi other embodiments, the biological samples are rodent samples (e.g., mice or rat) or primate samples (e.g., monkey or human). hi certain embodiments, the biological samples are from rodents and wherein each of the five individuals is separated from the other individuals by at least 20 generations, hi certain embodiments, the biological samples are from primates and wherein each of the five individuals is separated from the other individuals by at least 4 generations, hi certain embodiments, the biological samples are from humans and each of the five individuals is from a different ethnic group.

In certain embodiments, the biological samples comprise endothelial cells, connective tissue cells, epidermal cells, hematopoietic cells, stem cells or differentiated daughter cells derived from stem cells, nervous system cells, endocrine cells, spermatogonial stem cells, tracheobronchiolar cells muscle cells, urogenital cells, cardiac cells, digestive tract cells, umbilical cord cells or cells from amniotic fluid. hi certain embodiments, the biological samples comprise liver cells or fibroblasts. hi certain embodiments, at least one of the biological samples comprises a primary cell, a stem cell, or a genetically modified cell. hi certain embodiments, at least one of the biological samples comprises a cell having at least one reporter gene, hi certain embodiments, each biological sample comprises a cell having the same reporter gene or, alternatively, at least two biological samples comprise a cell having different reporter genes. In certain embodiments, at least one of the biological samples is from a wild mouse, a laboratory mouse strain, a mouse disease model, a transgenic mouse, a mouse having a knock out gene mutation, a mouse having a knock in gene mutation, or a mouse having a chemically induced mutation. In certain embodiments, the genetic diversity of the biological samples of the panel may be determined using clustering analysis or a phylogenetic tree.

In an exemplary embodiment, the invention provides a panel comprising at least five genetically diverse biological samples isolated from a given species, wherein the biological samples are obtained from five individuals of the species that are genetically diverse based on clustering analysis or a phylogenetic tree, and wherein the biological samples comprise cells of the same cell type or tissues of the same tissue type. hi another aspect, the invention provides a panel comprising at least five genetically diverse biological samples comprising cells of a first cell type and at least five genetically diverse biological samples comprising cells of a second cell type, wherein each of the cells of the first and second cell types are from the same species.

In another aspect, the invention provides a method for producing a panel of genetically diverse biological samples, comprising: (i) determining the genetic relatedness of a plurality of biological samples from the same species; and (ii) selecting at least five biological samples based on the degree of genetic diversity among the biological samples, thereby producing a panel of genetically diverse biological samples.

In certain embodiments, the method may further comprise performing genotype and/or phenotype analysis on a plurality of biological samples from the same species.

In certain embodiments, genotype analysis is performed on at least 5, 10, 25, 50, 100, 250, 500, or more biological samples from the same species. Genotype may be carried out, for example, using SNP analysis, microsatellite markers, DNA microarray analysis, sequencing, or combinations thereof. hi certain embodiments, the genetic relatedness of the plurality of biological samples is determined using bioinformatic analysis to construct a dendrogram, using clustering analysis, using parsimony analysis, or PHYLIP analysis. In an exemplary embodiment, at least one biological sample from each main branch of a common node is selected.

In certain embodiments, at least 5, 10, 20, 30, 40, 50, or 100 biological samples that are genetically diverse are selected. In an exemplary embodiment, the biological samples are provided in a standard multi-well plate format, e.g., 4, 6, 12, 24, 48, 72, 96, 384, or 1536 well plate format.

In certain embodiments, the biological samples are vertebrate samples (such as, for example, fish, mice, rat or human samples), plant samples or algae samples. In an exemplary embodiment, the biological samples are mammalian samples. hi certain embodiments, at least one of the biological samples is from an inbred individual or an outbred individual. hi certain embodiments, the biological samples are from different individuals of a given species having a common trait. hi another aspect, the invention provides a method for evaluating the effect of at least one compound on a genetically diverse population, comprising: (i) contacting a panel of biological samples with the compound, wherein the panel comprises at least five genetically diverse biological samples from the same species; (ii) observing at least one response of each of the biological samples to the compound; and (iii)comparing the responses of each of the biological samples, thereby evaluating the effect of the compound on a genetically diverse population.

In certain embodiments, the toxicity, effectiveness, or selectivity of the compound is evaluated.

In certain embodiments, the compound is a chemical, small molecule, nucleic acid (such as, for example, DNA, RNA, an oligonucleotide, a nucleic acid analog, an artificial nucleic acid, a peptide nucleic acid (PNA), a morpholino, a glycerol nucleic acid (GNA), a threose nucleic acid (TNA), pseudo-complementary DNA (pcDNA), a locked nucleic (LNA) acid and variants and homologs thereof, an antisense nucleic acid, RNAi compound, or enzymatic nucleic acid), polypeptide (such as, for example, a protein, enzyme, peptide, antibody, or alternative binding protein), a virus, a bacteria or mixtures of any of the foregoing. Compounds may be purified or part of a mixture, such as for example, a cellular extract, fractionated cell extract, a virus or bacterial extract, etc.

In certain embodiments, the compound is a drug, clinical drug candidate, cosmetic agent, environmental pollutant, industrial chemical, vaccine, or bacterial toxin.

In certain embodiments, the compound is formulated in a pharmaceutically acceptable carrier.

In certain embodiments, the biological samples comprise cells having at least one reporter gene. In certain embodiments, the method further comprises correlating a response of at least one of the biological samples with a genetic marker. hi certain embodiments, a response of the biological samples is observed using an optical assay, a gene expression assay (for example, measuring production of mRNA or protein), a phenomenological assay, a physiological transport assay, a physiological secretion assay, an apoptosis assay, a cell proliferation assay, or a toxicity assay.

In certain embodiments, the biological samples are vertebrate samples (such as, for example, fish, mice, rat or human samples), plant samples, or algae samples. In an exemplary embodiment, the biological samples are mammalian samples. hi certain embodiments, a plurality of compounds is evaluated simultaneously in a high throughput format.

In another aspect, the invention provides a method for producing a panel of phenotypically diverse biological samples, comprising: (i) performing a phenotypic clustering analysis for a plurality of biological samples from the same species; and (ii) selecting at least five phenotypically diverse biological samples based on the clustering analysis, thereby producing a panel of genetically diverse biological samples.

In another aspect, the invention provides a kit comprising a panel comprising at least five genetically diverse biological samples from a given species. In certain embodiments, the kit may comprise a panel comprising at least 10,

20, 30, 40, 50, or 100 genetically diverse biological samples. In an exemplary embodiment, the biological samples are provided in a standard multi-well plate format, e.g., 4, 6, 12, 24, 48, 72, 96, 384, or 1536 well plate format. In certain embodiments, the biological samples may be cryopreserved or may be cell or tissue cultures. The biological samples may be provided in separate containers, such as, for example, a multiwell tissue culture plate. The kit may additional comprise one or more of the following: culture media, instructions, or reagents for a screening assay.

The appended claims are incorporated into this section by reference.

BRIEF DESCRIPTION OF THE FIGURES The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIGURE 1. Effect of acetaminophen on hepatocytes from different mouse strains. FIGURE 2. Analysis of cell viability of mouse embryonic fibroblasts (MEF) after treatment with acetaminophen (APAP).

FIGURE 3. An example of a phylogenetic tree constructed by a clustering program like PHYLIP.

FIGURE 4. Cluster analysis of the human population (reproduced from the world wide web at dnatribes.com).

FIGURE 5. Phylogenetic tree of 102 inbred mouse strains (modified version of Figure 3 of Petkov et al. (2004)).

FIGURE 6. Analysis of cell viability of mouse embryonic fibroblasts (MEF) from 5 different strains after treatment with acetaminophen (APAP).

DETAILED DESCRIPTION Panels

Provided herein are panels of genetically diverse biological samples that can be used as model systems reflecting the degree of natural genetic variation in a population. Biological samples include cells or tissue from an organism. The panels provide a quasi-unlimited and consistently reproducible resource. Also provided are methods of using the panels for evaluating the differential response of genetically diverse biological samples to a compound. The source of biological samples used in the panel may be chosen according to an intended use, such as, for example, toxicity screening, efficacy screening, drug repositioning, pathway identification, or mechanism of action studies. The panels provided herein comprise at least five genetically diverse biological samples from the same species. In certain embodiments, the panels may comprise at least 5, 10, 25, 50, 100, 150, 200, 250, 500, 1000, or more genetically diverse biological samples from the same species. The number of samples to be used in a panel may be determined based on a variety of considerations including, for example, the desired use of the panel, the type of biological samples being used, the depth of genotypic analysis, the source species of the biological samples, the availability of samples, the range of genetic diversity available, etc.

The panels provided herein comprise genetically diverse biological samples that are of the same species. The samples may be obtained from any organism including, for example, mammals, plants, algae, fish, amphibians, birds or invertebrates. Exemplary mammals include humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats). Exemplary plants include monocots and dicots, such as crop plants, ornamental plants, and non-domesticated or wild plants. Invertebrates include, for example, arthropods, annelids, nematodes, and molluscs. Suitable biological samples include any sample obtained from an organism, plant or algae. For example, biological samples from an organism may include, for example, a cell containing body fluid sample, a cell sample, or a tissue sample. Suitable biological samples include, but are not limited to, samples of pleural fluid, pulmonary or bronchial lavage fluid, synovial fluid, peritoneal or ascites fluid, pericardial fluid, bone marrow aspirate, lymph, cerebrospinal fluid, amniotic fluid, liposuction fluid, mucosal tissue, foreskin material, cadaver material, sputum, bladder washes, semen, urine, saliva, tears, cord blood, blood, serum, plasma, bone marrow, abortus material and the like. Biological samples from plants may include, for example, samples of leaf (e.g., petiole and blade), root, stem (e.g., tuber, rhizome, stolon, bulb, and corm) stalk (e.g., xylem, phloem), wood, seed, fruit (e. g., nut, grain, fleshy fruits), or flower (e.g., stamen, filament, anther, pollen, carpel, pistil, ovary, ovules). In certain embodiments, the biological samples may be algae samples or yeast samples. In exemplary embodiments, the biological samples comprise cell and/or tissue samples from an organism, plant or algae. hi certain embodiments, biological samples may be obtained from cadavers. Cadavers may be a source for cells or tissue samples of a wide variety including, for example, cornea, retina, optic nerve, bone marrow, spinal cord, bone, cartilage, brain, olfactory tissue, pancreas, heart, lung, kidney, liver, intestine, testis, skin, tongue, or skeletal muscle. hi certain embodiments, the biological samples comprise cells. The cells may be a purified population of cells or may be a mixture of cells. For example, a cell sample having a mixture of cells may be purified using any of a variety of techniques known in the art including, for example, Fluorescence Activated Cell Sorting (FACS) to isolate a specific cell population out of the cell mixture or to enrich the cell mixture for a specific cell type. Alternatively, the cell mixture may be used directly in the panel. When using a cell mixture it is generally desirable that at least a portion of the samples used in the panel is similar in composition. For example, a variety of cell mixtures may be isolated from a given source, e.g., a specific organ, a specific region within an organ, or a specific tissue, etc., from a variety of individuals and thus would have a similar composition. Alternatively, a variety of similar cell mixtures could be produced by treating a variety of cell samples with the same conditions. For example, a variety of stem cell samples could be treated to the same differentiation conditions to produce a mixture of cell types which would be similar among the different samples. Alternatively, a variety of stem cell samples could be treated to the same differentiation conditions to produce a single cell type which would be similar among the different samples. In certain embodiments, techniques known in the art such as, for example, FACS, gradients, picking, differential attachment, migration, laser capture microdissection or magnetic bead separation could be used to characterize a given cell mixture, alter the mixture so that it is similar to another mixture, or even to separate an individual cell type from a given cell mixture. hi certain embodiments, the biological samples comprise tissue samples.

