Methods for Conditional and Inducible Transgene Espression to Direct the Development of Embryonic, Embryonic Stem, Precursor and Induced Pluripotent Stem Cells

Abstract
Methods are disclosed in which the expression of a specific gene, or combinations of genes, is controlled spatially and temporally to develop intra- and interspecies chimeras. A transgenic EC/ES/P/iPS cell line is created which conditionally expresses a suicide or compromiser gene configured to compromise all cell lineages except that corresponding to a target tissue/organ. The EC/ES/P/iPS cell line is injected into donor embryos having a specific target gene deficiency or embryos genetically engineered to be complementary compromised in lineages corresponding to the target tissue/organ cell lineages of the EC/ES/P/iPS line. One or more stimuli is provided to the embryo to activate compromiser genes for ablation of non-target tissues/organs of the EC/ES/P/iPS line and target tissues/organs of the host embryo, resulting in a chimeric animal having target tissues/organs derived from the genotype of the transgenic cell line and all remaining tissues/organs derived from the donor embryo.
Description
BACKGROUND

The present disclosure relates to methods to direct the development of embryonic cells, embryonic stem, precursor and induced pluripotent stem (EC/ES/P/iPS) cells to any cell type, tissue or organ system in vitro or in vivo in an exclusive manner, particularly for the creation of chimeras.


The human and mouse genome sequences together created an unprecedented opportunity to develop new, genetically engineered animal models to expedite the development of new treatment modalities to address and relieve human pain and suffering due to diseases. The differentiation program of EC/ES/P/iPS cells is one of the central questions in biology. Furthermore, isolation of tissue-specific stem cells presents a potentially powerful opportunity to develop effective therapeutics to facilitate repair of damaged or diseased organs. The best hope for more rapid discovery of effective prevention and treatment of cancer, cardiovascular disease, diabetes and other catastrophic human diseases, is via enhanced animal models of human health and disease.


Transplantation of organs is a well-known and accepted life-saving procedure for many of these human diseases, such as end-stage kidney, liver, heart and lung diseases. From both a medical and an economic point of view, organ transplantation is often preferable to alternative forms of therapy. But, the insufficient number of donor organs limits the application of this technique and can lead to unnecessary loss of life when other procedures prove ineffectual. Experimental techniques, such as xenotransplantation, have become increasingly more important to develop new methods of creating organ availability.


In past years several kinds of EC/ES/P/iPS cells have been isolated and their differentiation potential has been tested both in vivo and in vitro. However, none of these early studies addressed the “true” physiological fate of such stem cells and progenitor cells as a part of normal development. Several years ago, a novel cell-mapping system was developed which is based on expressing Cre or Flp recombinase in a stem cell or progenitor cell population. See, Dymecki and Tomasiewicz, Dev. Biol. 201:57-65 (1998); Gu et al., Development 129:2447-2457 (2002); and Zinyk et al., Curr. Biol. 8:665-668 (1998). Cre-mediated excision of the “floxed” sequences (i.e., loxP-flanked termination sequences) or Flp-mediated excision of the FRT-flanked sequences in the reporter constructs was shown to result in the permanent expression of the reporter in all the descendant cells. Since Cre or Flp can be introduced into these cells transgenically by using stem cell (or progenitor cell) specific promoter and/or enhancer elements in mice, this strategy permits analysis of the fate of these precursor cells throughout the cells' life in complex organ systems in vivo. A good example of the power of this new recombination-based fate-mapping system is the fate determination of Flk1+ cells in mice and proof that Flk1+ cells also exhibit a differentiation potential for the other mesodermal lineages than endothelial cells. See, Motoike et al., Genesis 28:75-81 (2003).


Matsumura et al. (2004) reported a new transgenic mouse model with a lineage-specific cell disruption system to express DT which was silent and harmless without the co-expression of Cre recombinase. This mouse provided a model for a variety of studies addressing the consequences of specific cell-type ablations produced by activation of DT expression when it was bred with lineage/cell-specific Cre-expressing mice. See, e.g., Brockschnieder et al., Genesis 44:322-327 (2006) and Kisanuki et al., Developmental Biology 230,230-242 (2001). However, these conditional gene targeting systems have a number of limitations, as they are either spatially controllable or temporally controllable—but not both.


A mutant ligand binding domain of the human estrogen receptor has also been fused to the Cre recombinase by Metzger and Chambon (2001). In transgenic mouse lines produced with this modification, the nuclear localization of the Cre recombinase leads to action that is tamoxifen dependent. These mice have been used to generate cell/organ specific spatio-temporally controlled somatic mutations. The system has been also used in enriching for desired cell types in stem cell differentiation studies.


Two predominant methods have been developed for introducing ES cells into pre-implantation-stage embryos: the so-called injection chimeras and aggregation chimeras. The injection of embryonic cells directly into the cavity of blastocysts is one of the fundamental methods for generating chimeras. ES cells can also be injected into blastocysts, which is probably the most common method for introducing genetic alterations performed in ES cells into mouse by producing germ-line-transmitting chimeras (Bradley et al., Nature 309:255-256 (1984)). Chimeras can also be created by aggregation of embryonic cells with morula-stage embryos. Although ES cells are typically established from the blastocyst stage, they are still capable of integrating one day earlier into the eight-cell-stage embryos. By taking advantage of this property, a relatively simple way of introducing ES cells back into embryonic environment has been developed (Nagy and Rossant, Gene Targeting: A Practical Approach, pp. 177-206 Oxford University Press (1999). Thus, ES cells can also be aggregated with morula-stage embryos to generate chimeras.


SUMMARY

According to the present method, a novel combination of known genetic tools are used to provide genetically engineered cell, embryo or animal models in which embryonic cells, embryonic stem, precursor and induced pluripotent stem (EC/ES/P/iPS) cells can be precisely directed into desired cell types in intra- or interspecies chimeric composition with differently altered cells in vitro or in vivo. Using this method the expression of a specific gene, or combinations of genes, can be controlled spatially and temporally to develop intra- and interspecies chimeras.


In a preferred embodiment, the method comprises three steps. The first step is to make a transgenic EC/ES/P/iPS cell line which conditionally expresses a suicide or cell progression/existence compromiser gene. Suitable suicide/compromiser genes include Diphtheria Toxin A (DT A), Herpes Simplex Virus-Thymidine Kinase (HSV-TK) or hypoxanthine phosphoribosyltransferase (hprt), although other such genes are contemplated. In the context of the present method, the suicide/compromiser gene is operable to kill target cells or place the target cells at a disadvantage once it is expressed. The time and the type of target cells, i.e., when and where the compromiser gene expression occurs, are controlled by using genetic tools. In certain embodiments, suitable genetic tools include the Cre/loxP, Flp-FRT, and the Tet-inducible recombination systems. In this step, the location of the compromiser gene expression is determined by the gene lineage corresponding to target tissue or organ cells to be derived from the transgenic cell line. Specifically, the compromiser gene is configured to compromise all lineages except that corresponding to the target tissue/organ.


The second step is to aggregate/inject these EC/ES/P/iPS cells into donor embryos. The embryos may have specific gene deficiencies (i.e., knock-out embryos) corresponding to the target lineage. Alternatively, these embryos may be genetically engineered to be complementary compromised in lineages where the EC/ES/P/iPS cells component would be expected to colonize—i.e., the lineage corresponding to the target tissue/organ. The embryo will be a host for the introduced EC/ES/P/iPS cells, establishing the part of the organism where its cells are not compromised. The EC/ES/P/iPS cell contribution may not or may be withdrawn by specific compromiser expression. The complementing part in the organism will be derived exclusively from the introduced EC/ES/P/iPS cells.


The last step of the present embodiment is to apply one or more stimuli to activate the compromiser gene(s) for ablation of undesired tissues/organs of the EC/ES/P/iPS cells and of the host embryo. The stimuli may include exposure of the embryos to a recombination control, such as a particular drug. In a specific example, a suitable drug is a tetracycline.


The present method provides a genetic engineering system for whole organism- or cell-based approaches which can specifically and precisely direct the development of EC/ES/P/iPS cells to desired cell types, tissues or organ systems in vitro or in vivo in an exclusive manner. Using this method, the expression of a specific gene, or combinations of genes, can be controlled spatially and temporally to develop intra- and interspecies in vivo or in vitro chimeric conditions. In these chimeras, a specific cell type, tissue and/or organ system will come exclusively from one component (genotype) and the other cells, tissues and organs are originated from the other component (genotype). For example, this method allows the establishment of a human vasculature (blood vessels) and hematopoietic (blood) system in non-human species such as the mouse or the pig. The method will also enable new approaches to increase the precision of gene therapy methods by differentiating EC/ES/P/iPS cells to specific cell lineages.


According to an alternative embodiment, the method may use genetically modified early cleavage stage embryos or morula embryos (embryonic cells) instead of genetically modified EC/ES/P/iPS cells, in combination with counterpart early cleavage stage or morula embryos instead of blastocysts. These complementary genetically modified cells can then be physically aggregated to produce a viable embryo chimera which can then be transferred to a recipient animal host for gestation and production of live offspring (Nagy et al., Manipulating the Mouse Embryo: A Laboratory Manual, 3d Ed. (2003). A further variation of this method can be to make EC/ES/P/iPS embryonic cell aggregates.





DESCRIPTION OF THE FIGURES


FIG. 1 is diagram showing the steps of one embodiment of the methods disclosed herein.



FIG. 2 depicts the construction of the LoxP-tet-O-DT-A-pA-loxP [SEQUENCE NO. 1] plasmid used in one embodiment of the method.



FIG. 3 depicts the construction of the HSC-SCL-Cre-ERT-pA plasmid [SEQUENCE NO. 2] used in one embodiment of the method.



FIG. 4 depicts the construction of the Endothelial-SCL-Cre-ERT-pA plasmid [SEQUENCE NO. 3] used in one embodiment of the method.





DETAILED DESCRIPTION OF THE EMBODIMENTS

Specific language is used to describe several embodiments of this invention to promote an understanding of the invention and its principles. It must be understand that no specific limitation of the scope of this invention is intended by using this specific language. Any alteration and further modification of the described methods or devices, and any application of the principle of this invention are also intended that normally occur to one skilled in this art.


The methods disclosed herein provide genetically engineered animal models that will be extremely helpful to provide new treatment modalities to address human diseases. These animal models may provide a foundation for producing transplantable human organs or tissues, or make such organs and tissues available for drug testing, for instance. In this model, the development of embryonic, embryonic stem, precursor and induced pluripotent stem (EC/ES/P/iPS) cells in an in vitro and in vivo chimeric organism can be precisely directed to any cell type, tissue or organ system in an exclusive manner. In one example, this method allows the establishment of a human vascular endothelium (blood vessels) and hematopoietic (blood) system in non-human species such as the mouse or the pig.


The present method first makes use of cell depletion due to compromiser genes. Examples of suitable compromiser genes include: diphtheria toxin A (DT A), as demonstrated by Ivanova et al., in the article “In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A”, Genesis 43:129-135 (2005), the disclosure of which is incorporated herein by reference; or Herpes Simplex Virus-Thymidine Kinase (HSV-TK). The present method further makes use of certain genetic tools such as: Cre/LoxP as disclosed by Sauer et al., in U.S. Pat. No. 4,959,317, the disclosure of which is incorporated herein by reference; or Flp/FRT, as described by Wahl et al., in U.S. Pat. No. 5,654,182, the disclosure of which is also incorporated herein by reference. These tools further include recombination systems, such as the recombination system demonstrated by Nagy in the article “Cre recombinase: the universal reagent for genome tailoring”, Genesis 26:99-109 (2000), the disclosure of which is incorporated herein by reference.


In a final step of the method, inducible gene expression system are implemented, such as the tetracycline inducible system described by Bujard et al., in U.S. Pat. No. 5,814,618, the disclosure of which is incorporated herein by reference; or by Belteki et al., in the article “Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction”, Nucleic Acids Research 33, No. 5 (2005), the disclosure of which is also incorporated herein by reference. Using a combination of these tools, the present method contemplates precisely spatially and temporally controlling the expression of cell-specific genes (compromiser) during the development or differentiation processes.


