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The inventions listed below are owned by an agency of the U.S. Government and are available for licensing in the U.S. in accordance with 35 U.S.C. 207 to achieve expeditious commercialization of results of federally-funded research and development. Foreign patent applications are filed on selected inventions to extend market coverage for companies and may also be available for licensing.
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The TGF-beta-related superfamily plays an important role in multiple biological systems including embryogenesis. TGF-beta ligands activate specific receptors, which interact with specific Smad proteins, which in turn form a complex with a common partner, Smad4, that conveys the signal to downstream targets. Exon 8 of the
The fibroblast growth factor receptor 4 (
The five Muscarinic Acetylcholine (ACh) receptors are G-protein coupled receptors (M1R–M5R). M1R, M3R and M5R selectively couple to Gq/G11; M2R and M4R selectively couple to Gi/Go. M5R knockout mice are viable and fertile, and have no major morphological abnormalities.
M5 muscarinic ACh receptors are located in the central nervous system and may contribute to the cognitive-enhancing effects of ACh. M5R knockout mice show deficits in two hippocampus-dependent cognitive tasks, and exhibit reduced cerebral blood flow in the cerebral cortex and hippocampus, consistent with the observation that M5Rs mediate ACh-mediated dilation of cerebral blood vessels. M5R agonists or agonists for mixed M1/M5 receptors may be effective in the treatment of Alzheimer's disease and related memory disorders. The M5R knockout mutation also appears to exert a stabilizing effect on sensorimotor gating in intact mice, which is decreased in schizophrenia. Analysis of M5R knockout mice also has shown that the lack of M5Rs reduces drug-seeking behavior.
The signal transducer and activator of transcription (STAT) family of transcription factors conveys signals from membrane receptors to the nucleus, where they activate diverse genetic programs. Stat5a and Stat5b are highly conserved proteins that are activated by many cytokines, erythropoietin, prolactin and growth hormone. Despite their similarities, they have many unique functions. Stat5a deficiency results in the loss of prolactin-dependent mammary gland development, but does not affect body growth. Inactivation of Stat5b does not adversely affect mammary development and function, but leads to severe growth retardation. To study the effects of combined deficiency of Stat 5a and 5b before and during pregnancy, loxP was added to the ends of a DNA fragment that contains the two genes which are located within a stretch of 110 kb on chromosome 11 in a head to head orientation with no other genes between them. The loxP-flanked fragment was introduced into the genome using homologous recombination, and deleted using two transgenic lines expressing Cre in mammary epithelium at different times. Deletion of Stat 5 before pregnancy prevents epithelial proliferation. Ductal characteristics are retained but differentiation into secretory alveoli does not occur. When deletion of Stat5 occurs late in pregnancy after differentiation has started, differentiation is halted and premature death occurs.
Prolactin induces mammary gland development and lactogenesis. Binding of Prolactin to its receptor leads to the phosphorylation and activation of STAT (signal transducers and activators of transcription) proteins. Two Stat proteins, Stat5a and Stat5b, are expressed in mammary tissues during pregnancy. Stat5a null mice developed normally, and were indistinguishable from hemizygous and wild-type littermates in size, weight and fertility. Mammary lobulo-alveolar outgrowth during pregnancy was reduced and females failed to lactate after parturition. Stat5b, despite 96% similarity to Stat5a, could not compensate for the loss of Stat5a. Stat5a is the principal and obligate mediator of mammopoietic and lactogenic signaling.
The heterotrimeric G protein G
G
The Sirtuins (Sirt1–7), a family of seven proteins related to yeast Sir2, are histone deacetylases that regulate many critical biological processes including genomic stability, adaptation to calorie restriction and aging. Mice with a targeted disruption of Sirt6 had very low levels of blood glucose (and paradoxically, low insulin levels) and died shortly after weaning. Hypoglycemia, attributed to increased sensitivity to insulin, was the major cause for lethality.
Because of the post-weaning mortality of Sirt6 null mice, liver-specific Sirt6 conditional knockout mice were constructed using Cre-Lox technology to study the effects on glucose and lipid metabolism. Hepatic-specific Sirt6 deficient mice exhibited increased glycolysis and triglyceride synthesis, resulting in the development of fatty liver. Sirt6 is a potential therapeutic target for treating fatty liver disease, the most common cause of liver dysfunction.
