WO2002061073A2 - Recherche genetique a haut rendement de lipide et traitement du cholesterol a l'aide de composes fluorescents - Google Patents

Recherche genetique a haut rendement de lipide et traitement du cholesterol a l'aide de composes fluorescents Download PDF

Info

Publication number
WO2002061073A2
WO2002061073A2 PCT/US2002/002766 US0202766W WO02061073A2 WO 2002061073 A2 WO2002061073 A2 WO 2002061073A2 US 0202766 W US0202766 W US 0202766W WO 02061073 A2 WO02061073 A2 WO 02061073A2
Authority
WO
WIPO (PCT)
Prior art keywords
fish
agent
lipid
cell
fluorescent
Prior art date
Application number
PCT/US2002/002766
Other languages
English (en)
Other versions
WO2002061073A3 (fr
Inventor
Steven Farber
Michael Pack
Original Assignee
Thomas Jefferson University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/974,550 external-priority patent/US20020049986A1/en
Application filed by Thomas Jefferson University filed Critical Thomas Jefferson University
Publication of WO2002061073A2 publication Critical patent/WO2002061073A2/fr
Publication of WO2002061073A3 publication Critical patent/WO2002061073A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/40—Fish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes

Definitions

  • the present invention generally relates to the fields of biochemistry and pharmacology and to a mutant fish having a metabolic defect affecting phospholipid and cholesterol processing, and more particularly to the use of a mutant fish labeled with fluorescent lipids to screen for drugs and genetic alterations related to phospholipid and/or cholesterol metabolism and, more particularly, to the use of a mutant zebrafish in conjunction with tagged or quenched lipids for studying lipid metabolism in vivo,.
  • the fluorescent phospholipase A 2 (PLA 2 ) substrates described in the present invention are the first prototypes in this class of reagents. Although lipid metabolism in the digestive tract is complex and involves multiple organs the present invention discloses a method of assaying this pathway since gall bladder fluorescence represents one of the last steps in lipid processing.
  • the fluorescently-tagged reagents of the instant invention provide a sensitive assay for a wide range of digestive developmental and physiological processes including, but not limited to, swallowing; lipid digestion, absorption, and transport; esophageal sphincter function; intestinal motility; organogenesis of the mouth and pharynx, esophagus, intestine, liver, gallbladder and biliary system, and exocrine pancreas and ducts; and the cellular and molecular biology of PLA 2 regulation, polarized transport, and secretion.
  • zebrafish mutagenesis screens using lipid reporters can be used to identify genes with functions relevant to human lipid metabolism and disease.
  • the high throughput screens and fluorescent lipids disclosed in the instant invention can be employed using a variety of vertebrate model systems, including but not limited to, rodents, amphibia, and fish.
  • the present invention involves utilizing fluorescent lipids to screen for phenotypes representing perturbations of lipid processing; to screen for mutations of specific genes that lead to disorders of phospholipid and/or cholesterol metabolism; and to screen for compounds designed to treat disorders of phospholipid and/or cholesterol metabolism, such as, but not limited to cancer, inflammatory and cardiovascular disease, and congenital and acquired diseases of the intestine and liver.
  • the precise mechanism of phospholipid and cholesterol processing is unknown at this time, there exists a need in the art for models to study such metabolism.
  • one of the best approaches is to generate genetic mutant organisms, such as fish, that have a metabolic defect affecting phospholipid and cholesterol processing.
  • This approach allows the creation of new fish strains for physiological study as well as tissues and cell lines for in vitro examinations.
  • the present invention is further directed to a mutant fish having a metabolic defect affecting phospholipid and/or cholesterol processing.
  • the invention is useful for the study of phospholipid and/or cholesterol metabolism and, hence, for the development of treatments of diseases or disorders related to phospholipid and/or cholesterol metabolism.
  • this invention relates to isolated cells derived from these fish and to the use of these cells in assays for assessing treatments of diseases or disorders related to phospholipid and/or cholesterol metabolism.