Tissue samples may be obtained using art recognized techniques such as, for example, a needle biopsy, skin biopsy, surgical resection, etc. The tissue samples can be obtained, for example, from brain, retina, spinal cord, gut, liver, kidney, ovary, testis, pancreas, spleen, thymus, adrenal gland, heart, prostate, mammary gland, breast, lung, nasal, endometrium, cervix, skin, bone marrow, small intestine, large intestine, biliary tract, bladder, bone, cartilage, cervical, lymph node, muscle, myocardium, nerve tissue, spinal cord, salivary gland, synovium, tendon or tongue. Tissue samples can be used directly, cultured in vitro, or stored until use. In certain embodiments, tissue samples used in a panel may be tissue slices. Tissue slices are thin slices from fresh or cryopreserved tissue. Tissue slices can be isolated from embryonic or adult tissue and cultured in vitro. Exemplary methods for organotypic tissue slice cultures include (i) the roller tube technique (Gahwiler BH, J. Neurosci. Meth. 4.1981) and (ii) the semi-permeable membrane technique (Stoppini et al., J. Neurosci. Meth. 37: 173, 1991; Tauer et al., Appl. Neurobiol. 22:361-369, 1996). hi certain embodiments, the biological samples comprise primary cells from an organism, plant or algae. Primary cells are cells or cell lines obtained directly from an organism or tissue which have not been immortalized. Primary cells have a basically normal karyotype as compared to transformed cell lines which typically have abnormal karyotypes due to chromosomal rearrangements. Primary cells may be isolated from tissue or tissue fluids (e.g., blood, amniotic fluid, etc.) and cultured. Mammalian primary cells can be from embryonic tissue or from adult tissue.

Examples of embryonic-derived primary cells include, for example, embryonic stem cells or embryonic stem-like cells (such as, for example, morula, blastomer, blastomer-like, blastocyst-, gonad- or amniotic-derived cells), fibroblasts, keratinocytes, fetal liver cells, fetal thymus derived stroma cells, fetal cardiomyocytes, fetal gonads, primordial germ cells, bone marrow cells, bone marrow stromal cells, embryonic neuronal cells, and embryonic neuronal stem cells or derivatives of these cell types. Examples of adult-derived primary cells include, for example, fibroblasts (for example from skin, foreskin, ear punch, lung biopsy, aorta, ligament, periodont, gingival, meringeal, choroid plexus, cardia, conjunctiva, etc.), hepatocytes, liver cells (such as, for example, Kupffer cells, Ito cells, stellate cells), renal tubule cells, bone marrow cells, bone marrow stromal cells, monocytes, macrophages (such as, for example, histiocytes, Langhans giant cells, microglia), dendritic cells (such as, for example, Langerhans cells), T cells, B cells, granulocytes (such as, for example, neutrophil, eosinophil, basophil), megakaryoblasts, megakaryocytes, platelets, stroma cells, lymphocytes, endothelial cells, neuronal cells, neurons, glia cells (such as, for example, oligodendrocytes, Schwann cells), astrocytes, Purkinje cells, dorsal root ganglia, stem cells (such as, for example, mesenchymal stem cell, hematopoietic stem cell, neuronal stem cell, cord blood-derived stem cell, fat-derived stem cell, muscle-derived stem cell, skin- derived stem cells, spermatogonial stem cells, epithelial stem cells, keratinocyte stem cells), keratinocytes, melanocytes, follicle dermal papilla cells, epithelial cells, stromal cells, chondrocytes, bone cells (such as, for example, osteoblasts, osteocytes, osteoclasts), adipocytes, preadipocytes, islet cells, endoderm-derived cells, myocytes, cardiomyocytes, smooth muscle cells, cornea cells, epithelial cells (such as, for example, mammary, bronchial, or prostate), connective tissue cells, epidermal cells, endocrine cells, lung cells, urogenital cells, cardiac, digestive tract cells, oocytes, umbilical cord cells or amniotic fluid cells. hi an exemplary embodiment, the biological samples comprise stem cells. Suitable stem cells include adult stem cells or embryonic stem cells. Adult stem cells include but are not limited to mesenchymal stem cells, hematopoietic stem cells, neuronal stem cells, spermatogonial and epithelial stem cells. Stem cells can be isolated, for example, from amniotic fluid, cord blood, blood, bone marrow, liposuction fluid, synovial fluid, mucosal tissue, testis, foreskin, biopsy, abortus or cadaver material. Stem cells can be used directly, or after culturing and expanding, after cryopreservation, as undifferentiated cells or as differentiated cells. For example, non-hematopoietic mesenchymal or hematopoietic stem cells from the bone marrow may be mobilized with granulocyte colony stimulating factor (G-CSF) treatment. Cells isolated from blood or bone marrow may be sources for a variety of cells including erythrocytes, leukocytes (granulocytes (neutrophils, basophils, and eosinophils), agranulocytes (lymphocytes, monocytes, and macrophages)), platelets, stromal cells or fat cells. Foreskin cells may be used as a source of primary fibroblasts. Stem cell populations are desirable because they represent a quasi- unlimited resource and have a normal karyotype. It has also been reported that adult stem cells can be changed by dedifferentiation into embryonic-like stem cells which in turn can be driven to differentiate into all known cells types (see M. Raff, Annu. Rev. Cell Dev. Biol. 2003. 19:1-22; R. Hass, Signal Transduction 2005, 5 (3), 93- 102; R. Lewis 2004, The Scientist, 18(16): 20; Wagers, A. J. & Weissman, I. L. Plasticity of adult stem cells, Cell 116, 639-648 (2004)). Dedifferentiation is a process whereby a differentiated cell is caused to change to an earlier precursor cell or earlier stem cell. For example, Dr Kiminobu Sugaya (Stem Cell Laboratory, University of Central Florida) has presented data showing adult mesenchymal stem cells can be dedifferentiated by the expression of nanog in embryonic-like stem cells (see Sugaya, K. et al. 2006 Panminerva Medica 48 (2), pp. 87-96; M. Perkel, Science 2007: 316, 463-468). Stem cells can also be generated by a technique called "induced pluripotent stem cells" (iPS) using just 3 or 4 factors to generate stem cells from fibroblasts (see Takahashi et al., Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors, Cell 126: 663- 676 (2006); Maherali et al., Directly Reprogrammed Fibroblasts Show Global Epigenetic Remodeling and Widespread Tissue Contribution, Cell

Stem Cell 1: 55-70 (2007); Wernig et al., In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state, Nature 448: 318-324 (2007); Okita, et al., Generation of germline-competent induced pluripotent stem cells, Nature 448: 313- 317 (2007); Nakagawa et al., Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts, Nature Biotechnology 26: 101-106 (2007)). Alternatively, stem cells can be created by somatic cell nuclear transfer (see Byrne et al., Nature 450: 497-502 (2007)).

In developing panels of diverse cells for biological assays, the embryonic stem cells offer the highest usage capabilities as they can be essentially differentiated into all cell types. Thus the isolation or production of these cells from individuals (animals and humans) offers the greatest opportunity in screening panels. Another approach known in the art to generate such stem cells is somatic cell nuclear transfer (SCNT), which allows the transfer of a somatic cell nucleus (for example a cell nucleus isolated from a differentiated adult cell like a fibroblast or other cell from the body) into an enucleated oocyte. After further development into the 4-cell stage, 8-cell-stage 16- cell-stage, 32-cell-stage, morula or blastocyst or early embryogenesis (genital ridge formation stage), these can be used as a source to generate embryonic stem cells or primordial germ cells. For example, a skin fibroblast nucleus can be used to generate an embryonic stem cell-like cell from any individual (see Yang, X. et al. 2007, Nature Genetics, 39, 295-302; Hwang, W. S et al. 2005, Science, 308, 1777-1783). In certain embodiments, the panels may comprise plant stem cells (see e.g.,

Ben Scheres, Nature Reviews Molecular Cell Biology 8, 345-354 (May 2007)).

In certain embodiments, the primary cells may be from inbred, recombinant inbred, hybrid, genetically modified, or outbred populations. For example, inbred mice are genetically homogeneous and homozygous at all loci. The International Committee on Standardized Nomenclature for Mice and Rats has ruled that a strain of mice or rats can be considered inbred at generation F₂₀. Hybrid mouse strains are produced by crossing two inbred strains including mixed inbred strains. Outbred populations are produced by breeding schemes that avoid crosses between closely related individuals in order to maintain the maximal level of heterozygosity in the offspring.