Sirtuin 1 (Sirt1), a homolog of yeast Sir 2, is an NAD-dependent histone and protein deacetylase. It has a wide range of biological functions, ranging from DNA damage repair to effects on glucose metabolism. Sirt1 null mice die before birth due to chromosomal aberrations and impaired DNA damage repair. Sirt1 is thought to affect energy metabolism, but the mechanism remains poorly understood. In order to study tissue-specific metabolic effects of Sirt1, floxed Sirt1 was constructed so that exons 5 and 6 would be deleted using the Cre-Lox strategy. In contrast to a previously reported deletion of Sirt1 exon4, no truncated (and potentially active) Sirt1 forms were detected when exons 5 and 6 were deleted.
Hepatic exon 5–6 null Sirt1 mice were generated when Floxed Sirt1 exon 5 and 6 mice were mated with mice that expressed the Cre-recombinase in liver. The hepatic exon 5–6 null Sirt1 mice developed fatty liver under normal feeding conditions. This was accompanied by increased expression of the carbohydrate responsive element binding protein, which is a major
Endochondral ossification is a major mode of bone formation. Cartilage proliferates, undergoes hypertrophy, begins to calcify, undergoes a program of cell death, and is replaced by osteoblasts. Fibroblast Growth Factor Receptor 3 (FGFR3) is expressed in cartilage rudiments of a wide variety of bones, and dominant missense mutations in the human FGFR3 gene cause achondroplasia, a common form of human dwarfism characterized by minimal proliferation of the growth plate cartilage in long bones. To determine the effect of complete absence of FGFR3 on bone development in mice, targeted disruption of the FGFR3 gene was accomplished by homologous recombination in embryonic stem cells. Remarkably, the vertebral column and long bones of FGFR3 null mice were extremely long, suggesting that in normal development, FGFR3 restrains cartilage promotion and limits bone elongation so that the endochondral ossification program can proceed. Restraint of cartilage growth by FGFR3 provides a plausible explanation for the role of FGFR3 missense mutations in human achondroplastic dwarfs.
Fibroblast Growth Factor Receptor 2 (FGFR2) is a high affinity receptor for several members of the FGF family. The FGFR2 gene was inactivated by deleting the entire immunoglobulin-like domain of the receptor which is critical for FGF binding and FGFR2 activity. Embryos that lack this domain die at E10–11.5 owing to a failure in chorioallantoic fusion or placental formation. The deletion also blocks limb bud initiation, establishing FGFR2 as the major receptor that mediates FGF signals during limb induction.
The E. Coli tetracycline operon regulatory system was used to generate a liver-specific transcription activation system that was inhibited by tetracycline. The transcription activator was a fused protein consisting of a tetracycline repressor gene (tetR) that was only active in the presence of tetracycline and a herpes simplex virus protein (VP–16) transcription activating domain. Transcription was induced only in the absence of tetracycline (Tet-Off). A liver-specific promoter such as mouse albumin determined that the tetracycline-regulated transcriptional activator (tTA) would be expressed specifically in liver. To study the effect of the transcription activator on a target gene (for example, Simian Virus 40 (SV4) large tumor (T) antigen (TAg)) specifically in liver, Alb-tTA mice were mated with transgenic mice in which the Target gene (TAg) was controlled by the E. Coli Tetracycline Operator (Tet-O). In this example, TAg was expressed in hepatocytes in the absence of Tetracycline, leading to hepatoma formation. When the mice were treated with tetracycline, TAg was not expressed and hepatomas did not form.
The E. Coli tetracycline operon regulatory system was used to generate a liver-specific transcription activation system that was inhibited by tetracycline. The transcription activator was a fused protein consisting of a
Erythropoietin, acting by binding to Erythropoietin receptors (EpoR) on erythroid progenitor cells, is required for erythropoiesis. Absence of erythropoietin or the EpoR in mice interrupts erythropoiesis in the fetal liver and results in death at embryonic day 13.5. An 80-kb human EpoR transgene bred onto a mouse EpoR null background (provided by F. Constantini of Columbia University) restored effective erythropoiesis in the EpoR null mouse. Erythropoietin preparations made utilizing recombinant DNA technology are used in the treatment of anemia in chronic kidney disease and other critical illnesses. The mouse EpoR null mouse containing the human EpoR transgene can be used to define the potency of erythropoietin preparation in humans.