  • PDA 2 means "phospholipase A 2
  • PLB means "phospholipase B"
  • PED means "phospholipase D”
  • PED6 means "N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1 -palmitoyl-2-
  • FRET fluorescence resonance energy transfer
  • PC phosphatidyicholine
  • cPLA 2 means "cytoplasmic PLA 2
  • API means "adenomatous polyposis coli”
  • SLR means "single locus rate
  • IVF means "in vitro fertilization
  • BAC means "bacterial artificial chromosome”
  • PAC means "P1 -derived artificial chromosome”
  • YAC means "bacterial artificial chromosome”
  • CSGE means "conformation sensitive gel electrophoresis"
  • VLDL means "very low density lipoprotein”
  • EM means "embryo medium”
  • NBD cholesterol means "nitrobenzoxadiazole cholesterol”
  • agent refers to any molecule, compound, or treatment that assists in the treatment of a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism.
  • screening or “to screen” refers to a process in which a large number of potentially useful agents are processed in the methods of the invention.
  • Fig. 1 Schematic of PED6, a quenched PLA 2 substrate.
  • A. The dinitrophenol on the phospholipid headgroup effectively quenches any emission resulting from excitation at 505 nm of the BODIPY-labeled acyl chain.
  • B. When the BODIPY-labeled acyl chain is liberated by PLA 2 mediated cleavage, the quencher is separated from the fluorophore and emission (515 nm) is observed.
  • C NDB-labeled cholesterol in which the fluorophore replaces the terminal segment of cholesterol's alkyl tail.
  • PED6 a fluorescent lipid reporter.
  • A Bright-field image of a 5 dpf larva soaked in PED6 (3 ug/ml, 2 hr).
  • B Corresponding fluorescent image, with intestinal (arrow) and gall bladder (arrowhead) labeling.
  • C Larva soaked in BODIPY-C5-PC (0.2 ug/ml). In contrast to B, unquenched fluorescent lipid labels the pharynx (arrowhead), confirming that lipid is swallowed before gall bladder labeling (arrow).
  • FIG. 5 Lipid processing.
  • D. Gall bladder fluorescence after processing (t 30 min) of PED6 (1 ug), administered by gavage. Symbols: gb, ball bladder; d, common bile duct; Iv, liver. Scale bars, 1.0 mm (C and D), 200 urn (other images).
  • BODIPY FR-PC reveals both the substrate and PLA 2 cleavage product.
  • excitation at 505 nm results in an emission at 568 nm (orange) due to fluorescence resonance energy transfer
  • Fig. 7 Fluorescence emission spectrum of mixed micelles of 0.05 mol % BODIPY FR -PC in mixed-lipid vesicles.
  • the fluorescence emission spectrum of BODIPY FR -PC (0.5 ⁇ M in ethanol; excitation, 505 nm) showed peaks at 514 nm and 568 nm with a ratio of 1.0 indicating FRET.
  • An excitation scan (emission, 568 nm) showed peaks at 507 and 560 nm with a ratio (507/560) of 1.2.
  • Solid line before addition of PLA 2 .
  • Dashed line after addition of N. naja PLA 2 .
  • Vesicles were prepared by sonication of the dried lipids (from chloroform-methanol solution); 1.1 M BODIPY FR -PC (0.05 mol %), 0.1 mM dimyristoylphosphatidycholine (46 mol %), 0.12 mM ditetradecylphospha-tidylmethano! (54 mol %), buffer (50 mM Tris, pH 8, 100 mM NaCI, 1 mM CaCI 2 ); excitation, 505 nm.
  • BODIPY FR-PC is effective in paramecium. Paramecium incubated (1 hr) in BODIPY FR -PC (2.5 ug/ml) results in labeling of lipid droplets (orange). Digestion of the lipid results in green fluorescence. Excitation 505 nm, emission LP520.
  • BODIPY FR -PC localizes to the intestinal epithelium. Fish were incubated (1-4 hr) in BODIPY FR -PC (2.5 ug/ml). BODIPY FR -PC metabolites are observed in gall bladder (green). Excitation 505 nm, emission LP520. A. Brightfield image. B. and C. Visualization of antigen presenting enterocytes in segment II of the larval zebrafish intestine after only 1 hr of labeling. Arrows mark segment II domain. D. 4 hr labeling with BODIPY FR -PC results in FRET effect throughout intestine. Fig. 10. Lipid processing in intestinal mutants. A and C.
  • B and D Fluorescent images corresponding to A and C. Normal lipid processing of PED6 (0.3 ug/ml, 2 hr) in mlt larva (arrowhead marks gallbladder).