In certain embodiments, the primary cells may be genetically modified or may carry naturally occurring mutations. For example, the primary cells can be modified by mutagenesis agents including chemically induced mutations (e.g. ENU or EMS), radiation induced mutations, or spontaneous mutations (including, for example, spontaneous mutations/modifications maintained on mouse or rat strains such as C57BL/6, 129, FVB, C3H, NOD, DBA/2, BALB/c, or CD-I). Exemplary genetic modifications include, for example, gain of function, loss of function, deletion, or disruption of a specific gene or DNA region, replacement of DNA sequences by homologous recombination methods, overexpression of one or more genes, and RNA interference (or RNAi), siRNA knockdown, shRNA, microRNA or silencing. In certain embodiments, the primary cells may be obtained from transgenic, knockout, knockin, knockdown, or other genetically modified animals. In certain embodiments, the primary cells can be modified to include one or more reporter genes (e.g., genes that produce detectable signals such as fluorescence, bioluminescence, or positron emission tomography (PET) signals), such as, for example, luciferase, green fluorescent protein (GFP), GFP derivatives, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (ECFP, Cerulean, CyPet), yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet), chloramphenicol acetyltransferase (CAT), beta- galactosidase, or alkaline phosphatase. Reporter genes can be utilized, for example, as a read-out for various assays or as a means to examine a signaling pathway (for example GPCR signaling, TGF-beta signaling, kinase activation, or other specific pathways). Reporter genes may be introduced into primary cells using any method known in the art, such as, for example, lipofection, calcium phosphate coprecipitation, electroporation or viral vectors (e.g., adenovirus, lentivirus, retrovirus etc.). hi various embodiments, primary cells may be used at an early passage, or after further manipulation or differentiation into specific or multiple cell types. For example, the primary cells may be differentiated in vitro to increase the variety of primary cell types available and the complexity of the process examined. For example, embryonic stem cells can differentiate into mesoderm, endoderm-like or ectoderm-like cells, etc; embryonic fibroblasts can differentiate into adipocyte-like cells; and a myocyte can differentiate into a myotube, etc. hi exemplary embodiments, the stem cells may be mouse, rat, primate or human stem cells, hi various embodiments, primary cells or tissue samples may be used directly or may be stored before use. For example, primary cells or tissue samples may be cryopreserved using a cryopreservative medium (e.g. 10 % DMSO, 90 % FBS or 20 % DMSO, 20 % FBS, 60 % DMEM) and stored at -196 ⁰C in liquid nitrogen. hi an exemplary embodiment, the biological samples may be from inbred mice, inbred mouse embryos, outbred mice, transgenic mice, mutagenized mice, genetically engineered outbred mice, congenic mouse strains, recombinant congenic mouse strains, or combinations thereof. Examples of suitable mouse strains are provided below in Table 1.

Table 1. Exemplary mouse strains.

In another embodiment, the biological samples may be from inbred rats, inbred rat embryos, outbred rats, transgenic rats, mutagenized or genetically engineered outbred rats, congenic rat strains, or combinations thereof. Examples of suitable rat strains are provided below in Table 2.

Table 2. Exemplary rat strains.

In another embodiment, the biological samples included in a panel may be non-human primate samples such as, for example, samples from old world monkeys (including baboons, gibbons and rhesus macaque, cynomolgus monkeys), new world monkeys, lemurs, gorilla and chimp (Pan troglodytes).

In another embodiment, the biological samples included in a panel may be human samples, including samples comprising primary human cells or viable human tissue. Biological samples from humans may be obtained directly from individuals (e.g., biopsy, blood or bone marrow samples), abortus material, from banks of human tissue, cell, or blood samples, or from cadavers, or combinations thereof. In an exemplary embodiment, the biological samples from humans comprise stem cells, including iPS cells. In another embodiment, the biological samples are plant, yeast or algae samples. Plant cell or tissue samples may be used directly upon isolation or after culturing, expanding, cryopreservation, etc. Plant cell culture methods are well established (see for example Loyola-Vargas and Vazquez-Flota 2005).

Suitable plant samples may be obtained from a variety of families of plants including, for example: Acanthaceae, Aizoaceae, Amaranthaceae, Amaryllidaceae, Anacardiaceae, Annonaceae, Apocynaceae, Araceae, Araliaceae, Aristolochiaceae, Asclepiadaceae, Basellaceae, Begoniaceae, Berberidaceae, Betulaceae, Bignoniaceae, Bombacaceae, Boraginaceae, Buddlejaceae, Buxaceae, Cactaceae, Campanulaceae, Capparaceae, Caprifoliaceae, Caryophyllaceae, Celasteraceae, Celastraceae, Chenopodiaceae, Cistaceae, Compositae, Connaraceae,

Convolvulaceae, Crassulaceae, Cruciferae, Cucurbitaceae, Dioscoreaceae, Dipsacaceae, Droseraceae, Epacridaceae, Ephedraceae, Equisetaceae, Ericaceae, Euphorbiaceae, Gentianaceae, Ginkgoaceae, Gramineae, Guttiferae, Labiatae, Lardizabalaceae, Lecythidaceae, Leeaceae, Leguminosae, Liliaceae, Linaceae, Loganiaceae, Malvaceae, Meliaceae, Menispermaceae, Monimiaceae, Moraceae, Myrtaceae, Nolanaceae, Nyctaginaceae, Oleaceae, Onagraceae, Orchidaceae, Orobanchaceae, Papaveraceae, Passifloraceae, Phytolaccaceae, Pinaceae, Plumbaginaceae, Polemoniaceae, Polygonaceae, Portulacaceae, Proteaceae, Ranunculaceae, Rosaceae, Rubiaceae, Rutaceae, Sapotaceae, Scrophulariaceae, Solanaceae, Sphagnaceae, Srcophulariaceae, Stemonaceae, Sterculiaceae, Taxaceae, Tiliaceae, Turneraceae, Umbelliferae, Valerianaceae, Verbenaceae, Violaceae,

Vitaceae, Zingiberaceae, Zygophyllaceae. Examples of the Leguminosae family include, for example: Adenantherapavonina L., Anthyllis vulneraria L., Astragalus australis (L.) Lam., Astragalus bisculatus (Hook.) A. Gray, Astragalus bisculatus (Hook.) A. Gray, Astragalus pectinatus (Hook.) G. Don, Astragalus racemosus Pursh, Caesalpinia pulcherima (L.) Sw., Calliandra tweedii Benth., Coronilla coronata L., Coronilla vaginalis Lam., Crotalaria scasselati, Crotalaria verrucosa, Cytisus canadensis (L.) O. Kuntze, Cytisus purpureus Scop., Cytisus scoparius (L.) Link, Delonix regia (Boj. ex Hook.) Raf, Genista pilosa L., Genista tinctoria L., Glycine max (L.) Merr., Glycine max (L.) Merr. cv. Mandarin, Leucaena glauca (Willd.) Benth., Leucaena latisiliqua (L.) Gillis, Leucaena leucocephala (Lam.) de Wit, Lotus ornithopodioides L., Lupinus albus L., Lupinus hartwegii Lindl., Lupinus hartwegii Lindl., Lupinus hartwegii Lindl., Lupinus mutabilis Sweet., Lupinus nootkatensis Sims., Lupinus polyphyllus Lindl., Lupinus polyphyllus Lindl., Lupinus polyphyllus Lindl., Lupinus polyphyllus Lindl., Lupinus polyphyllus Lindl., Lupinus polyphyllus Lindl., Lupinus sativum Gaertn., Medicago sativa L. cv. Europe, Onobrychis viciifolia Scop.,

Parkinsonia aculeata L., Phaseolus aureus Roxb., Phaseolus coccineus L., Phaseolus lunatus L. var. Jackson Wonder, Phaseolus vulgaris L., Poinciana pulcherrima L. subsp. caesalpinia, Sophora japonica L., Spartium junceum L., Tamarindus indica L., Tetragonolobus maritimus (L.) Roth, Trifolium repens L., Vicia dumentorum L., Vicia ervilia (L.) Willd., Vicia faba L., Vicia oroboides WuIf., Vicia sativa L., Vigna dolomitica R. Wilczek and Vigna radiata (L.) Wilczek (see world wide web at cabri.org).

Examples of the Leguminosae family include, for example: Alyssum argentum All., Alyssum wulfenianum Bernh., Arabidopsis thaliana (L.) Heynh., Armoracia rusticana (Lam.) Ph. Gaertn., B. Mey. et Scherb., Brassica napus L. emend. Metzg. var. napus cv. Bronowski, Brassica napus L. emend. Metzg. var. napus cv. Liratop, Brassica napus L. emend. Meztg. var. napus cv. Darmor, Brassica napus L.emend. Metzg. var. napus , Brassica rapa f. autumnalis (DC.) Mansfeld, Cochlearia armoracia L., and Erucastrum nasturtiifolium (Poir.) O.E. Sch. In another embodiment, the biological samples are algae or microalgae samples. Over 3000 strains of algae are available for culture including, for example, prokaryotic and eukaryotic algae, freshwater algae, salt plains algae, snow algae etc. Culture collections are available from a variety of sources (see e.g., world wide web at wdcm.nig.ac.jp/hpcc.html; world wide web at bacterio.cict.fr/collections.html; world wide be at dsmz.de; world wide web at

217.114.171.142/alginet/databasel .html). In another embodiment, a panel of genetically diverse biological samples may be a disease specific panel. For example, a panel may comprise samples from mouse or rat strains carrying specific disease phenotypes within different genetic backgrounds (e.g. diabetes, obesity, neurodegenerative diseases, etc.).

In another embodiment, a panel of genetically diverse biological samples may be a disease susceptibility-specific panel. For example, a panel may comprise samples from mouse or rat strains carrying specific susceptibility genes within different genetic backgrounds.

In another embodiment, a panel of genetically diverse biological samples may be a pathway-specific panel. For example, a panel may comprise samples from mouse or rat strains having a genetic composition permitting examination of a signaling pathway. Suitable mouse or rat strains for use in a pathway-specific panel include, for example, mouse or rat strains lacking one or more genes in a pathway and/or mouse or rat strains overexpressing one or more genes in a pathway.

Exemplary genes include, for example, kinases (src, trk, lck, fyn), p53, caspases, beta-catenin, TGF-beta, TNF, EGF, Insulin, VEGF, interleukins, GPCR, PPAR, NF- kappaB, Erk, TLR, Akt, ion channels, multidrug resistance, p450, nitric oxide, protein phosphorylation, or cell cycle genes, etc.

Genetic Diversity A panel having suitable genetic diversity may be selected on the basis of phenotype information, genotype information, or a combination thereof. The phenotype and/or genotype information may be obtained from publicly available sources and/or generated using art recognized techniques. The genetic diversity between various biological samples may be determined by analyzing the genotype information using standard bioinformatics techniques such as, for example, clustering tools or computational phylogenetic tools that can be used to construct phylogenetic trees or dendrograms representing genetic relationships between samples (see Joseph Felsenstein (2003), Inferring Phytogenies, Sinauer Associates, ISBN-10: 0878931775).