By way of example the method disclosed herein allows the establishment of a human vasculature (blood vessels) and hematopoietic (blood) system in a non-human species such as the mouse or the pig. First, a novel mouse embryonic stem cell (ESC) line will be created which combines all the required genetic tools and inducible systems. In this ESC line, tetracycline inducible compromiser genes are flanked by recombinase attachment sites, such as loxP sites, so that recombinase will delete the compromiser in the lineage of its specificity of expression. A novel transgenic mice line will be produced which is specific gene deficient or in which the inducible compromiser has exactly complementing specificity of expression. This can be achieved by making the reverse tetracycline transactivator recombinase excision conditional, as described by Gossen et al., in the article “Transcriptional activation by tetracyclines in mammalian cells”, Science 23 Jun. 1995 268:1766-1769 (1995), the disclosure of which is incorporated herein by reference.


Chimeras will be formed between these ESC and embryos and the chimeras will be incubated or will be transferred to pseudo-pregnant recipients, such as in a manner described by Voncken in “Genetic modification of the mouse: Transgenic mouse—methods and protocols”, Methods in Molecular Biology, Volume 209 (2003), the disclosure of which is incorporated herein by reference. By administering inducible drugs to the recipient mice, such as doxycycline (a derivative of tetracycline), at specific times in development of the embryo, the expression of recombinase and compromiser genes in the chimeric embryos/fetuses will be regulated. This method will be used to establish chimeras in which, by way of non-limiting example, there is a vascular endothelium and hematopoietic system from one genotype (i.e., from the donor ESCs) with all other tissues from another genotype (i.e., from the recipient), as depicted in the diagram of FIG. 1.


EXAMPLES

The following examples will serve to illustrate the application of the methods described herein.


Example 1
Spatial and Temporal Regulation of Endothelial and Hematopoietic-Specific Gene Expression and its Application in Mouse Esc-Mouse Chimeras

FLK1 is a receptor tyrosine kinase and the main signaling receptor for Vascular Endothelial Growth Factor-A (VAGF-A) during embryonic development and adult neovascularization. (Millauer et al., Cell 72:835-846 (1993), Nature 367:576-579 (1994); Goede et al., Lab Invest. 78:1385-1394 (1998)). Analysis of FLK1 knock-out mice by Shalaby et al., (Nature 376:62-66 (1995), Cell 89:981-990 (1997)) revealed a central role of FLK1 in hematopoietic and endothelial development. Licht and co-workers created a novel transgenic mouse line of FLK1-Cre and then cross-bred with the LacZ report mouse line. (Licht et al., Development Dynamics 229:312-318 (2003)). They detected strong, reproducible LacZ staining primarily in the endothelium of blood vessels, but also in circulating blood cells. An almost complete vascular staining was found at mid-gestation and persisted in all organ systems examined in adult mice.


The stem cell leukemia gene (SCL) encodes a basic helix-loop-helix transcription factor with a pivotal role in both hematopoiesis and endothelial development. During mouse development, SCL is first expressed in extra-embryonic mesoderm, and is required for the generation of all hematopoietic lineages and normal yolk sac angiogenesis. SCL deficient embryos lacked yolk sac hematopoiesis and large vitelline vessels although endothelial capillary spaces were present in SCL-l-yolk sac, as demonstrated by Lorraine, et al. (Proc. Natl. Acad. Sci. USA, VOL. 92, pp. 7075-7079), and substantiated by Shivdasani et al. (Nature (London) 373:432-434 (1995)). To address that the lineage relationship between embryonic and adult hematopoietic stem cells (HSC) in the mouse exists, Joachim et al. (Blood 1 April, Vol. 105, No. 7 (2005)) generated transgenic mice which expressed the tamoxifen inducible Cre-ERT recombinase under the control of the stem-cell enhancer of SCL locus (HSC-SCL-Cre-ERT-pA) (Sanchez, et al. Development 126:3891-3904 (1999), Development 128:4815-4827 (2001); Gottgens, et al., EMBO J 21:3039-3050 (2002)). and proved that tamoxifen-dependent recombination occurred in more than 90% of adult long-term HSCs. This experiment was a clear demonstration of successful inducible genetic manipulation of HSCs in vivo.


The FLK1 and SCL play crucial roles in the establishment of hematopoietic and endothelial cell lineages in mice. Changwon et al. (Development and Disease 131:2749-2762 (2004)) have previously used an in vitro differentiation model of embryonic stem (ES) cells and demonstrated that hematopoietic and endothelial cells develop via sequentially generated FLK1+ and SCL+ cells.


Where the Cre recombinase expression specificity is determined by the endothelial and blood precursor specific promoters, cells derived from the ESC component of the chimeras and differentiated into all non-endothelium and non-hematopoietic (i.e., non-target) lineages will be eliminated by inducing the expression of compromiser genes. At the same time, cells derived from the donor ESC line that developed into target endothelium and hematopoietic lineages will not express the compromiser genes and therefore will survive. Reciprocally, the cells derived from embryo component of the chimeras and differentiated into endothelium and hematopoietic lineages will be eliminated by inducing the expression of compromiser genes. Conversely, cells derived from the embryo component and developed into all non-endothelium and non-hematopoietic lineages will not express the compromiser genes and therefore will survive. As a result, in these chimeras the ESC and embryo components will complement each other; the endothelium and hematopoietic cells will be built from the ESC component, while the embryo component will provide the remaining cells/structure of the chimera.


Applying the present method to this example, a new mouse ESC line will be created which contains LoxP-tet-O-DT-A-pA-loxP (FIG. 2 and SEQUENCE NO. 1), Rosa26-rtTA-IRES-EGFP-pA (Enhanced Green Fluorescent Protein, as disclosed in U.S. Pat. No. 5,625,048, the disclosure of which is incorporated herein by reference), FLK1-Cre-pA and HSC-SCL-Cre-ERT-pA (FIG. 3 and SEQUENCE NO. 2). Mouse SCL−/− recipient blastocysts will be created by breeding SCL−/+mice or mouse recipient blastocysts will be created which contain tet-O-DT-A-pA, Rosa26-LoxP-STOP-LoxP-rtTA-IRES-EGFP-pA, FLK1-Cre-pA and HSC-SCL-Cre-ERT-pA. The new ESC line will then be injected into recipient blastocysts and embryo transfer performed according to suitable techniques, such as that described by Voncken.


A Tet-On and Cre-LoxP system will be combined to regulate specific genes' expression by introducing a recombination control drug, such as tetracycline, into the host embryos. In the stem cells system, when endothelial/hematopoietic cell-specific promoters of FLK1 and SCL express, Cre recombinase will be expressed followed by excision of LoxP recognition sites which contain DT-A. Meanwhile, the lineages other than the target endothelial and hematopoietic lineage will express DT-A which kills the cells. In the recipient blastocysts system, SCL−/− blastocysts are hematopoietic and endothelial cells deficient which will be rescued by stem cells because in the blastocysts, this gene regulatory program is working in an opposite way relative to that in stem cell line. When FLK1 and SCL are expressed, Cre recombinase is expressed followed by excision of STOP gene which stops expression of rtTA. After this stop is removed, the tet-O system is activated and DT-A will be expressed. The result is that the recipient blastocysts will be hematopoietic and endothelial deficient and will be “rescued” by the cells coming from donor stem cell system.


By phenotyping the resulting chimeras to confirm different genotypes of the vascular endothelium and hematopoietic system vs. other tissues, it will be possible to identify if the endothelial and hematopoietic cells differentiated from the ESC line rescued the target lineage of the recipient blastocysts.


Alternatively, a stem cell line will be made with constructs of SCL-Cre and Rosa 26-loxP-TK-loxP. By injecting this cell line into SCL −/− embryos, the hematopoietic and endothelial system in the SCL −/− embryos will be replaced with the corresponding system from the stem cell line.


Example 2
Spatial and Temporal Regulation of Endothelial and Hematopoietic-Specific Gene Expression and its Application in Human ESC-Mouse Chimeras

The highly conserved basic helix-loop-helix (bHLH) transcription factor SCL has been shown in mice and zebrafish to play a crucial role in patterning of mesoderm into blood and endothelial lineages by regulating the development of the hemangioblast. See, for instance, Labastie et al., Blood 92:3624-3635 (1998) and Lorraine et al., EMBO J. 15:4123-4129 (1996), Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7075-7079 (1995). To address the role SCL plays in normal human developmental hematopoiesis, Elias's work (Elias, et. al, Blood 106:860-870 (2005)) provide insight into the role that key hematopoietic genes may play in human embryonic development. Elias' data revealed that SCL was the first and most dramatically up-regulated gene coinciding with emergence of primitive hematopoiesis and was expressed abundantly in all hematopoietic colonies.


The SCL gene is expressed in a subset of blood cells, endothelial cells, and specific regions of the brain and spinal cord. This pattern of expression is highly conserved throughout vertebrate evolution from zebrafish to mammals. Systematic analysis of the murine SCL locus has identified a series of independent enhancers, each of which directs reporter gene expression to a subdomain of the normal SCL expression pattern. Of particular interest is a 3′enhancer that directs expression to blood and endothelial progenitors throughout ontogeny. See, Sanchez, et al., Development 126:3891-3904 (1999). Joachim, et al. (Blood 104:1769-1777 (2004)) generated endothelial-SCL-Cre-ERT mice using inducible Cre recombinase driven by the 5-endothelial enhancer of the SCL locus. By intercrossing with Cre reporter mice, Joachim found Cre-mediated recombination in almost all endothelial cells of the developing vasculature.


Combining all this information, mouse-human chimeras can be made using the methods described in Example 1. A new human ESC line will be created which contains LoxP-tet-O-DT-A-pA-loxP (FIG. 2 and SEQUENCE NO. 1), Rosa26-rtTA-IRES-EGFP-pA and SCL-Cre-pA (FIG. 3 and SEQUENCE NO. 3). Meanwhile, mouse SCL−/− recipient blastocysts will be created, or alternatively recipient blastocysts will be created which contain tet-O-DT-A-pA, Rosa26-LoxP-STOP-LoxP-rtTA-IRES-EGFP-pA, and SCL-Cre-pA. The new ESC line will be injected into recipient blastocysts and embryo transfer will be performed.


The site-specific recombination systems will be activated at a pre-determined time in the development of the embryo by administration of a recombination control, such as the drug doxycycline. Expression of the suicide/compromiser genes in the ESC line and the donor embryo will result in reciprocal ablation of the non-target cells in the ESC line and the target cells in the donor embryo. The ESC line will thus provide the target cells, in this case vascular endothelium and hematopoietic tissues, for the developing chimeric mouse. The resulting chimeras can be phenotyped to confirm different genotypes of the vascular endothelium and hematopoietic system vs. other tissues. In these chimeras, the endothelial and hematopoietic cells will be human genome background while all the other tissues and organs will be mouse genome background.


Example 3
Spatial and Temporal Regulation of Endothelial and Hematopoietic-Specific Gene Expression and its Application in Human ESC-Pig Chimeras

The chronic shortage of human organs, tissues and cells for transplantation has inspired research on the possibility of using animal donor tissue instead of human donor tissue. Transplantation over a species barrier is associated with rejections which are difficult to control. Therefore, it is has been proposed that successful pig to human xenotransplantation requires donor pigs to be genetically modified. See, Prather et al. Theriogenology 59:115-123 (2003); and Kolber-Simonds et al. PNAS 101:7335-7340 (2004). Vascular endothelium is the most immediate barrier between the xenogeneic donor organ and host immune and non-immune defense systems. Thus, these cells are the prime targets for such genetic modifications.


Godwin et al. (Xenotransplantation 13(6):514-521 (2006)) cloned and characterized the regulatory elements of the pig intercellular adhesion molecule-2 (ICAM-2) gene. They observed that a 0.90-kb pig ICAM-2 promoter fragment had strong activity in pig endothelial cells but not in non-endothelial cells. Deletion analysis revealed that the majority of promoter activity was specified by a 0.48-kb sub-fragment with significant homology to the human ICAM-2 promoter. Significant enhancer activity was identified within the first intron of the pig ICAM-2 gene.


The Tie2 promoter and intron/enhancer element has been previously shown to drive reporter genes in vitro and in vivo. Inclusion of a Tie2 intronic enhancer element in conjunction with the Tie2 promoter in Tie2-βgal transgenic mice has resulted in expression in embryonic and adult endothelium as expected, as reported by Schlaeger et al. (Proc. Nat. Acad. Sci. USA 94:3058-3063 (1997)). This same type of promoter-element transgene design was used to generate Tie2-Cre and Tie2-GFP transgenic mice, and Tie2-GFP transgenic Zebrafish (Constien et al. Genesis 30:36-44 (2001); Motoike et al. Genesis 28:75-81 (2000)). Hao et al. (Transgenic Research DI 10.1007/s11248-00609020-8 (2006)) have generated transgenic Yucatan pigs that express the eNOS cDNA under the Tie2 endothelial-specific promoter and Tie2 intron/enhancer element and have demonstrated a similar expression profile in the endothelial compartment in the Tie2-eNOS transgenic swine by immunohistochemistry.