  • D and £ Abnormal lipid processing in PED6 labeled pie and slj larvae; fluorescence is present in the intestinal lumen (white arrowhead) and reduced in the gallbladder (red arrowhead).
  • Fig. 11 Phospholipid processing and transport.
  • Solvent 1 toluene, ether, ethanol, acetic acid; 25/15/2/0.2
  • Solvent 2 chloroform, methanol, acetic acid, water; 25/15/4/2).
  • Fig. 14 fat free larvae lack gall bladder fluorescence.
  • A Bright-field images of wild-type (WT) and mutant 5 dpf larvae.
  • B Corresponding fluorescent image of the gall bladder after PED6 labeling (0.3 ug/ml) (arrowheads).
  • C Biliary secretion of phenol red in fat free larvae. Mutant larvae identified by PED6 labeling were injected with phenol red (arrowhead, gall bladder).
  • D Reduced lipid processing in fat free mutants.
  • TLC of the pooled lipid fraction from eight larvae is shown after labeling with BODIPY C5- PC (12 hours, 0.16 ug/ml); red arrowhead denotes BODIPY-choIesteryl ester, black arrowhead indicates BODIPY C5-PC (lane 1 , WT; lane 2, fat free; lane 3, endogenous fluorescent lipids present in unlabeled WT larvae).
  • TLC solvent 1 (chloroform:methanol:water 35/7/0.7); solvent 2 (ethyl ether:bensene:ethanol:acetic acid; 40/50/2/0.2).
  • the present invention relates to a mutagenesis screen to identify genes that regulate lipid metabolism using fluorescently-tagged or quenched lipids such as cholesterol or lipids that are substrates for phospholipases such as PLA 2 and to a fish having a metabolic defect affecting phospholipid and cholesterol processing, which is identified using a mutagenesis screen.
  • fluorescently-tagged or quenched lipids such as cholesterol or lipids that are substrates for phospholipases such as PLA 2
  • a fish having a metabolic defect affecting phospholipid and cholesterol processing which is identified using a mutagenesis screen.
  • cleavage of quenched phospholipid substrates by PLA 2 results in an increase in fluorescence intensity or alters the spectral properties of fluorescent emission thus allowing lipid metabolism to be followed in vivo.
  • optically transparent zebrafish larvae exposed to the fluorescently-tagged or quenched lipids display intense gallbladder fluorescence, which reflects lipid cleavage by intestinal PLA 2 and subsequent transport of the fluorescent cleavage products through the hepatobiliary system.
  • the instant invention presents evidence demonstrating that in the context of a mutagenesis screen, fluorescent PLA 2 substrates and fluorescently-tagged cholesterol or other lipids have the potential to identify genes that affect many aspects of lipid metabolism. Consequently, when used in the context of a genetic screen these reagents provide a high throughput readout of digestive physiology that cannot be assessed using standard screening strategies. Given current understanding of the pathway of lipid processing in zebrafish, specifically its shared features with lipid processing in mammals, the present invention has relevance for biomedical research related to, among other things, cancer, inflammatory and cardiovascular diseases, and congenital and acquired diseases of the intestine and liver.
  • Fluorescent reagents to assess organ physiology in vivo Fluorescent reagents to assess organ physiology in vivo
  • the fluorescent lipids of the instant invention allow assaying of physiological processes.
  • the reagents are fluorescent analogues of compounds that could serve as modifiable substrates in important metabolic and signaling pathways.
  • the reagents of the instant invention were constructed by covalently linking fluorescent moieties to sites adjoining the cleavage site of phospholipids. Dye-dye or dye-quencher interactions modify fluorescence without impeding enzyme-substrate interaction (106). PLA 2 cleavage results in immediate unquenching and detectable fluorescence.
  • Quenched phospholipids [N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1 - palmitoyl-2-BODIPY-FL-pentanoyl-st7-glycero-3-phosphoethanolamine (PED6)] allow subcellular visualization of PLA 2 activity and reveal organ- specific activity (36).
  • the evidence presented in the instant invention demonstrates that zebrafish larvae 5 days post-fertilization (dpf) bathed in PED6 show intense gall bladder fluorescence and, shortly thereafter, intestinal luminal fluorescence.