Phenotype information may be obtained, for example, by examination of physical appearance and constitution, a trait, behavior, clinical parameters, cellular, biochemical or molecular parameters. Depending on the phenotype to be studied, appropriate parameters may be chosen by one of skill in the art based on the disclosure provided herein. For example, parameters for obesity may include weight or body mass index and parameters for type 2 diabetes may include impaired glucose tolerance or insulin resistance. Genotype information can be obtained using a variety of art recognized techniques including but not limited to single nucleotide polymorphism (SNP) analysis, simple sequence length polymorphism (SSLP) analysis, fragment analysis or partial, shotgun or complete sequencing approaches, allele specific hybridization (ASH), Single Base Chain Extension (SBCE), Allele Specific Primer Extension (ASPE), Oligonucleotide Ligation Assay (OLA), DNA microarrays or other assays using methods applying polymerase extension, oligonucleotide ligation, enzymatic cleavage, flap endonuclease discrimination, hybridization, sequencing, fluorescence, colorimetry, chemiluminiscence, mass spectrometry or combinations thereof.

Genotype data may be analyzed using bioinformatics tools known in the art to establish the genetic relatedness between samples. Suitable analytical tools include but are not limited to supervised and unsupervised cluster analysis tools, computational phylogenetic tools, PHYLIP (world wide web at evolution.genetics.washington.edu/phylip.html), maximum parsimony (MP) analysis, Fitch-Margoliash method, UPGMA (Unweighted Pair Group Method with Arithmetic mean) method, Akaike information criterion (AIC) analysis, Sankoff- Morel-Cedergren algorithm, likelihood ratio test (LRT), maximum likelihood, Bayesian inference, distance matrix and other likelihood methods, including bootstrapping and consensus trees. A typical clustering analysis involves two stages: (1) evaluating similarities using some defined distance measure among the subjects, and (2) grouping the subjects using the calculated distance matrix.

The results of such analyses can be displayed, for example, as phylogenetic trees or dendrograms representing the degree of genetic distance between a variety of samples. Haplotype blocks and genome wide association studies (GWAS) are exemplary tools that can used to facilitate selection of appropriate samples from a genetic analysis (see Bogue and Grubb, 2004; Pletcher et al. 2004; Zhang, J. et al 2005). For example a group of individuals for a study can be selected from inbred strains (each strain can be considered as a quasi immortal genetically stable living organism) or individuals selected from a population of outbred organisms, e.g., humans. Although not immortal, humans are long lived, and offer the potential of continual resampling via primary cells or potential "stem cells", e.g., hematopoietic stem cells, mesenchymal stem cell, amniotic stem cells, embryonic stem cells, blood, bone marrow cells, neuronal stem cells, spermatogonial cells, iPS stem cells, etc. These cells may be expanded, frozen or cryopreserved to generate long lived resources. Similarly, from one tissue many tissue slices can be prepared and also cryopreserved for later usage. The initial group can be a randomly selected group within a species or selected using some defined criteria, either phenotype or genotype (see e.g., the DNA tribe tree in Figure 4). A wide range of parameters may be chosen as phenotype criteria, such as, for example, a metabolic parameter, blood pressure, blood typing, enzyme activity, weight, body mass index, biochemical or biophysical, chemical parameters or gene expression data (RNA or protein). For genotype criteria, sequencing data, microsatellite marker data, DNA fingerprinting data, haplotype block data, SSLP marker analysis data, SNP analysis data, fragment analysis or partial, allele specific hybridization (ASH) data, whole genome hybridization data, Single Base Chain Extension (SBCE) data, Allele Specific Primer Extension (ASPE) data, Oligonucleotide Ligation Assay (OLA) data, hybridization data, PCR data, DNA microarray data or other genomic data can be used.

Alternatively individuals in the group may be selected to be included in a panel on the basis of the relationship of their genotypes, for example five or more individuals may be selected from the study group on the basis that their genotypes represent the maximum genetic diversity of the study group. For example, individual 1 is selected, then the genotype of individual 1 is compared to the genotypes of all others in the study group to identify the most unrelated individual, which is selected as individual 2. Then the genotypes of individuals 1 and 2 are compared to all other genotypes in the study group in order to select individual 3, etc.

Alternatively individuals in the group may be selected to be included in a panel on the basis of the relationship of their phenotypes, for example five or more individuals may be selected from the study group on the basis that their phenotypes represent the maximum phenotypic diversity of the study group. For example, individual 1 is selected, then the phenotype of individual 1 is compared to the phenotypes of all others in the study group to identify the most unrelated individual, which is selected as individual 2. Then the phenotypes of individuals 1 and 2 are compared to all other phenotypes in the study group in order to select individual 3, etc.

The initial sample group (e.g., 100 individuals) is then subjected to genotyping (for example, ranging from 10's of SNPs to millions of SNPs to complete DNA sequencing). Using this data, the initial sample group is subject to unsupervised clustering (e.g., using PHYLIP (the PHYLogeny Inference Package) or PAUP*). PHYLIP is available on the world wide web at evolution.genetics.washington.edu/phylip/getme.html. The clusters are composed of those individuals which are the most related based on genotyping data. Using the cluster method, for example, 100 individuals can be distributed into 5 groups, 10 groups, or more. These methods are without an a priori hypotheses and represent the most significant solution possible. At least one member from each group is selected. Any member of a given group can be used for constructing the panel. Figure 3 shows an example of a tree constructed by a clustering program like PHYLIP. The program groups the individual members analyzed according to their genetic relatedness. The user will define the parameter of how many groups are needed, e.g., 5 if a panel of 5 primary cells is to be made, 6 if the panel should contain 6 primary cells, 10 if the panel contains 10 primary cells and so on.

Using the genotype information of the initial sample group (e.g., 100 individuals) a dendrogram can be generated using computational algorithms, which include distance-matrix methods such as neighbor-joining or UPGMA, ClustalW, parsimony analysis, maximum likelihood and/or Bayesian inference. Such tools calculate the genetic distance from multiple sequence alignments. The calculated data can then be presented as a diagram, known as a dendrogram or phylogenetic tree. For example an unrooted tree can be constructed, which can be bifurcating or multifurcating. Closely related individuals are under the same interior node with the branch length displaying the distance between individuals. The distance is defined as the fraction of mismatches at aligned positions, which represents the number of genetic changes. More distantly related individuals are under different interior nodes. Preferably, first individuals from different nodes are chosen. When individuals from the same node are chosen, then the most distant on the branch are chosen with the branch length representing the distance. For example when the parsimony analysis results in five nodes, one individual from each node is chosen, resulting in a panel with five individuals. When a panel of five needs to be generated and the dendrogram has three nodes, then one individual from each node is selected and an additional two individuals from the most distant branches are chosen. When a panel of five needs to be generated and the dendrogram has seven nodes, then five individual from the most distant nodes are selected.

Instead of genotype data phenotypic data can be used to perform cluster analysis or create dendrograms as described for genotype data above. The selection of the individuals for a panel is done in the same way by selecting the most phenotypically diverse individuals for a panel.

Alternatively individuals may be selected to be included in a panel on the basis of the relationship of their genotypes or phenotypes or combinations thereof, for example, five or more individuals may be selected on the basis that their genotypes represent the maximum genetic or phenotypic diversity. For example, individual 1 is selected, then the genotype or phenotype of individual 1 is compared to the genotypes or phenotypes of all others to identify the most unrelated individual, which is selected as individual 2. Then the genotypes of individuals 1 and 2 are compared to all other genotypes in order to select individual 3, etc.

Genotype and phenotype information about a wide variety of mouse strains is publicly available. For example, a phylogenetic tree representing the genetic distance of 102 inbred mouse strains has been constructed using 1638 SNP markers

(see Petkov et al. 2004). Other examples of phylogenetic trees of inbred mouse strains are described in Beck et al. 2000 (see world wide web at informatics.jax.org/mgihome/genealogy/) and Tsang et al. 2005. As additional genotype information becomes available, a given phylogenetic tree may be added to or adjusted based on the new information. For example, there are over 450 inbred mouse strains and as new genotype information becomes available, e.g., new sequencing data, refinement of haplotype blocks, additional SNP or SSLP markers, additional mice strains may be added to the described phylogentic trees or used to construct new phylogenetic trees. New SNP data and databases are regularly released providing additional genotype information (Reuveni 2007, Agrafioti 2007). Genotype data is also available on the world wide web (e.g. atjax.org/phenome/; informatics.jax.org/strains_SNPs.shtml; snp.gnf.org; ncbi.nlm.nih.gov/SNP/; sanger.ac.uk/Projects/M_musculus/; well.ox.ac.uk/mouse/; broad.mit.edu/mouse/hapmap/; mouseibd.florida.scripps.edu:9865/snpstrainsl40k/index.jsp; and nervenet.org). Phenotypic data can be found on the world wide web, for example the mouse phenome database (PMD) (world wide web atjax.org/phenome) or other phenotype data sites (e.g. world wide web at nervenet.org; and functionalglycomics.org).

The phylogenetic tree constructed by Petkov et al. organized 102 mouse strains into seven groups. The data provided by Petkov et al. may be used to exemplify selection of genetically diverse sources for biological samples. For example, a genetically diverse panel may be chosen by selecting at least five different mice strains from among the seven groups represented on Petkov's phylogentic tree (see Figure 5). In Figure 5, the phylogenetic tree of Petkov et al. (2004) is modified to highlight the process of going from one group, to two groups etc. (see arrows in Figure 5). This process allows a logical stepped clustering of heterogeneous populations to groups which are held together by shared characteristics. The arrows highlight how to choose five genetically diverse groups wherein any of the individuals of those five groups can be included in the panel. As an example, a genetically diverse panel of five samples could comprise biological samples from: one line from group 1 (e.g., Balb/c) or group 2 (e.g., NOD/LtJ), one line from group 3 (e.g., NZW or NON/LtT), one line from group 4 (e.g., C57BL/6) or group 5 (e.g., 129Sl/SvImJ), one from group 6 (e.g., DBA) and one from group 7 (e.g., Cast/EiJ). As another example, a panel may comprise a biological sample from at least one mouse strain from each of the seven groups. In yet other examples, biological samples multiple strains from each group may be selected to produce panels having biological samples from at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or even all 102 of the mouse strains represented in Petkov's phylogenetic tree.

Genotype and phenotype information about a variety of rat strains is also publicly available and can be used in the construction of phylogenetic trees. For example, SNP analysis data for at least four different rat strains is available in the public domain (Zimdahl et al 2004, Guryev et al. 2004, Smits et al. 2004 and see world wide web at hgsc.bcm.tmc.edu/projects/rat/).