So far, there is no specific gene known which will regulate the differentiation of hematopoietic stem cells from embryonic stem cells in pig. But, it is known that the pattern of SCL gene expression is highly conserved throughout vertebrate evolution from zebrafish to mammals. Thus a promoter of SCL gene can be used to regulate the hematopoietic development in swine.


Consequently, pig-human chimeras can be made using the methods described in Example 1. A new human ESC line will be created which contains LoxP-tet-O-DT-A-pA-loxP, Rosa26-rtTA-IRES-EGFP-pA, SCL-Cre-pA and ICAM-Cre-pA/Tie2-Cre-pA. Concurrently, pig SCL−/− recipient blastocysts will be created or alternatively recipient blastocysts will be created which contain tet-O-DT-A-pA, Rosa26-LoxP-STOP-LoxP-rtTA-IRES-EGFP-pA, SCL-Cre-pA and ICAM-Cre-pA/Tie2-Cre-pA. The new ESC line will be injected into recipient blastocysts and embryo transfer will be performed.


The site-specific recombination systems will be activated at a pre-determined time in the development of the embryo by administration of a recombination control, such as the drug doxycycline. Expression of the suicide/compromiser genes in the ESC line and the donor embryo will result in reciprocal ablation of the non-target cells in the ESC line and the target cells in the donor embryo. The ESC line will thus provide the target cells, in this case vascular endothelium and hematopoietic tissues, for the developing chimeric pig. Finally, the resulting chimeras will be phenotyped to confirm different genotypes of the vascular endothelium and hematopoietic system vs. other tissues. In these chimeras, the endothelial and hematopoietic cells will be human genome background while all the other tissues and organs will be pig genome background.


Example 4
Spatial and Temporal Regulation of any Organ/Tissue-Specific Gene Expression and its Application in Chimeras

Based on the method described above, chimeras of any species can be for which EC/ES/P/iPS cells are available and for which the specific promoter/enhancer required to genetically control the chimeric characteristics is known. These chimeras can be created at various stages of embryonic development. In the present example this process can be used at a point in development in the formation of the initial three (triploblastic) tissue layers, namely the endoderm, ectoderm and mesoderm. In this example, inducing chimerism in one of these tissue lineages will result in all subsequent cells, tissues and organs that are derived from a different genotype.


For example, using this method, a pig with a human endoderm lineage can be made. In one specific embodiment, when a specific promoter/enhancer for endoderm is observed which might be called END, the new ESC line of any kind of background would be created which contains LoxP-tet-O-DT-A-pA-loxP, Rosa26-rtTA-IRES-EGFP-pA and END-Cre-pA. Meanwhile, END−/− recipient blastocysts would be created or alternatively blastocysts of any kind of background would be created which contain tet-O-DT-A-pA, Rosa26-LoxP-STOP-LoxP-rtTA-IRES-EGFP-pA, and END-Cre-pA. The new ESC line would be injected into recipient blastocysts and embryo transfer performed.


The site-specific recombination systems will be activated at a pre-determined time in the development of the embryo by administration of a recombination control, such as the drug doxycycline. Expression of the suicide/compromiser genes in the ESC line and the donor embryo will result in reciprocal ablation of the non-target cells in the ESC line and the target cells in the donor embryo. The ESC line will thus provide the target cells for the developing chimeric animal. Finally, the resulting chimeras would be phenotyped to confirm different genotypes of all the tissues/organs coming from endoderm layers vs. other tissues/organs. In these chimeras, the cells coming from endoderm layer will be one genome background and all the other tissues and organs will be the other genome background.


Example 5
Spatial and Temporal Regulation of Specific Gene Expression and its Application in Embryonic Cell Derived Chimeras In Vitro

Examples 1-4 described above contemplate spatial and temporal regulation of specific gene expression in vivo. In the present example, this method will be used in vitro as well. As in the prior examples, a new ESC line or ECs will be created which contains three transgenes: (1) loxP-tet-O-DT-A-pA-loxP, (2) Rosa26-rtTA-IRES-EGFP-pA, (3) FLK1-Cre-pA/HSC-SCL-Cre-ERT-pA. Instead of blastocysts injection, chimeras will be made by ES cell-diploid/tetraploid embryo aggregation and injection.


The new ESC line will be created to contain LoxP-tet-O-DT-A-pA-loxP, Rosa26-rtTA-IRES-EGFP-pA and END-Cre-pA. Meanwhile, END−/− recipient diploid embryos would be created or alternatively embryos of any kind of background would be created which contain tet-O-DT-A-pA, Rosa26-LoxP-STOP-LoxP-rtTA-IRES-EGFP-pA, and END-Cre-pA. ESC line will be aggregated with recipient embryos and cultured in vitro. Before embryo transfer, inducible drugs will be administered which will result in embryo chimeras having endoderm lineage that comes from the ESC line while the ectoderm and mesoderm lineages come from the recipient blastocysts.


The resulting chimeras would be phenotyped in vitro to confirm different genotypes of all the tissues/organs coming from endoderm layers vs. other tissues/organs. In these chimeras, the cells coming from endoderm layer will be one genome background and all the other tissues and organs will be the other genome background.


While the invention has been illustrated and described in detail in the drawings and foregoing description, the same should be considered as illustrative and not restrictive in character. It is understood that only the preferred embodiments have been presented and that all changes, modifications and further applications that come within the spirit of the invention are desired to be protected.