Yeast Sir2, a nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase, has been implicated in chromatin silencing, longevity and genome stability. Mammals contain a family of related deacetylases, the sirtuins, of which 7 have been identified. Sirt1 is the closest mammalian orthologue of yeast Sir 2. The Sirt1 gene in mice was disrupted by homologous recombination in embryonic stem cells. The majority of Sirt1 (−/−) embryos die between E9.5 and E14.5, displaying altered histone modification, increased chromosomal aberrations, and impaired DNA damage repair. Tumor formation was increased in mutant tissues in Sirt1(+/−): p53(+/−) double heterozygotes, indicating that full levels of Sirt1 are necessary for tumor suppression. Tumorigenesis is reduced by treatment with the polyphenol, resveratrol, which activates Sirt1. Sirt1 may act as a tumor suppressor by promoting DNA damage repair and maintaining genome integrity. Sirt1also is involved in the regulation of proteins involved in energy metabolism, and components of the circadian clock.
STAT1 is considered a tumor suppressor, but it is not known if this effect occurs directly in mammary cells or secondarily by disrupting interferon signaling through the JAK/STAT1 pathway to induce immune responses. ERBB2/neu-induced breast cancer appeared sooner in mice lacking STAT1 only in mammary cells than in wild-type mice, indicating that STAT1 tumor suppression was intrinsic to mammary cells and not secondary to an induced immune response.
The Cre recombinase from bacteriophage P1 excises intervening DNA sequences located between two unidirectional lox sites positioned on the same linear DNA segment, leaving one lox site behind. Through insertion of lox sites via homologous recombination into the gene of interest and targeting Cre recombinase expression to a specific cell type using a tissue-specific promoter, it is possible to introduce predetermined deletions into the mammalian genome. To delete genes specifically from mammary gland, transgenic mice were created carrying the Cre gene under the control of the whey acidic protein (WAP) gene promoter. Expression of WAP–Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally was observed in the brain. High-level transcriptional activity of WAP-based transgenes can be obtained at every stage of mammary development.
The Cre recombinase from bacteriophage P1 excises intervening DNA sequences located between two unidirectional lox sites positioned on the same linear DNA segment, leaving one lox site behind. Through insertion of lox sites via homologous recombination into the gene of interest and targeting Cre recombinase expression to a specific cell type using a tissue-specific promoter, it is possible to introduce predetermined deletions into the mammalian genome. To delete genes specifically from mammary gland, transgenic mice were created carrying the Cre gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). In adult MMTV–Cre mice, expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. In contrast to WAP–Cre, however, Cre expression under control of the MMTV LR resulted in recombination in all tissues.
Bcl-x is a pro-survival protein that opposes the pro-apoptotic action of Bax which interacts with mitochondria to activate the caspase 9 pathway. Mice in which the Bcl-x gene is inactivated die at E12.5. To be able to study lineage-specific activities of Bcl-x at different stages of development, the Cre-LoxP recombination system was used. Homologous recombination was used to flank the promoter, exon1, and major coding exon2 of the Bcl-x gene with loxP sites. The targeted allele contained a loxP flanked (or floxed) neomycin cassette in the Bcl-x promoter, and an additional loxP site in intron 2. Floxed Bcl-x has been used to study the balance between Bcl-x and Bax in primordial germ cells that undergo controlled levels of cell reduction due to apoptosis, the induction of hemolytic anemia and splenomegaly following conditional deletion of the Bcl-x gene from erythroid cells, the protection of hepatocytes from apoptosis and ensuing fibrotic response by Bcl-x, and the demonstration that Bcl-x is critical for the survival of dendritic cells, important regulators of immune function.
Di- and tri-methylations on histone H3 lysine 27 (H3K27me2 and H3K27me3) are epigenetic marks for gene repression. UTX (ubiquitously transcribed X chromosome protein), also known as Kdm6a (lysine (K)-specific demethylase 6a) is a histone demethylase that specifically removes H3K27me2 and H3K27me3. UTX knockout mice are embryonic lethal, so we have generated UTX conditional knockout mice (UTX-flox) in which exon 24 is flanked with loxP sites. UTX has been found to be a tumor suppressor gene mutated in a wide variety of human cancers. The UTX-flox mice provide a valuable tool to study how