  • Substrate modification allows localization of enzymatic activity by altering the emission spectrum of the fluorescent compounds. When used in the context of a genetic screen these fluorescent lipids provide a high throughput readout of organ function.
  • the reagents of the instant invention facilitated the development of genetic screens that are more sensitive than the whole-mount in situ and antibody based screening protocols now used to assay gene expression.
  • the fluorescent reagents are simpler to use since they can be administered to and assayed in a wide range of organisms, including, but not limited to rodents and teleosts, and they offer the opportunity to screen for hypomorphic mutations that alter gene function but do not affect levels of gene expression.
  • these reagents can be used to identify mutations that affect more than just the single gene responsible for substrate modification. Visualization of the fluorescent signal also is dependent upon the delivery and uptake of the substrate as well as the storage, metabolism or secretion of its modified metabolites.
  • PLA 2 s were chosen as the "target" enzymes to assay because of the important role these enzymes play in the generation of lipid signaling molecules (32-34).
  • PLA 2 s are a large family of enzymes that can be categorized according to their cellular distribution, molecular weight, and calcium dependence (33). Some PLA 2 s also exhibit a preference for phospholipids with arachadonyl sn2 acyl side chains (34, 37, 38). Given the wide range of processes the PLA 2 family of enzymes is known or thought to regulate, the advantage of the fluorescent lipids as screening reagents is significant.
  • cPLAp the advantage of the fluorescent lipids as screening reagents is significant.
  • cytoplasmic PLA 2 regulates eicosanoid production.
  • Eicosanoids are ubiquitous signaling molecules generated by the biochemical modification of arachidonic acid, the principal fatty acid liberated from membrane phospholipids by cPLA 2 s (42-44).
  • cPLA 2 s have a strong preference for arachidonyl phospholipids and cPLA 2 cleavage of these phospholipids is known to be the rate-limiting step in eicosanoid synthesis (39, 41 , 44-47).
  • secretory PLA 2 s sPLA 2 s
  • sPLA 2 s exhibit little substrate preference and unlike cPLA 2 , the arachidonic acid generated through sPLA 2 activity is not directly coupled to eicosanoid production (37, 46).
  • Prostaglandins are a large family of signaling molecules that are synthesized through the action of cyclooxygenases (COX) and prostaglandin-isomerases on PLA 2 generated arachidonic acid (48). Prostaglandins are especially important in vertebrate physiology as they regulate a myriad of physiological processes, including hemostasis, cell proliferation, fertility, and inflammation (48-50). The importance of prostaglandin in humans is underscored by the widespread use of COX inhibitors (e.g., aspirin) as medicinal agents. Given the focus of this invention, there is special interest in intestinal prostaglandins.
  • COX inhibitors e.g., aspirin
  • COX inhibitors such as aspirin
  • intestinal prostaglandins regulate epithelial cell proliferation 56-58.
  • COX inhibitors are known to have a chemopreventitive effect on several human digestive cancers, and COX-2 has been identified as a genetic modifier of APC, an important colon cancer gene. Because arachidonic acid release is the rate-limiting step in eicosanoid production, characterization of the cell specific regulation of cPLA 2 s is important (33, 34).
  • sPLAp Another PLA 2 gene family member, sPLA 2 , plays a role in inflammation, host defenses, digestion, and cell proliferation (43, 70, 71 ). In comparison to cPLA 2 s, sPLA 2 s show little preference for arachidonic acid over other phospholipids sn2 acy ⁇ side chains (34).
  • sPLA 2 serum levels of sPLA 2 are increased in inflammatory conditions such as Rheumatoid Arthritis, Crohn's Disease, endotoxic shock, and atherosclerosis (32, 72-76).
  • sPLA 2 cleavage of arachidonyl phospholipids has been shown to augment cPLA 2 mediated eicosanoid production (46, 71 , 77).
  • sPLA 2 independent of its enzymatic activity, sPLA 2 has been shown to bind to a specific cellular receptor that plays a role in endotoxic shock (78) and other aspects of the inflammatory response (43, 79, 80).
  • endotoxic shock 78
  • other aspects of the inflammatory response 43, 79, 80.
  • the regulation of inflammatory sPLA 2 s (Groups II and V) is an important area of biomedical research.