SEQUENCE NO. 1









Restriction analysis on pMC-loxp-tight-DTa-(R).seq



Methylation: dam-No dsm-No


Enzymes with >3 sites are not shown





Screened with 51 enzymes, 64 sites found


AstII GACGT/C 1: 5117


Acc651 G/GTACC 1: 532


ApaI GGGCC/C 1: 466


ApaLI G/TGCAC 3: 220, 3616, 4862


BamHI G/GATCC1: 1682


BglI GCCNNNN/NCCG 3: 294, 461, 4315


BglII A/GATCT 1: 468


BsaBI GATNN/NNATC 1: 1876


BssHII G/CGCGC 1: 1357


ClaI AT/CGAT 1: 475


EcoICRI GAG/CTC 2: 2552, 2909


EcoRI G/AATTC 3: 445, 719, 2517


EcoRV GAT/ATC 1: 482


HindIII A/AGCTT 2: 486, 2944


HpaI GTT/AAC 1: 1775


KpnI GGTAC/C 1: 536


loxp 2: 514, 2867


MscI TGG/CCA 2: 1042, 1983


NcoI C/CATGG 2: 1392, 2504


NdeI CA/TATG 1: 227


NheI G/CTAGC 1: 2845


NotI GC/GGCCGC 1: 2892


PmeI GTTT/AAAC 1: 2902


PstI CTGACA/G 3: 816, 1013, 2940


PvuI CGAT/CG 2: 322, 4565


PvuII CAG/CTG 3: 351, 1066, 3127


SacI GAGCT/C 2: 2554, 2911


ScaI AGT/ACT1: 4675


SmaI CCC/GGG 2: 458, 2927


SpeI A/CTAGT 1: 2913


StuI AGG/CCT 3: 591, 662, 2567


XbaI T/CTAGA 2: 538, 1883


XbaI <Methy> T/CTAGATC 1: 538


XhoI C/TCGAG 2: 450, 2919


XmaI C/CCGGG 2: 456, 2925


XmnI GAANN/NNTTC 2: 2881, 4794





Non Cut Enzymes


Acc65I<Methy>AflII      ApaI<Methy> BstEII     BstXI       ClaI<Methy>


I-PpoI      I-SceI     MscI<Mety> NruI        NruI<Methy> SacII


SalI












ORIGIN




    1
CTGCCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA CACATGCAGC TCCCGGAGAC


   61
GGTCACAGCT TGTCTGTAAG CGGAGCCGGG AGCAGACAAG CCCGTCAGGG CGCGTCAGCG


  121
GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG CGGAGTGTAC


  181
TGGCTTAACT ATGCGGCATC AGAGCAGATT GTACTGAGAG TGCACCATAT GCGGTGTGAA


  241
ATACCGCACA GATGCGTAAG GAGAAAATAC CGCATCAGGC GCCATTCGCC ATTCAGGCTA


  301
CGCAACTGTT GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC TATTACGCCA GCTGGCGAAG


  361
GGGGGATGTG CTGCAAGGCG ATTAAGTTGG GTAACGCCAG GGTTTTCCCA GTCACGACGT


  421
TGTAAAACGA CGGCCAGGGC CAGTGAATTC TCGAGCCCGG GGGGCCCAGA TCTATCGATG


  481
ATATCAAGCT TGGTACTATA ACTTCGTATA GTATACATTA TACGAAGTTA TGGTACCTCT


  541
AGATCGACAG TGTGGTTTTG CAAGAGGAAG CAAAAAGCCT CTCCACCCAG GCCTGGAATG


  601
TTTCCACCCA ATGTCGAGCA GTGTGGTTTT GCAAGAGGAA GCAAAAAGCC TCTCCACCCA


  661
GGCCTGGAAT GTTTCCACCC AATGTCGAGC AAACCCCGCC CAGCGTCTTG TCATTGGCGA


  721
ATTCGAACAC GCAGATGCAG TCGGGGCGGC GCGGTCCCAG GTCCACTTCG CATATTAAGG


  781
TGACGCGTGT GGCCTCGAAC ACCGAGCGAC CCTGCAGCCA ATATGGGATC GGCCATTGAA


  841
CAAGATGGAT TGCACGCAGG TTCTCCGGCC GCTTGGGTGG AGAGGCTATT CGGCTATGAC


  901
TGGGCACAAC AGACAATCGG CTGCTCTGAT GCCGCCGTGT TCCGGCTGTC AGCGCAGGGG


  961
CGCCCGGTTC TTTTTGTCAA GACCGACCTG TCCGGTGCCC TGAATGAACT GCAGGACGAG


 1021
GCAGCGCGGC TATCGTGGCT GGCCACGACG GGCGTTCCTT GCGCAGCTGT GCTCGACGTT


 1081
GTCACTGAAG CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG TGCCGGGGCA GGATCTCCTG


 1141
TCATCTCACC TTGCTCCTGC CGAGAAAGTA TCCATCATGG CTGATGCAAT GCGGCGGCTG


 1201
CATACGCTTG ATCCGGCTAC CTGCCCATTC GACCACCAAG CGAAACATCG CATCGAGCGA


 1261
GCACGTACTC GGATGGAAGC CGGTCTTGTC GATCAGGATG ATCTGGACGA AGAGCATCAG


 1321
GGGCTCGCGC CAGCCGAACT GTTCGCCAGG CTCAAGGCGC GCATGCCCGA CGGCGAGGAT


 1381
CTCGTCGTGA CCCATGGCGA TGCCTGCTTG CCGAATATCA TGGTGGAAAA TGGCCGCTTT


 1441
TCTGGATTCA TCGACTGTGG CCGGCTGGGT GTGGCGGACC GCTATCAGGA CATAGCGTTG


 1501
GCTACCCGTG ATATTGCTGA AGAGCTTGGC GGCGAATGGG CTGACCGCTT CCTCGTGCTT


 1561
TACGGTATCG CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT TGACGAGTTC


 1621
TTCTGAGGGG ATCGGCAATA AAAAGACAGA ATAAAACGCA CGGGTGTTGG GTCGTTTGTT


 1681
CGGATCCGTC GAGGCAGTGA AAAAAATGCT TTATTTGTGA AATTTGTGAT GCTATTGCTT


 1741
TATTTGTAAC CATTATAAGC TGCAATAAAC AAGTTAACAA CAACAATTGC ATTCATTTTA


 1801
TGTTTCAGGT TCAGGGGGAG GTGTGGGAGG TTTTTTAAAG CAAGTAAAAC CTCTACAAAT


 1861
GTGGTATGGC TGATTATGAT CCTCTAGACT CACACCACAG AAGTAAGGTT TCCTTCACAA


 1921
AGAGATCGCC TGACACGATT TCCTGCACAG GCTTGAGCCA TATACTCATA CATCGCATCT


 1981
TGGCCACGTT TTCCACGGGT TTCAAAATTA ATCTCAAGTT CTACGCTTAA CGCTTTCGCC


 2041
TGTTCCCAGT TATTAATATA TTCAACGCTA GAACTCCCCT CAGCGAAGGG AAGGCTGAGC


 2101
ACTACACGCG AAGCACCATC ACCGAACCTT TTGATAAACT CTTCCGTTCC GACTTGCTCC


 2161
ATCAACGGTT CAGTGAGACT TAAACCTAAC TCTTTCTTAA TAGTTTCGGC ATTATCCACT


 2221
TTTAGTGCGA GAACCTTCGT CAGTCCTGGA TACGTCACTT TGACCACGCC TCCAGCTTTT


 2281
CCAGAGAGCG GGTTTTCATT ATCTACAGAG TATCCCGCAG CGTCGTATTT ATTGTCGGTA


 2341
CTATAAAACC CTTTCCAATC ATCGTCATAA TTTCCTTGTG TACCAGATTT TGGCTTTTGT


 2401
ATACCTTTTT GAATGGAATC TACATAACCA GGTTTAGTCC CGTGGTACGA AGAAAAGTTT


 2461
TCCATCACAA AAGATTTAGA AGAATCAACA ACATCATCAG GGTCCATGGT GGCGGCGAAT


 2521
TCTCCAGGCG ATCTGACGGT TCACTAAACG AGCTCTGCTT ATATAGGCCT CCCACCGTAC


 2581
ACGCCTACCT CGACATACGT TCTCTATCAC TGATAGGGAG TAAACTCGAC ATACGTTCTC


 2641
TATCACTGAT AGGGATAAAC TCGACATACG TTCTCTATCA CTGATAGGGA GTAAACTCGA


 2701
CATACGTTCT CTATCACTGA TAGGGAGTAA ACTCGACATA CGTTCTCTAT CACTGATAGG


 2761
GAGTAAACTC GACATCGTTC TCTATCACTG ATAGGGAGTA AACTCGACAT ACGTTCTCTA


 2821
TCACTGATAG GGAGTAAACT CGACGCTAGC ATAACTTCGT ATAGCATACA TTATACGAAG


 2881
TTATTCTAGC GCGGCCGCGT TTAAACGAGC TCACTAGTCT CGAGCCCGGG ATCGACTGCA


 2941
GCCAAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC


 3001
AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT


 3061
GAGGTAACTC ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC


 3121
GTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGCGC


 3181
TCTTCCGCTT CCTCGCTCAC TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA


 3241
TCAGCTCACT CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG


 3301
AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG


 3361
TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG


 3421
TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG


 3481
CGCTCTCCTG TTCCGACCCT GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA


 3541
AGCGTGGCGC TTTCTCAATG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC


 3601
TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT


 3661
AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT


 3721
GGTAACAGGA TTAGCAGAGC GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG


 3781
CCTAACTACG GCTACACTAG AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT


 3841
ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT


 3901
GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT


 3961
TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG


 4021
GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT


 4081
AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT


 4141
GAGGCACCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC


 4201
GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG


 4261
CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC


 4321
GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG


 4381
GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACA


 4441
GGCATCGTGG TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA


 4501
TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT


 4561
CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG


 4621
CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCA


 4681
ACCAAGTCAT TCTGAGAATA GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATA


 4741
CGGGATAATA CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT


 4801
TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT


 4861
CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA


 4921
ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC


 4981
ATACTCTTCC TTTTTCAATA TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA


 5041
TACATATTTG AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA


 5101
AAAGTGCCAC CTGACGTCTA AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG


 5161
CGTATCACGA GGCCCTTTCG TCTTCAAGAA//











SEQUENCE NO. 2









HSC-CRE-ERt



LOCUS Untitled 13033 bp DNA linear SYN 03-JAN.-2008


DEFINITION.


ACCESSION.


KEYWORDS.