Genotype and phenotype information about a variety of primates is publicly available and can be used in the construction of phylogenetic trees. For example, sequence data is available on the world wide web at genome.wustl.edu/ or ncbi.nih.gov/Genbank. A large amount of phenotype and genotype information for humans is also available. For example, the international HapMap project describes the common patterns of human genetic variation for four populations: 30 adult-and-both-parents trios from Ibadan, Nigeria (YRI); 30 trios of U.S. residents of northern and western European ancestry (CEU); 44 unrelated individuals from Tokyo, Japan (JPT); and 45 unrelated Han Chinese individuals from Beijing, China (CHB) (see world wide web at hapmap.org/; ncbi.nlm.nih.gov/SNP; snp.cshl.org/; snpnet.jst.go.jp/top e.html; and International HapMap Consortium. (2007) A second generation human haplotype map of over 3.1 million SNPs, Nature Vol. 237, p. 851- 861). Additionally, publicly available databases currently contain over 6 million SNPs (see for example NCBI, HapMap, SeattleSNP and Celera) and this number is steadily growing. Other genotype information can be found at dnatribes.com on the world wide web where genetic studies have demonstrated that humans on the African continent are the most genetically diverse (see Race, Ethnicity, and Genetics Working Group 2005). When creating a panel of human samples, a phylogenetic tree including the different tribes or ethnic groups may be used or a phylogenetic tree including individuals from one tribe or ethnic group may be used depending on the intended use of the panel.

The term "ethnic group" or "tribe" as used herein, refers to a group of individuals defined by common genealogy or ancestry and sharing common behavioral or biological traits. For example different ethnic groups or tribes include, for example, Arabian, Alaskan, Athabaskan Northeast Amerindian, Salishan, South Amerindian, Mestizo, Han Chinese, Zhuang Chinese, Gaoshan, Korean, Mongol, Tibetan, Indian, North African, Sub-Saharan African, Finno-Ugrian, Basque, Eastern European, Mediterranean, Northwest European, Japanese, Polynesian, Australian, Southeast Asian, North Indian and South Indian.

The Race, Ethnicity, and Genetics Working Group (Am J Hum Genet. 2005; 77(4): 519-532) have completed a clustering analysis of that is representative of the human population (see Figure 4 and world wide web at dnatribes.com). This clustering analysis can be used to select a panel that is representative of the genetic diversity of the human population. For example, Figure 4 shows a clustering analysis of the different tribes and the grey line indicates how five genetically diverse groups can be chosen using the phylogenetic tree. A higher resolution can be obtained by increasing the number of groups selected.

In certain embodiments, a panel of human samples that reflects the genetic diversity within a single tribe may be selected.

To achieve a higher resolution for genotype analysis, a greater number of SNPs may be used (see e.g., Roses et al. 2007). For example, it may be possible to use at least 1,000 SNPs, 5,000 SNPs, 10,000 SNPs, 100,000 SNPs, 500,000 SNPs or more. Other types of genotype information may be used in addition to, or instead of SNP data, such as complete or partial sequence data. Genetic relatedness can be determined by cluster analysis and/or the generation of a phylogenetic tree or dendrogram or other bioinformatics tools capable of relating the samples to each other and establishing a hierarchy of relatedness.

Genotype information about plants and algae is publicly available or may be easily obtained using standard genotyping approaches (for example see Hazen & Kay 2003 and Weir 2007). In an exemplary embodiment, genetically diverse samples may be obtained from individuals that are determined to be genetically diverse based on the number of generations by which they are separated. For example, biological samples may be obtained from primate, e.g., human or monkey, individuals wherein each individual is separated from the other individuals by at least four generations (e.g., no common great grandparents), or by at least 5, 6, 7, 8, 9, 10, 15, 20 or more generations. Biological samples from rodents, e.g., mice or rats, may be selected from individuals that are separated by at least 5, 10, 15, 20, 25, 30, 40, 50 or more generations. Alternatively, biological samples from humans may be selected from human individuals wherein each individual is from a different ethnic group.

Methods of Use

The panels provided herein may be used for a wide variety of applications including, for example, toxicity screening, absorption, distribution, metabolism and excretion (ADME) screening, identification of adverse effects, identification of positive effects, efficacy screening, drug repositioning, pathway identification, mechanism of action studies, pharmacokinetic and/or pharmacodynamic studies.

In certain embodiments, it may be desirable to contact the biological samples with a compound and observe a response of the sample to the compound. For example, it may be desirable to treat the biological samples with a chemical compound, a polypeptide (including, for example, a protein, protein domain, peptide, aptamer etc.), a polypeptide analog (including, for example, a peptidomimetic), a nucleic acid (including, for example, RNA, RNAi, small hairpin RNA (shRNA), siRNA, microRNA, uaRNA, piwi-interacting RNA (piRNA), DNA, oligonucleotides, etc.), a drug, a formulated drug or combinations thereof.

Examples of proteins that may be used in accordance with the methods described herein include, for example, a growth factor, a cytokine, an antibody, antibody fragment, an enzyme (e.g. kinase, phosphatase, protease, etc.), a glycoprotein, an alternative binding protein, or a non-immunoglobulin antigen- binding scaffold (e.g., an antibody substructure, minibody, adnectin, anticalin, affibody, affilin, avibodies, DARPin, knottin, fynomer, glubody, C-type lectin-like domain protein, tetranectin, kunitz domain protein, phylomer, SMIP, versabodies, thioredoxin, cytochrome b562, zinc finger scaffold, Staphylococcal nuclease scaffold, fibronectin or fibronectin dimer, tenascin, N-cadherin, E-cadherin, ICAM, titin, GCSF-receptor, cytokine receptor, glycosidase inhibitor, antibiotic chromoprotein, myelin membrane adhesion molecule PO, CD8, CD4, CD2, class I MHC, T-cell antigen receptor, CD 1 , C2 and I-set domains of VCAM- 1 , 1 -set immunoglobulin domain of myosin-binding protein C, 1-set immunoglobulin domain of myosin-binding protein H, I-set immunoglobulin domain of telokin, NCAM, twitchin, neuroglian, growth hormone receptor, erythropoietin receptor, prolactin receptor, interferon-gamma receptor, /3-galactosidase/glucuronidase, β- glucuronidase, transglutaminase, T-cell antigen receptor, superoxide dismutase, tissue factor domain, cytochrome F, green fluorescent protein, GroEL, or thaumatin).

A response of the biological sample to a compound may be determined using a variety of art recognized methods. For example, suitable assays that can be used in accordance with the methods described herein include, for example, cell viability, proliferation, apoptosis, cell death, toxicity, kinase activity, GPCR assay, gene expression (e.g., RT-PCR, microarray analysis, in situ hybridization, Western blot, antibody detection, etc.), reporter gene, enzymatic, ion flux assays, measurement of cytokines, phosphorylation status, oxidative stress, mitochondrial functionality, metabolite production, metabolic activity and specific metabolic pathway reporter constructs, a physiological transport assay, a physiological secretion assay and/or a phenomenological assay.

A wide variety of assays are available that can be used to analyze a response of a biological sample to a compound. For example, effects on cell proliferation can be determined, for example, using a BrdU incorporation assay, ³H-Thymidine assay, MTT assay, WST assay, MST assay or XTT assay. Cell viability can be determined by measuring the incorporation of specific dyes such as Trypan blue, ethidium dye, propidium dye, or cell trace calceins. Apoptosis may be determined using a variety of assays such as a caspase assay, mitochondrial assay, TUNEL-assay, annexin V assay, calpain activity assay, cathepsin protease activity, activation of apoptosis- inducing factor, ATP level detection or DNA ladder analysis. Toxicity can be assayed by interrogating specific pathways such as P450 system, Bcl-2 pathway, NAD/NADH quantification assay, cytochrome c release from mitochondria. Other cell assays like migration or invasion may be used to monitor the behavior of a biological sample in response to a compound.

In certain embodiments, it may be desirable to correlate a response of one or more biological samples to a genetic marker, such as, for example, a sequence, a haplotype block, or a SNP. Bioinformatics techniques well known in the art can be used to conduct such an analysis. This can allow the mapping of a region, regions, gene or genes which are involved in the phenotype response. One method which can be applied is referred to as Genome-wide association studies (GWAS) and may be used to identify disease susceptibility genes or genetic factors underlying health or disease.

In certain embodiments, the panels described herein may be used to identify a pathway through which a compound is acting, reveal the mechanism of action of a compound, and/or reveal a previously unknown activity of a compound. In certain embodiments, the panels described herein may be used to study the pharmacology of a compound in a defined, stable genetically diverse population.

In certain embodiments, the panels described herein may be used to perform

ADME/toxicity analysis including, for example, liver toxicity, cardiac toxicity and reproductive toxicity studies. In certain embodiments, the panels described herein may be used to perform assays which allow the analysis and/or identification of idiosyncratic drug reactions. In certain embodiments, a panel of plant samples may be used for toxicity screening or for identifying plants suited for specific culture zones (e.g., drought, heat, cold, soil conditions, etc.) or specific purposes (e.g., nutrition, oil, biodiesel, hydrogen production, biomass, methane, livestock feed, recombinant protein production, ethanol production, etc.).

In certain embodiments, the panels described herein may be used in high throughput assays. For example, panels may be provided in multi-well plates in convenient formats such as 96, 384 or 1536 well plates. Kits

In another aspect, the invention provides any of the compositions described herein in kits, optionally including instructions for use of the compositions (e.g., using the diverse cell panel for screening, links to computer analysis systems, CD-ROM or other computer memory storage device for genetic analysis and mapping of genes and/or pathways). For example, the kit can include a description of methods for using a cell panel in a wide variety of methods as described further herein. A "kit," as used herein, typically defines a package, assembly, or container including one or more of the components of the invention, and/or other components associated with the invention, for example, cell lines or tissue slices, and culture media as previously described. The samples for the diverse panel may be provided in frozen form (e.g., cryopreserved) or in live form (e.g., as cultures in tissue culture plates). The samples may be provided in any appropriate format, e.g., individual plates, 4-well, 6-well, 12-well, 48-well, 96-well, 384-well or 1536-well plates, micro fluidic chamber, cell or tissue chamber slides.