  • Group IIA sPLA 2 recently has been shown to play a role in colon cancer tumorigenesis through its genetic interaction with APC in the min mouse, a colon cancer animal model (66-68). High levels of this gene are expressed in intestinal Paneth cells where it is also believed to function as an antimicrobial agent because of its ability to digest bacterial cell wall phospholipids (38, 76, 81 ). The precise mechanism of sPLA 2 's mitogenic effect is unclear. Since COX activity also has been shown to modify colon tumorigenesis in the min mouse, sPLA 2 may be acting via prostaglandins (69). This hypothesis is counter-intuitive, however, because in the min mouse, loss of sPLA 2 function is associated with a tumor promoting effect. An alternate hypothesis suggests that in the colon, sPLA 2 also may function as a cell-signaling molecule, independent of its enzymatic activity, through its interaction with the sPLA 2 receptor.
  • pancreatic sPLA 2 is the prototypical low molecular weight sPLA 2 - its proenzyme is secreted into the intestinal lumen where it is activated and cleaves dietary phospholipids that have been modified by bile salts (83).
  • PLA 2 activity also is present in the intestinal brush border and undoubtedly contributes to the digestion of dietary phospholipids (84, 85).
  • Intestinal PLA 2 activity is present in the form of PLB, an enzyme that can function as a high molecular weight, calcium independent PLA 2 as well as a lysophospholipase (86-89). Regulation of lipid absorption and transport is an important area of biomedical research that has implications for metabolic, inflammatory, and cardiovascular diseases.
  • dietary phospholipid is cleaved by intestinal and pancreatic PLA 2 s to form free fatty acid, lysolipid, and phosphoglycerol (82). These molecules are absorbed by enterocytes, presumably by simple diffusion (lysolipid, phosphoglycerol) and receptor mediated processes (fatty acid) (90). Within the enterocyte, the free fatty acids are processed according to their size: long carbon chain fatty acids are re-esterified to form triglycerides and phospholipids, packaged into Iipoprotein particles (chylomicrons or VLDL) and enter the general circulation via the lymphatics; shorter chain fatty acids presumably can enter the circulation directly via the portal vein (90-92). Lipoprotein bound phospholipids enter cells in the periphery by binding to specific receptors or via endocytosis (90, 91).
  • the fate of the phospholipid derived lysolipid and phosphoglycerol cleavage products are less certain.
  • mammals they are largely re-esterified to form phospholipids, which then are incorporated into lipoproteins and absorbed via the lymphatics, like triglycerides (90-92).
  • Vertebrate lipid absorption is further complicated by the existence of other intestinal phospholipases (PLD, PLAi ) that cleave the polar head group or sn1 fatty acid from lysolipids and phospholipids. This raises the possibility that phospholipids undergo extensive re-arrangement within the intestinal epithelium prior to absorption.
  • PLAi intestinal phospholipases
  • Zebrafish, Danio rerio were housed in a separate facility consisting of approximately 500 tanks of varying sizes (1 liter, 3.75 liter, and 9 liter). Environmental conditions were carefully monitored for disease prevention and to maintain fish in perpetual breeding condition. Male and female fish were reared at a density of no more than 8 fish per liter at a constant temperature and light cycle (27-29° C with the light/dark cycle kept at 14/10 hours) in pre- treated water (heated, charcoal-filtered and UV-sterilized). Fish were fed twice daily with a variety of dried and live foods.
  • Zebrafish provide a relatively simple model system for more complex vertebrates, such as humans. They are small in size, easy to maintain and breed, and produce large numbers of progeny on a daily basis. Their embryos develop rapidly and are optically clear, permitting direct observation of the developing digestive system. Being vertebrates, zebrafish contain orthologues for almost all human genes. The species also is amenable to genetic methods so that one can screen for mutations that disrupt organ function or development. It is possible, therefore, to identify genes important for intestinal development and function by examining fish that carry random mutations. In addition, many techniques have been worked out for manipulating zebrafish, including in vitro fertilization, production of haploids and parthenogenic diploid embryos, mutagenesis, cell lineage and cell transplantation.
  • a family of fluorescent phospholipids was generated with such phospholipids serving as substrates for PLA 2 .