FEATURES Location/Qualifiers


BASE COUNT 3087 a 31210 c 3427 g 3399 t












ORIGIN




    1
ACGACTGGAG AGATGGCTCA CTGGTTAAGA GCACTGACTA TTCTTCCAGA GGTCCTGAGT


   61
TCAATTCCCA ACAACCACAT GGTGGCTCAG AACCATCTGT AATGGGATCT GATGCCCTCT


  121
CCTAGTGTGT CTGAAGGCAG CCACAGTGTG TGTGTGTGTG TGTGTGTGTG TGTATACATA


  181
TACATATATA TGTATATATA TAATTTTTGC ATATTAAATC TATAAAAAAA AAACCCAGTG


  241
AGATCCGAGT TCTGTGTATT GAGAATACCA AGGTGTATGG TGTGTGTGTG TGGGGGGGAA


  301
GAGGACACTT CATTGGAATA ATTCAAGGAA GAGCTTTCTT TATATTTTCT CCATCAGGAG


  361
GGGAGCCCAG ATTCTAGTGA CTTCTGGAGC ACTTTCCCAA GTCTTAAGAG TCCAGCTGAG


  421
CAGAATGGGG TGGAGTGTGA AGGGTAGTAG GACCAGAATC CAGGATTAGC TTCAGTCCTT


  481
GACTCCCTTT CTTATGATAG GGTAGCTACT TGCAGAATAC AACGGTGGGT TTGCTTAGTG


  541
TAGGCTGCTT TCCTCTTGGC CGGGAATATT TCTGACATCC TTGGTTGAAT AGAGCAGAGT


  601
TCTTGCAGCT TCCACACCCT ACTTCACCAC CATAGTCTTT CTGGGTGTAT ATTTGCAGCG


  661
CATGTGTGTA GCAGTAGATC GGGAGAGGGT TCCTATAGCA CTGGACAGAT TCCCCGCCAA


  721
AACCAAAAGG GGGGCGGGAA GGACACGCTT GCTCGGGGGA TTAGTTCCCT CCCCTTCCCC


  781
TGTGGCCTAA GAAGGAGGGA CTGGGTGATC TTTCTCTTCT CTGTGCATTT CCTTCCTCCT


  841
TTTTCCCGTC GATTTTTGTC TCTCTGCCTG TATTCCTTTT CTCCCAAGGT TTCTGCCATC


  901
TTTCTCCAGC ACAATTCCTA CCCTTGGACA CTGTGCCTTC CGGGCTTGTC CCACCCTTTT


  961
CTTCCAATCT AGAGACACCC CCACATTGCT CCAGCTCCAG GCCTGTGGGC CTTCACGCCA


 1021
GCAGGGTTGG GGTGTGCGTC CACGTGGTGC TGAGTTTGTC CTGTCCGCTT TTCAGGTTTC


 1081
AGTGCGTGAT CTCCTCTCTG CCCCTTACCC TGTTACAGGA TGACGGAGCG GCCGCCGAGC


 1141
GAGGCGGCAC GCAGTGACCC GCAACTAGAG GGACAGGACG CGGCCGAGGC CCGCATGGCC


 1201
CCCCCGCACC TAGTCCTGCT CAACGGCGTC GCCAAGGAGA CGAGCCGCGC AGCCCCGGCT


 1261
GAGCCCCCCG TCATCGAGCT AGGAGCGCGC AGCGGCGCGG GGGGCGGCCC TGCCAGTGGG


 1321
GGCGGTGCCG CGAGGGACTT AAAGGGCCGC GACGCAGTAG CAGCCGAAGC TCGCCTTCGG


 1381
GTGCCCACCA CCGAGCTGTG CAGACCTCCC GGACCCGCCC CGGCGCCCGC GCCCGCTTCG


 1441
GTTCCTGCAG AGCTGCCTGG AGACGGCCGC ATGGTGCAGC TGAGCCCGCC CGCGCTGGCA


 1501
GCCCCTGCCG GCCCCGGCCG AGCGCTGCTC TATAGCCTTA GCCAGCCGCT CGCCTCACTA


 1561
GGCAGGTGAG CATCCCGGTC CCCTGCGGCG TTCTGGGTGC AGGCGAGGGT CGAGAGGAGG


 1621
GGGTGGTGGC TTAAGATTCC AAGAGGAACG AGCCCAGAGA CCAGAGTCTC TCCCGCAACC


 1681
CTCCCGCTAG TGGGAAAGGG GTCCCCTGTG AGACAGACTG TCAGGAAGGA CCGGTGGTCA


 1741
GGGGACGACA GTTGTGTAGA AACCGGGGGT GGTCGCCTGC ACTGTTGAGG GTGCGGGTCT


 1801
GTGGGTGAGT GTAAAAAGCT GCAGAGGTTG CTGACTACTG TTGAGTAGGC GGGATTCTTT


 1861
AATATGAGTT CTGGGCCAGT GTCTGAATGC CCCTCTGCAG CAGAGGTGAG GTTCGCCACA


 1921
AAGGGTGAAC TCTTCAGGAA GCTGCCGCGG TGGGTGGACA GGCTGGAGAG AAAGATCTAA


 1981
GGCCGTTGCT GAGGGCAGCT CTTCTCAGCC TCTGCTAGGA TGCAGTGAGC GACACTGTCA


 2041
TCCGCTCCTA ATCCTTCTGT CCCTTACCTG CGTGGTTGGT CTCCTTGCTG GGCCCTGTGG


 2101
TGAGGGAAGC TGAATGGCCA GCAGAGTGTA GGACAGGCGG TAGGAAAGAA TTATAGGACA


 2161
ACACGATGGT AGAGCAGTAG GGAGCGCTGT CAAGGGTTGG TGAGTGGGAG GTGGGGGGTG


 2221
GTGCCGATCT GTGATCAGAG AGTGATGGTC GGTGAGGTCT GAGGGGACAA TGTGAGACCC


 2281
TTTGTGGTGT GGGAGTTCTC TACTAGCACT TCCATCCCTC ACGTGTTGTC CTGTGTAGGT


 2341
ACTTGTCTCT GAGCAAAGGT CTACCAGGAT TGAAGGAGAT TTTGTGTGTG TGTGTGTGTG


 2401
TGTGTGTGTG TGTGTGTGTG TGTGTGTGTA CTTCAGCACA GGAATACGCC GCCTTGCCCC


 2461
TCCCATTTAT GTATTGTTCC ATATATTCAC CCTCTTCGCT TCTGTGAATG CATGCATACT


 2521
CAATTCAATC TGCATTTTAA GTGTGCAGGA GCAGGGGGTG CCTTAGCAGG AGGGGACTGA


 2581
AGACACACAG GGAGAATCCA TCTAAGGAGT CTTTTTGTCT TTAACCTCAT TGTGATCTAC


 2641
CTTCTCTTTC CATAGTGGGT TCTTTGGGGA ACCGGATGCC TTCCCCATGT TCACCAACAA


 2701
CAACCGGGTG AAGAGGAGGC CCTCCCCATA AATTCCACCA TGTCCAATTT ACTGACCGTA


 2761
CACCAAAATT TGCCTGCATT ACCGGTCGAT GCAACGAGTG ATGAGGTTCG CAAGAACCTG


 2821
ATGGACATGT TCAGGGATCG CCAGGCGTTT TCTGAGCATA CCTGGAAAAT GCTTCTGTCC


 2881
GTTTGCCGGT CGTGGGCGGC ATGGTGCAAG TTGAATAACC GGAAATGGTT TCCCGCAGAA


 2941
CCTGAAGATG TTCGCGATTA TCTTCTATAT CTTCAGGCGC GCGGTCTGGC AGTAAAAACT


 3001
ATCCAGCAAC ATTTGGGCCA GCTAAACATG CTTCATCGTC GGTCCGGGCT GCCACGACCA


 3061
AGTGACAGCA ATGCTGTTTC ACTGGTTATG CGGCGGATCC GAAAAGAAAA CGTTGATGCC


 3121
GGTGAACGTG CAAAACAGGC TCTAGCGTTC GAACGCACTG ATTTCGACCA GGTTCGTTCA


 3181
CTCATGGAAA ATAGCGATCG CTGCCAGGAT ATACGTAATC TGGCATTTCT GGGGATTGCT


 3241
TATAACACCC TGTTACGTAT AGCCGAAATT GCCAGGATCA GGGTTAAAGA TATCTCACGT


 3301
ACTGACGGTG GGAGAATGTT AATCCATATT GGCAGAACGA AAACGCTGGT TAGCACCGCA


 3361
GGTGTAGAGA AGGCACTTAG CCTGGGGGTA ACTAAACTGG TCGAGCGATG GATTTCCGTC


 3421
TCTGGTGTAG CTGATGATCC GAATAACTAC CTGTTTTGCC GGGTCAGAAA AAATGGTGTT


 3481
GCCGCGCCAT CTGCCACCAG CCAGCTATCA ACTCGCGCCC TGGAAGGGAT TTTTGAAGCA


 3541
ACTCATCGAT TGATTTACGG CGCTAAGGAT GACTCTGGTC AGAGATACCT GGCCTGGTCT


 3601
GGACACAGTG CCCGTGTCGG AGCCGCGCGA GATATGGCCC GCGCTGGAGT TTCAATACCG


 3661
GAGATCATGC AAGCTGGTGG CTGGACCAAT GTAAATATTG TCATGAACTA TATCCGTAAC


 3721
CTGGATAGTG AAACAGGGGC AATGGTGCGC CTGCTGGAAG ATGGCGATCT CGAGCCATCT


 3781
GCTGGAGACA TGAGAGCTGC CAACCTTTGG CCAAGCCCGC TCATGATCAA ACGCTCTAAG


 3841
AAGAACAGCC TGGCCTTGTC CCTGACGGCC GACCAGATGG TCAGTGCCTT GTTGGATGCT


 3901
GAGCCCCCCA TACTCTATTC CGAGTATGAT CCTACCAGAC CCTTCAGTGA AGCTTCGATG


 3961
ATGGGCTTAC TGACCAACCT GGCAGACAGG GAGCTGGTTC ACATGATCAA CTGGGCGAAG


 4021
AGGGTGCCAG GCTTTGTGGA TTTGACCCTC CATGATCAGG TCCACCTTCT AGAATGTGCC


 4081
TGGCTAGAGA TCCTGATGAT TGGTCTCGTC TGGCGCTCCA TGGAGCACCC AGGGAAGCTA


 4141
CTGTTTGCTC CTAACTTGCT CTTGGACAGG AACCAGGGAA AATGTGTAGA GGGCATGGTG


 4201
GAGATCTTCG ACATGCTGCT GGCTACATCA TCTCGGTTCC GCATGATGAA TCTGCAGGGA


 4261
GAGGAGTTTG TGTGCCTCAA ATCTATTATT TTGCTTAATT CTGGAGTGTA CACATTTCTG


 4321
TCCAGCACCC TGAAGTCTCT GGAAGAGAAG GACCATATCC ACCGAGTCCT GGACAAGATC


 4381
ACAGACACTT TGATCCACCT GATGGCCAAG GCAGGCCTGA CCCTGCAGCA GCAGCACCAG


 4441
CGGCTGGCCC AGCTCCTCCT CATCCTCTCC CACATCAGGC ACATGAGTAA CAAAGGCATG


 4501
GAGCATCTGT ACAGCATGAA GTGCAAGAAC GTGGTGCCCC TCTATGACCT GCTGCTGGAG


 4561
ATGCTGGACG CCCACCGCCT ACATGCGCCC ACTAGCCGTG GAGGGGCATC CGTGGAGGAG


 4621
ACGGACCAAA GCCACTTGGC CACTGCGGGC TCTACTTCAT CGCATTCCTT GCAAAAGTAT


 4681
TACATCACGG GGGAGGCAGA GGGTTTCCCT GCCACAGTCT GAGAGCTCCC TGGCGGAATT


 4741
CGGATCTTAT TAAAGCAGAA CTTGTTTATT GCAGCTTATA ATGGTTACAA ATAAAGCAAT


 4801
AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC ATTCTAGTTG TGGTTTGTCC


 4861
AAACTCATCA ATGTATCTTA TCATGTCTGG TCGAGATCTA AGGAAGACCC TGAATTCTGT


 4921
TCTCATACTC CATACCCCAT ATCTTTCTTC CTCTGTGTCT TCCTTGCCCT TAAAGAAATT


 4981
GCAGCATTCC AAGAACAATA TCTGTACAAA GGGGGAAATG TAAGCATGAG AAAACATTAA


 5041
AAAAAAAAAA CAGTGATGAA CATAACCACA GAGAGAATCC CACCCTTCAA GAATAATTCA


 5101
TGTTTATTTG TGGTGGCAAA TAACAAAATG GTACAACCTT TATCCTTTTC CAGAAACAAA


 5161
AACCAAGGGC ACAGCAACTA GAGTGAGCTG ACAGCTATTT TGGCCTTTTT GGTGGGTCTA


 5221
GCCGTACTTG GGATCCCAGT GGTACATGAC CCTCTGCCGA AGGCTTGCCT CAGTCTGTGT


 5281
ACATAGCACG CCATGTCTGT GGGCAAGCCC AGCACTTTGC GTCAGTGTCG TACTGTATGT


 5341
AATGAACTGT GTTGGTCTCT GTGTTTTTTT TTTCTGAAGA AGAGGAGTAA CTACTCCGGG


 5401
TACCTTGATA TTTGTACAGC CTATAGGCCA ACACTGCGGG CGTGTGACTC TTTATTGAAA


 5461
AACAAAAACA AAAAAATACC AGTGTGGTGA TGATAGTGTG TGTATATATA TATAAGGTTA


 5521
TATGGGGAAG ATTTCTAAAT AAAAGTTTTA CAAAGGGGCC TGGACTTTGT ACTTGGACTT


 5581
TGCCCCCTAG AGTCTGAGAA TGGGAACATC AAGGGGAAAG GCTGACAGCT TTTAGGAAGT


 5641
AGGATCTAGC TTCCAGTCTC AGCCTGTCGG GGAGGAAGGA GGCTACCCTA TGGGGGGGTT


 5701
TCCTTTTCCC CCCTTCTGCA AGGCTCCAAG GGCTTCAGTA TCCTGTCCTT GTGTTTGCAG


 5761
CCCTAGACAG CCTAGACCTC TCTGTGTAGG GTCAGCTTTC TCCTTGTTAG ATCACTTTCC


 5821
CAAGTTGGGA CCATTGCTCC CAGTGAGAGC TTAGGACAGA AAAATGTAGC TGTTATCCAC


 5881
CATTGGTGTC CATAGATTTC CTGATGACTC AGTGGGGGTT GCATCTTTTA CACTTGACTT


 5941
TTTTTTTAAA GGTTAAAAAA TATTTTATGT ATAGAGATGT TTTATGTGTA TAGGTACAGT


 6001
GCCCACAGAT GCCAGAAAAG GGAGTCGGAT TCCCTGGAAC TGGAGTTGCA AACCGTTGTG


 6061
AGTTGCCCTG TAGGTGCTGG AGTTTCATGA ATAGAATTTG GGAAAGAGAC TGGGTCTTGG


 6121
GGAGGCCATT ATGCATGGAC GTTTGGTCTC CTGGGAGTTT GTAAGCTGGG CATCTTCTGT


 6181
CTTCTCATTT AACAAGCATT TGCTGAGCTC CTGCTCTGGG CAGACACTGT TCTGTTGGGG


 6241
AGGGTTCAGC ATTGAATGAA ACAAGCATGG ATGCTCTCCA CTGCACCTTA CATTTTAGCA


 6301
GGGGGATGTT GAATGCAGAA ACACATACAA GTAGAGTTAA ATAGTTAGAA AGCAAATTAG


 6361
TATTAACCCA CAGTGAGTTT TATTCAGGCC AGCCTGGGCT ACAGTCTCAA AAACCAAAGC


 6421
CAAGAAAGGT GGTAAGGAAC AAAAGTGGGC AGATCAACAG GGATAGTTCA GGAAGGCCC


 6481
TAGGGTGCCA TCTTTTTCAT TCAGGATCAG ATGATTCCTG GTGTCAGAGA CAGTTTTGTC


 6541
CCAGGGACAG GTTGGGTCTT TCTATCTACA TGCCCTGAGA TGGCTTTTTT CTTTCTTCTT


 6601
CTCTGGACCT CAGTACTCAA CCCCAAATCT ACAGACATGG ACTAGCTCAG ATTCAACAAT


 6661
TGGGAGGGAA TTCAATAGTC TCACCGTTAA TTCCCAGCTG GCCTGTCTCT AGTCTCAGCT