In some cases, the kit includes one or more components, which may be within the same or in two or more receptacles, and/or in any combination thereof. The receptacle is able to contain a liquid, and non-limiting examples include bottles, vials, jars, tubes, flasks, beakers, plates, chamber slides or the like, hi some cases, the receptacle is spill-proof (when closed, liquid cannot exit the receptacle, regardless of orientation of the receptacle). hi some cases, the components of the kit may be contained within a suitable container, such as a cardboard box, a Styrofoam box, etc. The kit may be shipped at room temperature (about 25 ⁰C), chilled (e.g., at about 4 ⁰C), and/or any one or more of the components may be shipped frozen (e.g., between -20 ⁰C and -80 ⁰C, at about -150 ⁰C, etc.) or in liquid nitrogen (about -196 ⁰C). In some cases, one or more of the components are frozen and/or shipped on dry ice (about -80 ⁰C). hi some cases, the kit will include a cryogenic vessel which is a vessel suitable for containing materials at cryogenic temperatures, for example, liquid nitrogen. Those of ordinary skill in the art will be aware of suitable cryogenic vessels, for example, a Dewar flask (e.g., formed from stainless steel and/or aluminum, etc.), a vapor shipper, a stainless steel container, a Styrofoam container, or the like. Typically, cryogenic temperatures include temperatures below about - 150 ⁰C, below about -170 ⁰C, or below about -190 ⁰C. For instance, liquid nitrogen has a boiling point of about -196 ⁰C.

The kit may also contain a receptacle for cells or tissue slices. For example, the receptacle may be constructed so that it can withstand cryogenic temperatures without rupture or fracture. The receptacle may be pre-labeled in certain instances.

Examples of other compositions or components associated with the invention include, but are not limited to, diluents, salts, buffers, chelating agents, preservatives, drying agents, antimicrobials, growth factors, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, and the like, for example, for using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and/or preserving the components for a particular use. In embodiments where liquid forms of any of the components are used, the liquid form may be concentrated or ready to use. A kit of the invention generally will include instructions, directions to a website providing instructions, or other source of information in any form, for using the kit in connection with the components and/or methods of the invention. For instance, the instructions may include instructions for the use, modification, mixing, diluting, preserving, assembly, storage, packaging, and/or preparation of the components and/or other components associated with the kit. In some cases, the instructions may also include instructions for the assay to be performed with such a kit. The instructions may be provided in any form that is useful to the user of the kit, such as written or oral (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) and/or electronic communications (including Internet or web-based communications), provided in any manner.

As used herein, instructions can include protocols, directions, guides, warnings, labels, notes, and/or "frequently asked questions" (FAQs), and typically involve written instructions on or associated with the invention and/or with the packaging of the invention. Instructions can also include instructional communications in any form (e.g., oral, electronic, digital, optical, visual, etc.), provided in any manner (e.g., within or separate from a kit) such that a user will clearly recognize that the instructions are to be used with the kit. As an example, a kit as discussed herein may be shipped to a user, typically with instructions for use. For instance, the instructions may instruct the user to thaw the cells and plate the cells at a specific density, before performing a screening assay, e.g., apoptosis assay, proliferation assay, etc. As another example, the instructions may instruct the user to perform a screening assay (e.g., as described above).

In certain embodiments, kits provided herein comprise a panel of biological samples, hi certain embodiments, each sample is cryopreserved and stored in a separate container. In other embodiments, each sample is in culture and provided in separate container. For example, the biological samples may be stored in separate vials or in a high-throughput culture plate. The containers may comprise enough biological sample for multiple experiments or may be designed as single use panels. For single use panels, the samples may be plated so that approximately equal numbers of cells are in each container. For kits having algae or yeast samples, the biological samples may be provided in a lyophilized form. The kit may also include additional components, such as, for example, culture media, instructions, reagents for screening assay, and/or screening assay instructions.

EXEMPLIFICATION The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention in any way. EXAMPLE 1: Hepatocyte cell panel and treatment with acetaminophen (APAP) Five inbred mouse lines were selected on the basis phenotype and genetic diversity. Phenotype selection of mouse strains was based on published data (Hege et al. 2005). The phenotype selection was used to identify representative mouse strains which showed a wide range of APAP response in whole animal studies, e.g., from a highly insensitive strain to a highly sensitive strain, and three intermediate strains. Genotype selection was based on a mouse family tree generated from 102 mouse inbred lines (see Petkov et al. 2004). In the tree, seven major genetic groups have been identified and mice from 3 groups were chosen to examine whether a genetically diverse panel of biological samples can show differences in drug response. The panel contained the following mouse strains: from group 1, A/J, CBA/J and AKR/J, from group 4 C57BL/6J, and from group 6 DBA/2J. Group 1 splits into 3 subgroups and A/J, CBAJ] and AKR/J are each from a different subgroup. Mouse strains are available from The Jackson Laboratory (world wide web atjax.org). Hepatocyte Isolation

Hepatocytes were isolated from adult mouse livers from each of the five selected inbred strains (A/J, C57BL/6J, CBAJ], AKR/J and DBA/2J) using a collagenase perfusion method described by LeCluyse et al. (1996 and 2005). The hepatocytes were then treated with acetaminophen (N-acetyl-p-aminophenol also known as APAP), which is a commonly used as an over the counter analgesic and is well studied. APAP overdose causes an acute liver necrosis and is the cause of one third of acute liver failure in the USA (Lee 2003). Here APAP is used to demonstrate the effect of genetic background on cell viability. Hepatocyte Culture

The cell count and viability was determined using a haemocytometer following dilution of the cell suspension with 0.4% trypan blue. The hepatocytes were diluted to 1 x 10⁶ cells/mL in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with insulin, transferrin, selenium (ITS, e.g., from Invitrogen; Cat. No. 51500-056), 0.1 microM dexamethasone, and antibiotics (penicillin-G, streptomycin). Cells were plated into 24 well tissue culture treated plates for incubation.

The incubations were performed in duplicate at acetaminophen concentrations of 0, 2.5, 5.0, and 10.0 mM acetaminophen (APAP) at 37 ⁰C, 5% CO₂ with gentle shaking. Reactions were initiated with the addition of acetaminophen at 2X concentration such that the final cell concentration for each incubation was 0.5 x 10⁶ cells/mL in a volume of 1.0 ml. The samples were incubated for 0, 3, 6, 9, 12 and 24 hours. All acetaminophen stock solutions were prepared fresh in serum- free DMEM. Incubations were terminated by removing an aliquot from the incubation medium and subjecting the sample to either cell viability by Trypan Blue exclusion or intracellular ATP determination. Intracellular ATP Determination

Isolated hepatocytes were treated with either vehicle control or 2.5 mM, 5 mM, or 10 mM acetaminophen for 0, 3, 6, 9, 12, and 24 hours. Upon completion of treatment, ATP levels were determined using Promega's CellTiter-Glo™ Luminescent Cell Viability Assay (Cat. No. G75-70 from Promega) according to the manufacturer's instructions. Briefly, the CellTiter-Glo™ Reagent was prepared by reconstituting the lyophilized CellTiter-Glo™ Substrate in CellTiter-Glo™ Buffer. Cells were pelleted, and the supernatant was aspirated. One volume of Reagent equal to the amount of supernatant aspirated was added to each cell sample. After mixing for 2 minutes, and incubating at room temperature for 10 minutes, sample luminescence was detected using a FLUOstar OPTIMA luminometer (BMG Labtechnologies). Data Analysis

The average luminescent value determined in blank wells (cell-free wells) was subtracted from all sample values to yield corrected luminescent values.

Quantification of ATP levels was performed using a weighted (1/x²) linear least squares regression analysis generated from standard curves. The corrected and converted ATP values for each treatment group were reported as actual amounts and as a percent (%) change in ATP content compared to the solvent-treated control values.

Cell Viability Determination

Cell viability was determined by Trypan blue exclusion after incubating the hepatocytes with 0, 2.5 mM, 5 mM or 10 mM acetaminophen. The measurements were taken 0, 3, 6, 9, 12, and 24 hours after incubation. Briefly, 400 microliters of DMEM, 50 microliters of cell suspension, and 50 microliters of Trypan blue solution were added to a microcentrifuge tube and mixed by inversion. Samples were incubated at room temperature for one minute, then loaded onto a hemocytometer and counted. Each sample was counted twice to ensure accuracy, and three samples were counted at each time point. In Figure IA, the effect of acetaminophen on C57BL/6J hepatocytes is shown.

Determination of the Cytotoxic Potential of Acetaminophen in Cultured Hepatocytes

After In Vitro Exposure Experimental model. Fresh hepatocytes were isolated from five strains of mouse liver tissue. The mouse strains used were A/J, C57BL/6J, CBA/J, AKR/J and DBA/2J.

Experimental conditions. Hepatocytes were plated in collagen-coated tissue culture plates and were incubated in a collagen gel sandwich until the cultures were established. Hepatocytes were incubated with acetaminophen (at each of 3 concentrations: 1, 3 and 10 mM) or with control article (solution) in 24-well tissue culture plates for 6 hours (37°C, 5% CO₂). After this 6-hour exposure, the cells were lysed and the cellular ATP content was analyzed. The decrease of intracellular ATP in hepatocytes was used as an indicator of cell viability and cytotoxicity (Riss et al. 2004). ATP levels were determined using Promega's CellTiter-Glo™ Luminescent Cell Viability Assay according to the manufacturer's instructions. Briefly, the CellTiter-Glo™ Reagent was prepared by reconstituting the lyophilized CellTiter- GIo Substrate in CellTiter-Glo™ Buffer. Following removal of the supernatant, an equal volume of Reagent was added to each well. After mixing for 2 minutes, followed by a 10-minute incubation, sample luminescence was detected using a FLUOstar OPTIMA luminometer (BMG Labtechnologies). Data from the five different strains are shown in Figure IB. The y-axis shows the intracellular ATP level, which was calculated as ratio of control being 1. The x-axis shows the concentration of APAP used in the experiment, which was 0 mM APAP, 1 mM

APAP, 3 mM APAP and 10 mM APAP. Decrease in the ratio indicates a lower of level of ATP correlating with the number of live cells. This data shows that a hepatocyte cell panel consisting of cells from five genetically distinct mice displays different responses for the five different cell lines, with cells from the A/J strain being the most resistant.

EXAMPLE 2: Mouse Embryonic Fibroblasts Panel and Treatment with Acetaminophen

Five inbred mouse lines were selected to be genetically different. As a starting point, the mouse family tree generated from 102 mouse inbred lines was used (see Petkov et al. 2004). In this tree, seven major genetic groups have been identified and mice from 3 groups were chosen to examine whether a genetically diverse panel can show differences in drug response. The panel was selected as follows: from group 1, A/J and Balb/cJ, from group 3 NZW/LacJ (NZW), from group 4 C57BL/6J, and from group 6 DBA/2J. The mouse strains were obtained from The Jackson Laboratory (world wide web atjax.org). After selection of the genetically different inbred mouse lines, embryonic fibroblasts were isolated from the mouse lines and treated with acetaminophen.