 6721
GTGTTTTGTC CTCTTAGCTT CTATCCATCT ACAGGGAGAG GGTAGGATTC AGCCTGAGTG


 6781
TCAATATCTG ATCCAGCTAC TGGGAAGCTC CTCAGATATG CCTCTCTTTG GCCTAGGACA


 6841
AGGATGGTAG GATTTGGCCT TGGGGAGGGG AGAAAAATGG ATATTTAGGC TTATAGACCT


 6901
GAGGAACTAT CATGATAGGA GAGAAAGAAA GAGGACAGAG AAGGAAGAAT GTGTTTGGG


 6961
GTGGAGGAAG TGGCCAGTAT GCTCAGTACA ACTGAGGGGC CATGCACGGA AAGGCTGAGT


 7021
TAACTGGTTT GAGGCAGCTG GTGACTGGAA AGAGCTGCAG AGAGGAGTGA ATAGAGGTAG


 7081
TGACCTGAGG ACTCAGAGAT GTCACTTCCC ATCTTGTAAG ATTTTCCTCA GGAGAAATGA


 7141
AGCTTTCCAT GTAATGGTGA CAAAGAGAGC CCGAGGATTC TGATCACTCC CGGAGTTCAT


 7201
CGATGGGGCA GAGACCCAGA GAGAAAATGT CTTCTCAAGC CTTGTATCTC AGAGTGGTGT


 7261
GTAGGCAGGC CCATTCTCCC TGTCCCAAGA AAATGTTGTC TCTGAAGCCC AGAATCCCTG


 7321
ACTCCACAAG GGAAGAAAAG TGCCCTGAGG CCTGGCCTGA GGTGTTTTGC TGATCTGTTC


 7381
CCCTTTATTT CTTACCACTC CATTTGTGTG TGTGTGTGTG TGTGTGTGTG TGTTTGCTTA


 7441
TTTGTTTTTC TGAAACAGGG TCTCATGTGG CCTCAAACCC ACTAAGTTGC TGAGGCTGAC


 7501
TTTGAACTTC CGATCTTCCT GCCTCTGTCT CCAGAGTGCT GGGATTACAG GTGTGCACTA


 7561
GAATACCAGG TTTATTCAGT GACAGGAGCT AAATCCAGAG CTTTGTGCAT ATTAGGCAAG


 7621
CACTCTACAA CCAGACTGCA TCCCCACCCC ACGCCTCACT CTTTTGTGCC TACCGTACTA


 7681
GCTTTCTTCC TTTTTGTTTT AGACTGTTTT ATTGGTTTTT GACTCCCAGA TGTTGAATTT


 7741
TGGTTTATTT TTCACATAAC AGCCCATCTT CCTCTTTGCC CACTCTCATT TGGTTGAATT


 7801
GTCCCTGAAG TCCAGGAAGT TTTCCTGACT CCATGGGACT GGGTGCCTCC TTTGCATCCC


 7861
CATGGGACCC CAGGTATGCT GGCCCTTCCT GCCCTAACAT TTGCTTATTT AGTTGCTTCT


 7921
TCACTGAAAC ACAAACCCCT CAGAGCTGAA ACCAAGTCTG ATTAAGCCCT CTGCACCAGC


 7981
ACCTTAGGGT ACAGACACTC GGTTCTTTCC CCACTGGCCA TGAACAGCCC TTCTCCTCCC


 8041
ACTGGCTCTC TATTTTCTCT CTGGGCCTGG CGTCTGACCT GGCATCTGGC AAGGACCTGA


 8101
AAGGCTGGTA TAGAGTGGTG AAGACCAGGC ATGGAGGCTA TGGATCCAGT CAGCTGTCTG


 8161
GCCTCCTCAC GCCGGTCCCT ACCTGCTTCC TTTTTAATAA AATAAGTGTG TGTTCCTCAG


 8221
AAGCTGTCAC TGTGTCATTA GCTTCCTCGC ACCCCCTACC CGGACACACC CCCCTGCCCA


 8281
TGTAAACCTG TTACCTATTC ACAGAGCTTA ATTGTCATGA ATCTAAGTAA AGGGTTACCC


 8341
AGGGGAGGTG ACACAAAGCC CTGAGTTGGA AGGGGCTTGA GCAAGGTGAA GTAGGTGTGA


 8401
ATTCAGGGCG ACACCCAAGG TTAGAGATCC AGACCACATA GGAAGGTCAG GAAATAGAAG


 8461
AGGAGGCCAG TAGACAGCTA GAGTTCATAG AGAAAATGGC TTTACTTTCC TTATGGGCAA


 8521
GAGGGCTACA CAAATTTAGG CCCAGGACAG GTGGTGGTAG TGAAGAGCTT GCTGGCTGGA


 8581
GGACTGGCTC TGTGGATGAC CATGGGGACA GTGAGGAAGG ACAGTTGGTG TGGAACAGTT


 8641
GGTGAAGGGA GTAACTGGGG CCTGGGTGGA AGTGAGAAGA AAAGAGCAGC CAGGCTCTGG


 8701
AGGAGCTTGG CCTGGTCAGA ATCACTTGGG GCTTAAGGGC TTAAGTATTG CTACTGGGTG


 8761
TGCTGGCTTG TGACTTTGAG TGAGTCACTA TCATTCTGAG GTTTGGTTTC TTTATCTGTT


 8821
AAACAGAGAT GTTAACAGTC ATCTTCCAGG ACTGTCATGG GACTTCAGCA TAATATATGC


 8881
AAAGTATCTG TGTTTCATTA AAAAATGATT CTATAGAAAG AGCTACGGAA ATATCTATAA


 8941
GAAAGCATTC TTTTTCCAAG AAACAGGACC AGGAGGGATG GGACTGTCCT AACAGAAGAG


 9001
ACGAGGGAAG GACATGAGTG TGAGGGAATA TTAATCCCTC ACTCAACAGC AGGACTTTTG


 9061
TGTGCCTGTC TTATGTCAGG AAAGGAGGGG TAGCCAGTCT TGACCACCCA TTTTGACTTC


 9121
AGAGGCTGGA GAGCAGAGTG GAAGCTGGGA ATAGGAAGGA ATCCTAGAGG CAAGTGCTAT


 9181
GGGAGGAGCT TAGTGGTGTG GTGTGGGCAG CCTAGCTCTG ACAGTAAAGT CCCTGAGCAA


 9241
GTTGTGCTGA ACTGAACTGT CCTGAGGGGC AAGGTTGGGA GGTATCTGGG AGATTTCACA


 9301
TTCTGTCTTG AGCATTACCT AGTTTTCAGT GGTGGAGCGG GCTGGTCCAG GAATGCTGGC


 9361
TTCCTCCTGG GCCCCATACT CTTGCCAAGG CTACCTGGGG TGAGGCAATG CTCCCCCACC


 9421
TCACTTTGCC TTCCAGCTCC TACTTAAGCT CTCCCCACTG GTTTGCTCTG AGGCCTGCCC


 9481
CTCCCCAGCT CCTGGGCTTT CTCTCCACAC AATAACAGGA TGTGATCTTC GAAGAGAGGA


 9541
AGTGGGGGAG GACTGCTGTG CCGATAGCAG GGAAGGAGGG GGGCTTCTGA CTCTCCCCTC


 9601
TCCAGCCCTC CTTTGCTCTG TAGGCCAGCC CCTGCAGCTC CTTGATCCCC CTAAGCCCTA


 9661
CCTCAAGCTT CTATCTGAGA CAAGTAGGGA TGAAGGGTCT TTAGGCCCAT GTAGGACTGC


 9721
TTGCCTATGG AGAGACATGC CTTGGCCACA CCGTCTTCAG GATCTACCTT CTGGAGAGAC


 9781
TTGCTGGCCT AGCTTTAGAT GCTGGGTTGT TTTCTGCCCG GAGCTGCTGG AGTCTAAGGG


 9841
TGGGCAGGTG GGTCATTCTG TAGGGCTCCA TCTGTCCAGT GCACTCCCAA GTCCACACGA


 9901
GCATGATTCA GTGCAGGGAG TGCGTGATAG CATCAATCTA AAGGTCTATG TCAAATGCTG


 9961
GTTTGGCTTG CACAGTGTGT GTCAGGCTGC AAAAATGGAC AGTGAAATCC AGAAAGACAA


10021
GGAGCATGAG GAAGGAGCAA GGCTAGGCTG GAACCCAGCA CTAGGTCATT GGGTTACCGC


10081
CTCTTCGAGC CAGGGATGTT CTTAGAACTT CCAAAGTTGA TGGGAAAGTT TTAGATCGAG


10141
TCGACCGATG CCCTTGAGAG CCTTCAACCC AGTCAGCTCC TTCCGGTGGG CGCGGGGCAT


10201
GACTATCGTC GCCGCACTTA TGACTGTCTT CTTTATCATG CAACTCGTAG GACAGGTGCC


10261
GGCAGCGCTC TTCCGCTTCC TCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC


10321
GAGCGGTATC AGCTCACTCA AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG


10381
CAGGAAAGAA CATGTGAGCA AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AGGCCGCGT


10441
TGCTGGCGTT TTTCCATAGG CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA


10501
GTCAGAGGTG GCGAAACCCG ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT


10561
CCCTCGTGCG CTCTCCTGTT CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC


10621
CTTCGGGAAG CGTGGCGCTT TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG


10681
TCGTTCGCTC CAAGCTGGGC TGTGTGCACG AACCCCCCGT TCAGCCCGAC CGCTGCGCCT


10741
TATCCGGTAA CTATCGTCTT GAGTCCAACC CGGTAAGACA CGACTTATCG CCACTGGCAG


10801
CAGCCACTGG TAACAGGATT AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA


10861
AGTGGTGGCC TAACTACGGC TACACTAGAA GAACAGTATT TGGTATCTGC GCTCTGCTGA


10921
AGCCAGTTAC CTTCGGAAAA AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG


10981
GTAGCGGTGG TTTTTTTGTT TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG


11041
AAGATCCTTT GATCTTTTCT ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG


11101
GGATTTTGGT CATGAGATTA TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT


11161
GAAGTTTTAA ATCAATCTAA AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT


11221
TAATCAGTGA GGCACCTATC TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC


11281
TCCCCGTCGT GTAGATAACT ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA


11341
TGATACCGCG AGACCCACGC TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG


11401
GAAGGGCCGA GCGCAGAAGT GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT


11461
GTTGCCGGGA AGCTAGAGTA AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA


11521
TTGCTACAGG CATCGTGGTG TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT


11581
CCCAACGATC AAGGCGAGTT ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT


11641
TCGGTCCTCC GATCGTTGTC AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG


11701
CAGCACTGCA TAATTCTCTT ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG


11761
AGTACTCAAC CAAGTCATTC TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG


11821
CGTCAATACG GGATAATACC GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA


11881
AACGTTCTTC GGGGCGAAAA CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT


11941
AACCCACTCG TGCACCCAAC TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT


12001
GAGCAAAAAC AGGAAGGCAA AATGCCGCAA AAAAGGGAAT AAGGGCGACA GGAAATGTT


12061
GAATACTCAT ACTCTTCCTT TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA


12121
TGAGCGGATA CATATTTGAA TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT


12181
TTCCCCGAAA AGTGCCACCT GACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG


12241
TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT CCTTTCGCTT


12301
TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG TCAAGCTCTA AATCGGGGGC


12361
TCCCTTTAGG GTTCCGATTT AGTGCTTTAC GGCACCTCGA CCCCAAAAAA CTTGATTAGG


12421
GTGATGGTTC ACGTAGTGGG CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG


12481
AGTCCACGTT CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT


12541
CGGTCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG TTAAAAAATG


12601
AGCTGATTTA ACAAAAATTT AACGCGAATT TTAACAAAAT ATTAACGCTT ACAATTTGCC


12661
ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT


12721
TACGCCAGCC CAAGCTACCA TGATAAGTAA GTAATATTAA GGTACGTGGA GGTTTTACTT


12781
GCTTTAAAAA CCTCCCACAC CTCCCCCTGA ACCTGAAACA TAAAATGAAT GCAATTGTTG


12841
TTGTTAACTT GTTTATTGCA GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATT


12901
TCACAAATAA AGCATTTTTT TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATG


12961
TATCTTATGG TACTGTAACT GAGCTAACAT AACCCGGGAG GTACCGAGCT CTTACGCGTG


13021
CTAGCTCGAG ATC


//











SEQUENCE NO. 3









Endothelial-CRE-ERt



LOCUS Endo-pGL2-Promoter 12191 bp DNA circular SYN 12-DEC.-2007


DEFINITION.


ACCESSION.


KEYWORDS.


FEATURES Location/Qualifiers


BASE COUNT 3021 a 3013 c 2707 g 3451 t












ORIGIN




    1
CCCGGGAGGT ACCCTGGGCT ACACAGAGAT AGATGTCTTT TGCCACAGCT TCTCCTGGCA


   61
ACCCAAAGCT ACCTGGCAGA GTCCAGTCTG CCTAACACCT ATGAATCTAT GAGATACCTT


  121
AAAAAGCATA TCCTTCTTCT ATACATCTTT CCACTTCCTC CTCTTCTCCA CCCTATTCAT


  181
CAGACAACTG TCTCAGTCAG TGGGGAACAT GAAGAGGGGA TATGGATGCT TGCTTTCACA


  241
GGTGCCTCTG CATAAAGGGA GTTCTCAGTG AGCTGGAGCA GAGGCTATGG AGGAAGGAAG


  301
CAGAGATGAC AGATTAAAGA CAGATGCAGA GACAAGGCCT TTATACAGGA AAGAGGAGCA


  361
GATTCAGAGT TTGCCTGAGC CTAAGATAGA GACCGGAGAA ATGAAAGGCA GAGTGAGCAA


  421
GATAAGAGAT GAAAGGAATA GACCCCGGGG TTCCTCCACT GCATCCTCAT TACAGATAGG


  481
AAAACTGAAG GTCAGAGGAA GTGGTGGTTC TACTTCCTAC GGATGTATCC ATCACTCTTG


  541
TAAATACACT GGGTCAGGTC CTCCCTTCTC CAGCACTTTC CTCTTGCCCT TGTGCACTAG


  601
GACTGAGTAA CACAAGTGAC ACCCAGTGGG AGGCTCTTGG ACAAGTCAAC CAGGAAGAGG


  661
GAGAGAGGAG ACAGTGTAGA CAAAATAGAT TGACAGGGAA GTTTTTCTGG ACTAGGTAAG


  721
CTTGAAGAAG GGCACAGAGG GTGTAAACAA CTGTATTAAG GTATATGGTT TATGTGCAGT


  781
AAGATATACC ATTTCAGCAT CCAGTTAGCT GAGGTTTGAC AACACTTTTA TAGTCATTAC


  841
CACAATCAAT GTATAGAATA TGTCTTTGAT AGAAAGTTCC GTTGTGTCTC TTGTATCCAC


  901
TCCATGTTGC ATTTTATCCT TCTGAACTCT GTCATTGTAG ATTCATTTTG CTTTTCTTCC


  961
CCTAGGGTTT CATGTACATG TAGCTAGTTA CTATAGACAC TGTCTGATCG CTTTCACTCT


 1021
GCATGAATTG GAGGTTTTCT ATGTTTTTAC ATGTATCTGA AGTTATTTTT ATTGCTCAGC


 1081
CATATCCAGT TGCATGCCTG GACCAATATA TGTACCTATC TGTTGATATG CTTTGAAAGT


 1141
ATTTCTACTT TTTTTTTGTT TGTTTTTTGT TTTGTTTTTG AGACTATGTG GCTCTGGCTG


 1201
TCCTGGAACT CGAAATATAG GCCAGGCTGG TGTGGCACTC ACAGAGACTC ACCTGCTTCT


 1261
GTCTCTCACA TTCTGGGATT AAAGGTGTGG ACCATTATGC CCTGCTTATT TATTTTTTGA


 1321
TGCTATGGAT AAGTCTTTAT ATGCATATAT TCCTGTGGAT ATGTTTTTAT GTCTCTCAAT


 1381
AAATATGTAT TATTAACCAT ATGATTAAAG GGGCTCAAGT GTGTGTATTT TTCTCCAATA


 1441
TGGCTGAGTA GAGTTTGCAT TCCCTTCAGT GTACAAGAAT GCCAGTTGAC CTTTATTCCT


 1501
ATCAACACTT GGTCTTGTCT GTCTTTCACG TTCTAGCCAT ATTGACAGAC TTGTCATGGT


 1561
AGCTCTTTGT AGTTTGAATT TCTGTTTCTC TGGTGTAACT TTCTTTTCAT GAAAGTTTTG


 1621
TTTTCTAAGT TCATAAGGAT TTTCAACTCA GACACATTAC AGTGATACTC ACTGTTCGGC


 1681
TGAAGATTTT AAAGTAGTTC ACACAGGAAG AAATGTCATA TAGCCAACAG GGGTGGAGAG


 1741
GACAATAGGC CATGTTGTTC TAGGCTACAC AGCAGTTAAA TGACAAGAGT GAGCCTGCTT


 1801
TCTCACCTCC AAAGTAGCGT CACCAGGCGG CATGACACTG TCATTGTCTA CAGTCAGATG


 1861
ACAGGTGGAC ACAAGGGCAG AAGAGGTACA CACAGAGAGA TGCTCAGTAC ATGCATGTGC


 1921
AGGGCCTGGA GGCATATCTA CTGTCTTGAT GTGTGTCATA AACCTGGCCA CTGTCCTGAT


 1981
GACCATCGGC AGCTATTTGC GACAGAGTTG GTGGTTGTGC GTGTATTGTC TTCTAATGGC


 2041
TTGAACAAGT AAAACATTAA TGGCAGAATG CTCTCTCCTG AGGACAGAAA GCTTGGGAAC


 2101
ACAAACTGGG GACACAGCTT TGGTCCTCTG TGTACTTCTA GAAGATGCAT AGGTTGCACA


 2161
AGGAAGATAG GAGGCTAGAG AGCCCGCTGC CTTCTGCAGC TGCTCATTCA TTTTGCTTTG


 2221
GATTTTTTCC TTTCATTTCT CTTTCTTTCT TTCTTTCTTT CTTCCTTTCT CACCAATGGT


 2281
GCTCTAGTTC TTAAGCTGTG TGCTGCAGAC ATCATCCTGG AGGCTGGTGA AACACACCTG


 2341
GCCTCTCTTC CAGAGGAGCC TAGGGTCCCC TTCCAGAACT GACTTCTCTA AGGACATGGC


 2401
CCCTCCTTTG AAAGTCATAC ATTAGAGCAA AGCCCTTTCC ATCCCTGCAA ATGCTGATGG


 2461
CAAGGCTGGG ATAAGAACAT GGAAATGATT TCATCTGTGG GGTTCTGGGC TCAGCCTTGC


 2521
AAACTAGAAT GGCAGGGGCT CATTCCTAGT AAGGAACAGA GGCAAAATAT GGAGGACAGT


 2581
TATATGGAAA TGAATTGGAG CAGGTTATGA CATCTCCTTA AATGGGCATA TTTACCATCA


 2641
ATAAGTTTTA TAAAACCCAC TGTCAGGTAT GGGCAATTAT CACCTCCTCT TTACAGAGGA


 2701
GGAAAATGGA AGAGGCTATC TTGCCTATGC TCATGCAGCC CAGTGAGAAA GCAGGAATGA


 2761
GGGCTCAGAC ATGCTAGTCA ATGGTTCTGC TCTGCTGCCT GGAGGCACCA GAATGTCCCG


 2821
GCTGGGAATT CTTTATTCAC AGCAAGTTGC TTAGATGTCT GAGCTATCTA CTAAGTGGAA


 2881
GTCCCGACCT TCCCTACGTC TTTGAGCTGT TGTAAAATGA ACGGAATTGA CATTATGAAG


 2941
TGTTTAGGTC TGGCACGATA CAAATTCGTT ATAAACCCAT CTGCCCACCA GAGTGCTGGC


 3001
AGACCGAACT TCTCCAGGGG TGGAAGCTCA GAGATGGTAC AGCACCTGAA AACATTGCAA


 3061
ACCCTGGACT CTGGAGGGCG GACAACGTAG GCCCTGGGAG TGGAGGAGCC TGTCCCCTGC


 3121
TCTTGCCTAC CCGGGGCCAG ACTCCAGACT CCCTGGTTCC TCACCTCCCC GCCCCCTCAC


 3181
CACCCCCACC GAGGCGCTCC GAATTTCCTG CCCGACCGAG GCCCGGCTCG GGCGGGTGGA


 3241
GGAGGGCTGG CATTTCCTGG CCGCCGCGTC ACTGGCTCAG CGGTGCTCGG ACAAAGCGCT


 3301
GACCGACAGG CACCAGAAGC TATTTCAGGC GGCGCCCAGC TTAGCGCGCA GTTTCCGTTT


 3361
TTCCACCGTC GGAAACAGGG AACAGGGAGC TTGCAGACGT CACAAACCCC CAGCCTCAGG


 3421
CGTGGGTCCA GGGACCAGGA GAGGCAAGGC CCATGTGTTA GAAACAGGGT AGAGGCAGAC


 3481
GCTATCCCCG CACCTTCTAT CCAACCTTAC TCCTTAACTG TCCTTGGAAA CACCAGAGAA


 3541
GGCCATTTCA CACCCAGGAA AATGATCCAG TCGTCGTTGG TCAAGCCAAA TGCATAACCT


 3601
TTTCAAGCCC ATAAACCTCG AGACAGCCTT ACCCCATTCC CTCTCCTGAA TTAACTAACC


 3661
TGCCCCCAGA CATCCTGGAT TCTTCGATTT TCATTATTCA ACGGCGTCGT AGTTCTTCCA


 3721
AACTCAGTCT TAAATACCCT GTGCGAAACA TCTACCCCAC ACCTTCTCTT CCATCTCCTG


 3781
GAAGGAGAAT TAGAACAAGC TCTAACCTCT TTTCTCTGGT CACAGAACAC TTAGCCTTCA


 3841
CCTCCCAGCT CCCCACACCA ACACAGCCCC TACCGCCATT TCAACCCAAG GCTTTCCTTT


 3901
CCTTTCCTTT CCTTTCCTTT CCTTTCCTTT CCTTTCCTTT CCTTTCCTTT CCTTTCCTTT


 3961
CCTTTCCTTT CCTTTCCTTT CCTTTTTCCT TTTTTATTAG ATATTTTCTT TATTTACATT


 4021
TCAAATGTTA TCCCTTTCCT AGTTTCCCCT CCGAAAGTCC CCTATCCCTT CCCCCTCCCC


 4081
CTCCCCCTGC TCCCCAACCC ACCCACTCCT GCTTCCTGGC CCTGGCATTC CCCTATACTG


 4141
GGGCATAGAG CCTTCACAGG ACCAAGGGCC TCTCCTCCCA TTGATGACCA ATTAGGCCAT


 4201
ACTCATGGCT CTAACTGCAT ACTGCATATG CAGTTAGAGC CATGAGTCCC ACCATGTGTT


 4261
TTCTTTGATT GGTGGTTTAG TCCCAGGGAG CTCTGGGGGC ACTGGTTAGT TCATATTGTT


 4321
GTTCCTCCTA TGGGGCTGCA AACCCCTTCA GCTCCTTGGA TACTTTCTCT AGTTCCTTCA


 4381
TTCCAAGGGT TTTCTAAAAA AGCAAATCCG ATCTTACATA GGACAGCAAG CCCTTATGTA


 4441
AACACAGTGG TAAAAACAAA ACCCTCAATT CTTCCACCCA TACTGTACCA GTTTTCTGTT


 4501
TCTTACATTA ACTTTCCCCC TTTCTGTGTC AGCCCTTGGT CCAGGACGCC TGGCTTTCCT


 4561
GGGAAGCACA CCCAGTTAGC TCACATACAA TATAGTTAGC CCATATAACC AAGCGAAGGC


 4621
AGCACAGCTG GACTTTCATC AAATGTCACA GAGGAACAGA CAGGTGTAAC TAATACTCCA


 4681
TCTCCAGTAT TGGTCCTGAA ATCTAGGAGG GGCAGAACTC AAAACAGGTG CTACTCTTTG


 4741
GAATCAGCCC TTGACTGAGT CTCAGTCTGT GACCGGGTTC AGAGCTACTG GAAGGTCAAT


 4801
GCAGTTTGGG ATGCTTAGTG GGGTCTATGG AATGGAAATT GAACAAGAGA GTTCAGATAG


 4861
GTGCTGGCGT TACTCTAGCT ATAAGTTTGA TCAAGTTACA TCTCTTTGCC CCCTACTTTC


 4921
CTTTAACATT CTTAATTTCT GTATGGCAAC CAGACAAATG CCTATGATAT CTTATTAGCT


 4981
CCCTACCACC CACTTTTTAT ATTATTTCTC ATGTATATGA AACTTTGGCA TTTAAAATAT


 5041
TATTATTATT CATTTAGGCT TTTTGAGAAA GGGTTTCTCT GTGTAGTCTT GGCTGTCCTG


 5101
AAACTCCATA TGTAGATTAG GCTGGCCTCT AACTCAAAGG ATCTGCCTGC CTGCCTCAAT


 5161
GAGAGCTGGG ATTAAAGGTA TGTGCGTTTA TTCTTTGAGT ATTTTATAAA ATGAATTTTG


 5221
ATCATATTCA CCTCCTATTA CCCCTCATCT CCTCCTATCC TGATGTCTTT TTTTTTTTTT


 5281
TAAATCTACA GAGAGGCTAG AGAGATGGCT CAGTGGTTAA GAGCATTGGT TGCTCTTCTG


 5341
GAGGACCTGG ATTTGATTTC CAGTGTCTAT ATGGTAGCTC ACAAACTCCT GTTTCAGAGG


 5401
AGCTAATGTG TCTGCCTTCT TCTAGTCTCT GGATACAGTA CATAGACATT AATACAGGCA


 5461
AAACCAGGGA CCACACCCTT AAAAATGATT CCCCTTCTCA AGAAGTGTTC AACTGTCAAT


 5521
AGCTCTTCAG TTAGCGGTGA AGGCTCATGA ACACCCCCCC CCCACACACA CACACACACA


 5581
TACACCTACC TTCTGGCTAG AATCTTGACT GGCTTGATCC TGAGCAGGCA ACCACAGCTG


 5641
TGAGTTGGTC CTTTTCTGAC CAGAAGGTAG TCACTCTGGT CTTCCTTAAC TGCTCTTACT


 5701
ATCTTTTTCT TCTTTTCTTC TCTTCTGCTT CAAGGCAGGG TTTCACTATG AACCCGTGGC


 5761
TGGTGGCTGA CCTGGCTCTC TATACCAGGC TGTCTTTGAA TTCATGGGTG TCCACCTCCC


 5821
TCTGCCTCCC AAACACCACC ATGTGTCCAC CATGGTCTCA CTCACGTAGC CCAAACTGGC


 5881
CAAGAATTCT GCTATTTTTG CCCTTATCTT TTGGATTACA ACAGCCAGTT TCCACCATCA


 5941
TAGAAAGAAA AACCCAGATA CTCCTCAGCC CATTGGGTTC CTACAATGTA CCTATGGGCT


 6001
TCAATGTCAA ACTTCTTTCA AATCAGGCTT CTGTCCCTCA CTAGAAATTT AATATTGAGT


 6061
TATTGACACA GCCCTGTCAC CCCCTCCCCC CACTGTTTTC TCACCTATAA ATTAGGAATA


 6121
ATAAAAGCAC CAATGGGGAG AAGTTGGATG AAGGGGGAAA AGTATCACTA AAGCACACTA


 6181
TTATTCAAAG GTGCCCTAAT GATATCCAAT TGTATGGTAT TTAAAAAATA AAAAATAAAA


 6241
GCATTACGAA ACCGGGTGTG GTGGTGCATG CCTTTAATCC CAGCACTCCA GAGGCAGAGA


 6301
CAGGTGGATC TGCATCTCAA TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC


 6361
CCGCCCCTAA CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT


 6421
ATTTATGCAG AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA GTGAGGAGGC


 6481
TTTTTTGGAG GCCTAGGCTT TTGCAAAAAG CTCGATCCTG AGAACTTCAG GGTGAGTTTG


 6541
GGGACCCTTG ATTGTTCTTT CTTTTTCGCT ATTGTAAAAT TCATGTTATA TGGAGGGGGC


 6601
AAAGTTTTCA GGGTGTTGTT TAGAATGGGA AGATGTCCCT TGTATCACCA TGGACCCTCA


 6661
TGATAATTTT GTTTCTTTCA CTTTCTACTC TGTTGACAAC CATTGTCTCC TCTTATTTTC


 6721
TTTTCATTTT CTGTAACTTT TTCGTTAAAC TTTAGCTTGC ATTTGTAACG AATTTTTAAA


 6781
TTCACTTTTG TTTATTTGTC AGATTGTAAG TACTTTCTCT AATCACTTTT TTTTCAAGGC


 6841
AATCAGGGTA TATTATATTG TACTTCAGCA CAGTTTTAGA GAACAATTGT TATAATTAAA


 6901
TGATAAGGTA GAATATTTCT GCATATAAAT TCTGGCTGGC GTGGAAATAT TCTTATTGGT


 6961
AGAAACAACT ACATCCTGGT CATCATCCTG CCTTTCTCTT TATGGTTACA ATGATATACA


 7021
CTGTTTGAGA TGAGGATAAA ATACTCTGAG TCCAAACCGG GCCCCTCTGC TAACCATGTT


 7081
CATGCCTTCT TCTTTTTCCT ACAGCTCCTG GGCAACGTGC TGGTTATTGT GCTGTCTCAT


 7141
CATTTTGGCA AAGAATTGTA ATACGACTCA CTATAGGGCG AATTCCACCA TGTCCAATTT


 7201
ACTGACCGTA CACCAAAATT TGCCTGCATT ACCGGTCGAT GCAACGAGTG ATGAGGTTCG


 7261
CAAGAACCTG ATGGACATGT TCAGGGATCG CCAGGCGTTT TCTGAGCATA CCTGGAAAAT


 7321
GCTTCTGTCC GTTTGCCGGT CGTGGGCGGC ATGGTGCAAG TTGAATAACC GGAAATGGTT


 7381
TCCCGCAGAA CCTGAAGATG TTCGCGATTA TCTTCTATAT CTTCAGGCGC GCGGTCTGGC


 7441
AGTAAAAACT ATCCAGCAAC ATTTGGGCCA GCTAAACATG CTTCATCGTC GGTCCGGGCT


 7501
GCCACGACCA AGTGACAGCA ATGCTGTTTC ACTGGTTATG CGGCGGATCC GAAAAGAAAA


 7561
CGTTGATGCC GGTGAACGTG CAAAACAGGC TCTAGCGTTC GAACGCACTG ATTTCGACCA


 7621
GGTTCGTTCA CTCATGGAAA ATAGCGATCG CTGCCAGGAT ATACGTAATC TGGCATTTCT


 7681
GGGGATTGCT TATAACACCC TGTTACGTAT AGCCGAAATT GCCAGGATCA GGGTTAAAGA


 7741
TATCTCACGT ACTGACGGTG GGAGAATGTT AATCCATATT GGCAGAACGA AAACGCTGGT


 7801
TAGCACCGCA GGTGTAGAGA AGGCACTTAG CCTGGGGGTA ACTAAACTGG TCGAGCGATG


 7861
GATTTCCGTC TCTGGTGTAG CTGATGATCC GAATAACTAC CTGTTTTGCC GGGTCAGAAA


 7921
AAATGGTGTT GCCGCGCCAT CTGCCACCAG CCAGCTATCA ACTCGCGCCC TGGAAGGGAT