Isolation of Mouse Embryonic Fibroblasts (MEF)

Male and female mice from the strains C57BL/6, DBA/2, BALB/c, A/J and NZW were paired for time of pregnancy. E12.5 day pregnant female mice for each of the strains were sacrificed (EO.5 is the morning of finding a plug). The uteri were removed and placed into a dish with PBS and washed. The embryos were isolated free of extraembryonic tissue and washed in PBS. Each embryo was placed into a separate 14 ml tube containing 3 ml of media (DMEM, 10% FBS) and homogenized for 2 seconds. The homogenized tissues were put into 145 mm tissue culture plates (e.g. CellStar from USA Scientific cat.# 5663-9160) containing 20 ml of media (DMEM, 10% FBS). The cells were incubated at 37 ⁰C in 5% CO₂ until cells were confluent, which was on average from 5 to 7 days. The cells were expanded according to standard tissue culture methods using trypsin and the same passage number for each cell line was used for the experiment. All passage numbers used for the experiments were at least 2-3 passages before senescence. Treatment of Cells with Acetaminophen (APAP) and Viability Assay

Embryonic fibroblast cells (1x10⁵ cells) of the same passage number were plated into a 96-well plate and incubated overnight at 37 ⁰C in a 5% CO₂ tissue culture incubator. The next day cells were treated with acetaminophen (Sigma- Aldrich A7085) and assayed in triplicate with the following concentrations of acetaminophen: 0, 1, 3, 10, 30, 50 and 100 mM. The cells were incubated overnight at 37 ⁰C in a 5% CO₂ tissue culture incubator, washed with Dulbecco's PBS twice, and then 100 microliters of 5 % saponin was added. After 10 minutes, 100 microliters of a 2 micromolar Calcein AM stock solution (Molecular Probes L3224) in Dulbecco's PBS is added and incubated for 45 minutes. The fluorescence was measured in a 96 well spectrophotometer, e.g. Perkin Elmer 1420 Victor, using an excitation filter of 495 nm and an emission filter of 515 run. The data was plotted and the results are presented in Figure 2. The data show that cell viability after treatment for 24 hours with different concentrations of acetaminophen results in different responses with BALB/c being the most resistant when compared to DBA/2, A/J, C57BL/6 and NZW. This shows that a cell panel consisting of cells from five genetically different mice highlight different outcomes. EXAMPLE 3: Preparation of Adult Fibroblasts Panel

Fibroblast cells can be isolated from a number of sites from adult tissue: e.g., foreskin, ear punch, or skin from mammals. About 5 mm pinch punch biopsies are obtained (see Krathen R.A., Orengo I.F., How I do it. hi a pinch. Dermatol Surg. 2004 30(12 Pt 2): 1599). The tissue should be isolated sterilely, for example, for the human skin biopsies, the area can be cleaned with soap and then 70% ethanol. Samples are collected into a sterile dish in 5-10 volumes 4 mg/ml collagenase/dispase (Roche Applied Sciences) dissolved in Phosphate Buffered Saline (PBS). The tissues are then teased and effectively minced into small (lmm or less) fragments and incubated at 37⁰C for 1 to 2 hour. After 1-2 hours depending on the tissue, two volumes of medium (Dulbecco's modified Eagle medium (DMEM) supplemented with 10% serum (fetal bovine serum), monothioglycerol (150 microM) and antibiotics (e.g. penicillin and streptomycin)) are added and the cell suspension is pipetted gently a few times up and down. Leaving the larger fragments behind, the cell suspension is then transferred to tissue culture grade plates for 24 hours in a tissue culture incubator (37⁰C, 5% CO₂). The tissue culture cells may be coated with 0.1% collagen solution (bovine, swine or recombinant) or 0.1 % gelatin. After 24 hours the fibroblasts are adhered and can be expanded and cryopreserved or used directly in the experiments. EXAMPLE 4: Preparation of a Human iPS Cells Dermal fibroblast are isolated for example from a punch biopsy of the skin and cultured (see Example 3). To convert fibroblasts into induced pluripotent stem (iPS) cells, the cells are infected with multiple retroviral vectors which express OCT4, SOX2, C-MYC, NANOG, KLF4 (and optionally a reporter gene such as GFP as an infection efficiency indicator) in DMEM, 10% FCS, nonessential amino acids, L-glutamine, penicillin-streptomycin as described by Lowry et al. (PNAS

2008, VoI 105, No. 8, p 2883-2888). This infection is repeated at day 3 to increase infection rate. At day 7 whole cultures are passaged onto irradiated mouse embryonic fibroblasts (MEFs) feeders in DMEM F 12, L-glutamine, nonessential amino acids, penicillin-streptomycin, knockout serum replacement (Invitrogen)) plus 10 ng/ml bFGF which is conducive to iPS ES like cells. After about two weeks colonies exhibiting human ES cell morphology arise and are manually selected, gently mechanically disrupted to clumps and plated onto fresh MEFs in human ES cell media. The cells can be cultured and expanded, cryopreserved used for many assays over many years.

For genetic testing iPS cells are cultured two passages without MEF feeders and harvested for DNA isolation and analysis using standard methods. EXAMPLE 5: Production of an Adult Human Cell Panel from Cell Banks

To make human cell panel containing at least five different genetic samples a primary step is to locate the source of such samples and to determine their genetic relatedness. There are several sources available which can be used to isolate cells suitable for a cell panel. Biological material can be obtained from tissue banks, cell banks (stem cell banks, e.g. National Stem Cell Bank) or blood banks (e.g. umbilical cord blood, blood bank, bone marrow bank). For example the National Stem Cell Bank and Wicell currently offer 14 different human embryonic stem (hES) cell lines and plan to increase such to 19 different hES cell lines (world wide web at wicell.org/index.php?option=com_oscommerce&Itemid=132). The UK Stem CellBank (world wide web at ukstemcellbank.org.uk/) distributes 8 different hES cell lines. One can obtain for example the 22 different hES cell lines, genotype such using methods known in the art, e.g., by SNP analysis and/or sequencing. The genetic data is obtained and analyzed to establish the genetic relatedness of the individuals by applying standard bioinformatics tools, which may include cluster analysis tools and computational phylogenetic tools (e. g. PHYLIP (world wide web at evolution.genetics.washington.edu/ phylip.html). For example, the five genetically most distant samples are selected and the cells expanded for the manufacture of the cell panel. The cells can be provided in custom formats as live cells, as frozen cells in plates ready to use upon recovery; or as frozen vials of cells which can be recovered, grown up as needed and plated out into any form of cell based assay. EXAMPLE 6: Production of an Adult Human Cell Panel from Individuals

Human volunteers are recruited to obtain genetic material to be able to establish the genetic relatedness of the individuals. For example, genetic material is collected from about one hundred donors by means of a mouth swab. Any other method for collecting DNA known in the art may be applied. The DNA is analyzed using methods known in the art, like sequencing or SNP analysis, which are useful to compare DNA samples to each other and to establish a ranking for the relatedness of the individual samples. Standard bioinformatics tools, for example cluster analysis tools and computational phylogenetic tools (e. g. PHYLIP (world wide web at evolution.genetics.washington.edu/phylip.html), will be applied to rank the genetic information of the 100 individuals. For example 10 individuals, who are least related are chosen to obtain tissue or blood samples for cell isolation and production of the cell panel. For example dermal fibroblast can be easily isolated from a punch biopsy of the skin, cultured, propagated and cryopreserved. Further, these dermal fibroblasts can be readily converted into immortal normal cell type by using induced pluripotent stem cells (iPS) as described by Lowry et al. (PNAS 2008, VoI 105, No. 8, p 2883-2888 and see Example 4). Either the fibroblasts or better the iPS cells are used to make a cell panel containing cells from 10 different individuals. The cells will be genotyped to confirm their genetic identity. The cells can be provided in custom formats as live cells, as frozen cells in plates ready to use upon recovery; or as frozen vials of cells which can be recovered, grown up as needed and plated out into any form of cell based assay. The cells can be provided as part of kit for a specific compound assay, which may measure the proliferation rate, apoptosis or differentiation. EXAMPLE 7: Mouse Embryonic Fibroblasts Panel and Treatment with Acetaminophen

Mouse embryonic fibroblasts (MEF) are isolated as described in Example 2. The mouse strains used are CBA/J, A/J, BALB/cByJ, C57BL/6J and DBA/2J. The mouse strains CBA/J, A/J and BALB/cByJ (referred to a BALB/ByJ in Figure 6) belong to group 1 of figure 5, C57BL/6J to group 4 and DBA/2J to group 6. MEFs at greater than passage three were plated out into 96 well plates at Ixlθe4 cell/well in DME with 10% FCS. The cells were cultured overnight. Acetaminophen was dissolved in DME media, 10%FCS and added to triplicate wells at 0, 5, 20, 25, 30, 35, 40, 50, 60 or 10OmM concentration. Cells were incubated for a further 24 hours and then assayed using a Promega Celltiter-Glo Luminescent Cell Viability Assay kit (Cat number G7573 from Promega) for measuring cell viability. The assay measures the amount of ATP present in cells as an indicator of viability. As shown in Figure 6, the no drug control was set to 100%. The data shows that a cell panel consisting of cells from five genetically different mice highlight different outcomes, with CBA/J being less sensitive to acetaminophen when compared to the other mouse strains (see Figure 6). The data further shows that the genetic diversity in one subgroup is high enough to reveal different responses.

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EQUIVALENTS The present invention provides among other things panels of genetically diverse biological samples and methods of use thereof. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Claims

CLAIMS:

1. An in vitro method for evaluating the toxicity of at least one compound on a genetically diverse population, comprising:

(i) contacting a panel of biological samples in vitro with the compound, wherein the panel comprises at least five genetically diverse biological samples isolated from the same species, wherein the biological samples are obtained from five individuals of the species that are genetically diverse based on clustering analysis or a phylogenetic tree, and wherein the biological samples comprise cells of the same cell type or tissues of the same tissue type;

(ii) observing at least one response of each of the biological samples to the compound; and

(iii) comparing the responses of each of the biological samples, thereby evaluating the toxicity of the compound on a genetically diverse population.