 7981
TTTTGAAGCA ACTCATCGAT TGATTTACGG CGCTAAGGAT GACTCTGGTC AGAGATACCT


 8041
GGCCTGGTCT GGACACAGTG CCCGTGTCGG AGCCGCGCGA GATATGGCCC GCGCTGGAGT


 8101
TTCAATACCG GAGATCATGC AAGCTGGTGG CTGGACCAAT GTAAATATTG TCATGAACTA


 8161
TATCCGTAAC CTGGATAGTG AAACAGGGGC AATGGTGCGC CTGCTGGAAG ATGGCGATCT


 8221
CGAGCCATCT GCTGGAGACA TGAGAGCTGC CAACCTTTGG CCAAGCCCGC TCATGATCAA


 8281
ACGCTCTAAG AAGAACAGCC TGGCCTTGTC CCTGACGGCC GACCAGATGG TCAGTGCCTT


 8341
GTTGGATGCT GAGCCCCCCA TACTCTATTC CGAGTATGAT CCTACCAGAC CCTTCAGTGA


 8401
AGCTTCGATG ATGGGCTTAC TGACCAACCT GGCAGACAGG GAGCTGGTTC ACATGATCAA


 8461
CTGGGCGAAG AGGGTGCCAG GCTTTGTGGA TTTGACCCTC CATGATCAGG TCCACCTTCT


 8521
AGAATGTGCC TGGCTAGAGA TCCTGATGAT TGGTCTCGTC TGGCGCTCCA TGGAGCACCC


 8581
AGGGAAGCTA CTGTTTGCTC CTAACTTGCT CTTGGACAGG AACCAGGGAA AATGTGTAGA


 8641
GGGCATGGTG GAGATCTTCG ACATGCTGCT GGCTACATCA TCTCGGTTCC GCATGATGAA


 8701
TCTGCAGGGA GAGGAGTTTG TGTGCCTCAA ATCTATTATT TTGCTTAATT CTGGAGTGTA


 8761
CACATTTCTG TCCAGCACCC TGAAGTCTCT GGAAGAGAAG GACCATATCC ACCGAGTCCT


 8821
GGACAAGATC ACAGACACTT TGATCCACCT GATGGCCAAG GCAGGCCTGA CCCTGCAGCA


 8881
GCAGCACCAG CGGCTGGCCC AGCTCCTCCT CATCCTCTCC CACATCAGGC ACATGAGTAA


 8941
CAAAGGCATG GAGCATCTGT ACAGCATGAA GTGCAAGAAC GTGGTGCCCC TCTATGACCT


 9001
GCTGCTGGAG ATGCTGGACG CCCACCGCCT ACATGCGCCC ACTAGCCGTG GAGGGGCATC


 9061
CGTGGAGGAG ACGGACCAAA GCCACTTGGC CACTGCGGGC TCTACTTCAT CGCATTCCTT


 9121
GCAAAAGTAT TACATCACGG GGGAGGCAGA GGGTTTCCCT GCCACAGTCT GAGAGCTCCC


 9181
TGGCGGAATT CGGATCTTAT TAAAGCAGAA CTTGTTTATT GCAGCTTATA ATGGTTACAA


 9241
ATAAAGCAAT AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC ATTCTAGTTG


 9301
TGGTTTGTCC AAACTCATCA ATGTATCTTA TCATGTCTGG TCGACCGATG CCCTTGAGAG


 9361
CCTTCAACCC AGTCAGCTCC TTCCGGTGGG CGCGGGGCAT GACTATCGTC GCCGCACTTA


 9421
TGACTGTCTT CTTTATCATG CAACTCGTAG GACAGGTGCC GGCAGCGCTC TTCCGCTTCC


 9481
TCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC AGCTCACTCA


 9541
AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA


 9601
AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG


 9661
CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG


 9721
ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT


 9781
CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT


 9841
TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC


 9901
TGTGTGCACG AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT


 9961
GAGTCCAACC CGGTAAGACA CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT


10021
AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC


10081
TACACTAGAA GAACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA


10141
AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG GTAGCGGTGG TTTTTTTGTT


10201
TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT


10261
ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA


10321
TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA


10381
AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA GGCACCTATC


10441
TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT


10501
ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGACCCACGC


10561
TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT


10621
GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA


10681
AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTACAGG CATCGTGGTG


10741
TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT


10801
ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC


10861
AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT


10921
ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTC


10981
TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAATACG GGATAATACC


11041
GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA


11101
CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT AACCCACTCG TGCACCCAAC


11161
TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA


11221
AATGCCGCAA AAAAGGGAAT AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTT


11281
TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA CATATTTGAA


11341
TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT


11401
GACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG TGGTTACGCG CAGCGTGACC


11461
GCTACACTTG CCAGCGCCCT AGCGCCCGCT CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC


11521
ACGTTCGCCG GCTTTCCCCG TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT


11581
AGTGCTTTAC GGCACCTCGA CCCCAAAAAA CTTGATTAGG GTGATGGTTC ACGTAGTGGG


11641
CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT CTTTAATAGT


11701
GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT CGGTCTATTC TTTTGATTTA


11761
TAAGGGATTT TGCCGATTTC GGCCTATTGG TTAAAAAATG AGCTGATTTA ACAAAAATTT


11821
AACGCGAATT TTAACAAAAT ATTAACGCTT ACAATTTGCC ATTCGCCATT CAGGCTGCGC


11881
AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT TACGCCAGCC CAAGCTACCA


11941
TGATAAGTAA GTAATATTAA GGTACGTGGA GGTTTTACTT GCTTTAAAAA CCTCCCACAC


12001
CTCCCCCTGA ACCTGAAACA TAAAATGAAT GCAATTGTTG TTGTTAACTT GTTTATTGCA


12061
GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA AGCATTTTTT


12121
TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATG TATCTTATGG TACTGTAACT


12181
GAGCTAACAT AA


//





Claims
  • 1. A method of producing a transgenic animal comprising the steps of: providing a transgenic cell line which conditionally expresses a compromiser gene corresponding to a predetermined lineage complementary to a target lineage;providing a donor embryo having a specific gene deficiency corresponding to the target lineage or which conditionally expresses a compromiser gene corresponding to the target lineage;introducing the cell line into the donor embryo; andactivating the compromiser gene(s) at a predetermined time in the development of the donor embryo so that only the target lineage of the transgenic cell line survives and only the complementary lineage of the embryo survives.
  • 2. The method of claim 1, wherein the transgenic cell line is embryonic cells, embryonic stem cells, precursor or induced pluripotent stem cells [EC/ES/P/iPS cells].
  • 3. The method of claim 1, wherein the target lineage corresponds to the hematopoietic and endothelial system of the transgenic animal.
  • 4. The method of claim 1, wherein the target lineage corresponds to an organ of the transgenic animal.
  • 5. The method of claim 1, wherein the target lineage corresponds to tissue of the transgenic animal.
  • 6. The method of claim 1, wherein the transgenic cell line is human.
  • 7. The method of claim 6, wherein the donor embryo is a non-human animal.
  • 8. The method of claim 7, wherein the non-human animal is mouse or pig.
  • 9. The method of claim 1, wherein the donor embryo is a morula-stage embryo.
  • 10. The method of claim 1, wherein the introducing step is in vivo.
  • 11. The method of claim 1, wherein the introducing step is in vitro.
  • 12. The method of claim 1, wherein the compromiser gene is selected from Diphtheria Toxin A (DT A), Herpes Simplex Virus-Thymidine Kinase (HSV-TK) or hypoxanthine phosphoribosyltransferase (hprt).
  • 13. The method of claim 1, wherein the activating step includes a recombination control drug introduced into the host embryo.
  • 14. A method of producing a transgenic animal comprising the steps of: providing a transgenic cell line which conditionally expresses a compromiser gene corresponding to a predetermined lineage complementary to a target lineage;providing a donor embryo having a specific gene deficiency corresponding to the target lineage or a donor embryo which conditionally expresses a compromiser gene corresponding to the target lineage;introducing the transgenic cell line into the donor embryo; andactivating the compromiser gene(s) at a predetermined time in the growth of the donor embryo so that only the differentiated cells of the target lineage of the transgenic cell line will survive and only the differentiated cells of the complementary lineage of the embryo will survive.
  • 15. A method of directing the development of an embryo comprising the steps of: providing a transgenic cell line which conditionally expresses a compromiser gene corresponding to a predetermined lineage;introducing the cell line into a donor embryo having a specific gene deficiency or a compromiser gene corresponding to a complementary lineage; andactivating the compromiser gene(s) at a predetermined time in the growth of the donor embryo so that the complementary lineage of the transgenic cell line will substitute for the complementary lineage of the donor embryo as the embryo develops.
  • 16. A chimeric animal comprising: a target tissue and/or organ differentiated from the genotype of a transgenic cell line; andall remaining non-target tissues and/or organs differentiated from the genotype of a donor embryo.
  • 17. The chimeric animal of claim 16, wherein the transgenic cell line is embryonic cells, embryonic stem cells, precursor or induced pluripotent stem cells [EC/ES/P/iPS cells].
  • 18. The chimeric animal of claim 16, wherein the transgenic cell line is human.
  • 19. The chimeric animal of claim 17, wherein the donor embryo is a non-human animal.
  • 20. The method of claim 19, wherein the non-human animal is mouse or pig.
REFERENCE TO RELATED APPLICATION

This application claims priority to co-pending PCT application PCT/U.S.08/056,204, filed on Mar. 7, 2009, the disclosure of which is incorporated herein by reference, which claims priority to U.S. provisional application No. 60/690,169, filed on Mar. 9, 2007, entitled “A Novel Method for Conditional and Inducible Transgene Expression to Specifically and Precisely Direct the Development of Embryonic Cells, Embryonic Stem Cells and Precursor Cells”, the disclosure of which is incorporated herein by reference.

PCT Information
Filing Document Filing Date Country Kind 371c Date
PCT/US08/56204 3/7/2008 WO 00 9/9/2009
Provisional Applications (1)
Number Date Country
60906169 Mar 2007 US