2. An m vitro method for evaluating the effect of at least one compound on a genetically diverse population, comprising:

(i) contacting a panel of biological samples in vitro with the compound, wherein the panel comprises at least five genetically diverse biological samples isolated from the same species, wherein the biological samples are obtained from five individuals of the species that are genetically diverse based on clustering analysis or a phylogenetic tree, and wherein the biological samples comprise cells of the same cell type or tissues of the same tissue type;

(ii) observing at least one response of each of the biological samples to the compound; and

(iii) comparing the responses of each of the biological samples, thereby evaluating the effect of the compound on a genetically diverse population.

3. The method of claim 2, wherein the toxicity, effectiveness, or selectivity of the compound is evaluated.

4. The method of claim 1 or 2, wherein the biological samples are mammalian samples.

5 The method of claim 1 or 2, wherein the biological samples are rodent or primate.

6. The method of claim 1 or 2, wherein the biological samples are mice samples.

7. The method of claim 1 or 2, wherein the biological samples are human samples.

8. The method of claim 5, wherein the biological samples are from rodents and wherein each of the five individuals is separated from the other individuals by at least 20 generations.

9. The method of claim 5, wherein the biological samples are from primates and wherein each of the five individuals is separated from the other individuals by at least 4 generations.

10. The method of claim 7, wherein each of the five individuals is from a different ethnic group.

11. The method of claim 1 or 2, wherein the biological samples are plant samples.

12. The method of claim 1 or 2, wherein the biological samples are algae samples.

13. The method of claim 1 or 2, wherein the compound is a small molecule, nucleic acid, or polypeptide.

14. The method of claim 13, wherein the compound is an antisense nucleic acid, RNAi compound, or enzymatic nucleic acid.

15. The method of claim 13, wherein the compound is an antibody or alternative binding protein.

16. The method of claim 13, wherein the compound is a protein or peptide.

17. The method of claim 1 or 2, wherein the compound is a drug, clinical drug candidate, cosmetic agent, environmental pollutant, industrial chemical, vaccine, viral or bacterial component.

18. The method of claim 1 or 2, wherein the compound is formulated in a pharmaceutically acceptable carrier.

19. The method of claim 1 or 2, wherein a plurality of compounds is evaluated simultaneously in a high throughput format.

20. The method of claim 1 or 2, wherein the biological samples comprise cells having at least one reporter gene.

21. The method of claim 1 or 2, wherein a response of the biological samples is observed using an optical assay, a gene expression assay, a phenomenological assay, a physiological transport assay, a physiological secretion assay, an apoptosis assay, a cell proliferation assay, or a toxicity assay.

22. The method of claim 21, wherein the gene expression assay involves measuring production of mRNA or protein.

23. The method of claim 1 or 2, further comprising correlating a response of at least one of the biological samples with a genetic marker.

24. A panel comprising at least five genetically diverse biological samples isolated from a given species, wherein the biological samples are obtained from five individuals of the species that are genetically diverse based on clustering analysis or a phylogenetic tree, and wherein the biological samples comprise cells of the same cell type or tissues of the same tissue type.

25. The panel of claim 24, wherein the biological samples are vertebrate samples.

26. The panel of claim 25, wherein the biological samples are rodent or primate samples

27. The panel of claim 26, wherein the biological samples are mouse samples.

28. The panel of claim 26, wherein the biological samples are human samples.

29. The panel of claim 27, wherein at least one of the biological samples is from a wild mouse, a laboratory mouse strain, a mouse disease model, a transgenic mouse, a mouse having a knock out gene mutation, a mouse having a knock in gene mutation, or a mouse having a chemically induced mutation.

30. The panel of claim 26, wherein the biological samples are from rodents and wherein each of the five individuals is separated from the other individuals by at least 20 generations.

31. The panel of claim 26, wherein the biological samples are from primates and wherein each of the five individuals is separated from the other individuals by at least 4 generations.

32. The panel of claim 28, wherein each of the five individuals is from a different ethnic group.

33. The panel of claim 24, wherein at least one of the biological samples is from an outbred population.

34. The panel of claim 24, wherein the biological samples are from individuals of the same sex.

35. The panel of claim 24, wherein the biological samples are plant samples.

36. The panel of claim 24, wherein the biological samples are algae samples.

37. The panel of claim 24, wherein the biological samples comprise cells.

38. The panel of claim 24, wherein the biological samples comprise tissue.

39. The panel of claim 24, wherein at least one of the biological samples comprises a tissue slice.

40. The panel of claim 24, wherein the panel comprises at least 6, 10, 12, 20, 24, 30, 40, 48, 50, 60, 72, 96, 100, 384, or 1536 genetically diverse biological samples.

41. The panel of claim 24, wherein the biological samples comprise endothelial cells, connective tissue cells, epidermal cells, hematopoietic cells, stem cells or differentiated daughter cells derived from stem cells, nervous system cells, endocrine cells, tracheobronchiolar cells muscle cells, urogenital cells, cardiac cells, digestive tract cells, umbilical cord cells or cells from amniotic fluid.

42. The panel of claim 24, wherein the biological samples comprise liver cells.

43. The panel of claim 24, wherein the biological samples comprise fibroblasts.

44. The panel of claim 24, wherein at least one of the biological samples comprises a primary cell.

45. The panel of claim 24, wherein at least one of the biological samples comprises a stem cell.

46. The panel of claim 24, wherein at least one of the biological samples comprises a genetically modified cell.

47. The panel of claim 24, wherein at least one of the biological samples comprises a cell having at least one reporter gene.

48. The panel of claim 47, wherein each biological sample comprises a cell having the same reporter gene.

49. The panel of claim 47, wherein at least two biological samples comprise a cell having different reporter genes.

50. A panel comprising at least five genetically diverse biological samples comprising cells of a first cell type and at least five genetically diverse, isolated, biological samples comprising cells of a second cell type, wherein each of the cells of the first and second cell types are from the same species.

51. The panel of claim 50, wherein the biological samples are obtained from five individuals of the species that are genetically diverse based on clustering analysis or a phylogenetic tree.

52. A method for producing a panel of genetically diverse biological samples, comprising:

(i) determining the genetic relatedness of a plurality of biological samples from the same species; and

(ii) selecting at least five biological samples based on the degree of genetic diversity among the biological samples, thereby producing a panel of genetically diverse biological samples.

53. The method of claim 52, further comprising performing genotype analysis on a plurality of biological samples from the same species.

54. The method of claims 52 or 53, further comprising performing phenotype analysis on a plurality of biological samples from the same species.

55. The method of claim 53, wherein genotype analysis is performed on at least 5, 10, 25, 50, 100, 250, 500, or more biological samples from the same species.

56. The method of claim 53, wherein genotype analysis is carried out using SNP analysis, microsatellite markers, DNA microarray analysis, sequencing, or combinations thereof.

57. The method of claim 52, wherein the genetic relatedness of the plurality of biological samples is determined using bioinformatic analysis to construct a dendrogram or using clustering analysis.

58. The method of claim 57, wherein the genetic relatedness of the plurality of biological samples is determined using parsimony analysis or PHYLIP analysis.

59. The method of claim 57, wherein at least one biological sample from each main branch is selected.

60. The method of claim 52, wherein at least 6, 10, 12, 20, 24, 30, 40, 48, 50, 60, 72, 96, 100, 384, or 1536 biological samples that are genetically diverse are selected.

61. The method of claim 52, wherein the biological samples are vertebrate samples.

62. The method of claim 61, wherein the biological samples are mice, rat or human samples.

63. The method of claim 52, wherein the biological samples are plant samples.

64. The method of claim 52, wherein the biological samples are algae samples.

65. The method of claim 52, wherein at least one of the biological samples is from an inbred individual.

66. The method of claim 52, wherein at least one of the biological samples is from an outbred individual.

67. The method of claim 52, wherein the biological samples are from different individuals of a given species having a common trait.

68. The method claim 52, wherein the genetic relatedness of the biological samples is determined based on the degree of generational separation between the individuals from which the biological samples are obtained.

69. The method of claim 68, wherein the biological samples are selected from rodents and wherein each of the five individuals is separated from the other individuals by at least 20 generations.

70. The method of claim 68, wherein the biological samples are from primates and wherein each of the five individuals is separated from the other individuals by at least 4 generations.

71. The method of claim 68, wherein the biological samples are from humans and wherein each of the five individuals is from a different ethnic group.

72. A method produced by a method of any one of claims 52-71.

73. A panel comprising at least five phenotypically diverse, isolated, biological samples from a given species.

74. A method for producing a panel of phenotypically diverse biological samples, comprising:

(i) performing a phenotypic clustering analysis for a plurality of biological samples from the same species; and

(ii) selecting at least five phenotypically diverse biological samples based on the clustering analysis, thereby producing a panel of genetically diverse biological samples.

75. A kit comprising a panel comprising at least five genetically diverse, isolated, biological samples from a given species, wherein the biological samples are obtained from five individuals of the species that are genetically diverse based on clustering analysis or a phylogenetic tree, and wherein the biological samples comprise cells of the same cell type or tissues of the same tissue type.

76. The kit of claim 75, wherein the biological samples are from rodents and wherein each of the five individuals is separated from the other individuals by at least 20 generations.

77. The panel of claim 75, wherein the biological samples are from primates and wherein each of the five individuals is separated from the other individuals by at least 4 generations.

78. The panel of claim 75, wherein the biological samples are from humans and wherein each of the five individuals is from a different ethnic group.

79. The kit of claim 75, wherein the panel comprises at least 6, 10, 12, 20, 24, 30, 40, 48, 50, 60, 72, 96, 100, 384, or 1536 genetically diverse biological samples.

80. The kit of claim 75, wherein the biological samples are cryopreserved.

81. The kit of claim 75, wherein the biological samples are cell or tissue cultures.

82. The kit of claim 75, wherein the biological samples are provided in separate containers.

83. The kit of claim 75, wherein the biological samples are provided in a multiwell tissue culture plate.

84. The kit of claim 75, further comprising one or more of the following: culture media, instructions, or reagents for a screening assay.

85. A kit comprising a panel comprising at least five phenotypically diverse, isolated, biological samples from a given species.

86. The kit of claim 85, wherein the panel comprises at least 6, 10, 12, 20, 24, 30, 40, 48, 50, 60, 72, 96, 100, 384, or 1536 phenotypically diverse biological samples.

87. The kit of claim 85, wherein the biological samples are cryopreserved.

88. The kit of claim 85, wherein the biological samples are cell cultures, tissue cultures, or tissue slices.

89. The kit of claim 85, wherein the biological samples are provided in separate containers.

90. The kit of claim 85, wherein the biological samples are provided in a multiwell tissue culture plate.

91. The kit of claim 85, further comprising one or more of the following: culture media, instructions, or reagents for a screening assay.