  • characterization of the genetic regulation of PLA 2 is an active area of biomedical research (67, 68), and the reagents described were developed to function as in vivo biosensors of PLA 2 activity that can be assayed using microscopy or simple biochemical techniques. When administered to zebrafish embryos and larvae, these reagents provide a rapid readout of a wide range of developmental and physiological processes that are amenable to high throughput genetic analyses.
  • the fluorescent lipids described in this proposal are quenched fluorescent phosphatidylcholine analogues and NBD-labeled cholesterol (Fig. 1).
  • the phospholipid reagents differ in both their fluorescent emission and their specificity for different PLA 2 isoforms (93, 94). Cleavage of these phospholipids by PLA 2 generates a fluorescent fatty acid or lysolipid that can be used to localize and quantify PLA 2 activity in live fish (36, 94). It has been demonstrated that there is utility of these agents for revealing localized PLA 2 activity in developing zebrafish embryos (94).
  • the quenched fluorescent lipids were administered to zebrafish larvae at 5 dpf, a developmental stage when the major larval organ systems function (Fig. 2).
  • 5 dpf larvae soaked in PED6 uniformly developed intense gallbladder fluorescence 15 - 20 minutes after ingesting the lipid (Fig. 3).
  • the larval gallbladder fluorescence reflected ingestion of the lipid reagents followed by intestinal PLA 2 cleavage, intestinal absorption, transport to the liver and biliary excretion of the fluorescent cleavage product. This hypothesis was tested with three experiments.
  • EM embryo medium
  • BODIPY-FL-C 5 -PC 0.1 ug
  • PLA 2 activity was determined as described (1). Measurements were compared with activity of bile-depleted gallbladders. Third, this finding was confirmed by demonstrating the early appearance of fluorescent cleavage products in the liver of larvae exposed to PED6 compared with the gallbladder (Fig. 4). Larvae were labeled with PED6 (0.3 ug/ml) in EM, anesthetized (tricaine, 170 ug/ml), and placed in depression slides.
  • Fluorescent images were captured over 1 hr using a Zeiss Axiocam 2 mounted on a Leica MZFL-III. Because PLA 2 activity was not detected in bile and fluorescent PED6 metabolites underwent rapid hepatobiliary transport, labeling the liver before the gall bladder, PED6 must be cleaved within the intestine.
  • atorvastatin Lipitor, Parke-Davis
  • Fig. 5A a potent inhibitor of cholesterol synthesis in humans (108).
  • atorvastatin Lipitor, Parke-Davis
  • Fig. 5A a potent inhibitor of cholesterol synthesis in humans (108).
  • Bile was obtained from freshly killed tilapia (Orechromis mossambicus) and extracted with three volumes of methanol:chloroform (1 :2). The aqueous fraction was recovered, reduced to one volume under nitrogen, and added to EM (20 ul/ml)], and atorvastatin failed to inhibit processing of a water-soluble short-chain fatty acid (BODIPY-FL-C5, Molecular Probes Inc.) (105) (Fig.
  • BODIPY FR-PC a reporter of both substrate and cleavage product
  • BODIPY FR-PC (Fig. 6) is a substrate that can be used to determine the site of PLA 2 activity more precisely because it emits distinct fluorescent profiles before and after cleavage.
  • This phospholipid contains two dyes that interact via fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the fluorescence emission spectrum of BODIPY FR-PC was determined using mixed micelles of 0.05 mol % BODIPY FR-PC in dimyristoyl phosphatidylcholine (46 mol %) and ditetradecylphosphatidylmethanol (54 mol %) prepared in buffer (50mM Tris, pH 8, 100 mM NaCI, 1 mM CaCI 2 ) by sonication of the dried lipids.
  • BODIPY FR-PC behaves as anticipated in in vitro assays
  • its behavior in vivo was analyzed by exposing paramecia to the lipid for 1 hr, followed by excitation at 505 nm.
  • FRET emission (orange) was observed from lipid droplets within individual paramecium soon after exposure, followed by the gradual appearance of BODIPY emission at 515 nm (green fluorescence) indicative of substrate cleavage (Fig. 8).
  • BODIPY FR-PC To further demonstrate the behavior of BODIPY FR-PC in vivo, 5 dpf zebrafish larvae were bathed in BODIPY FR-PC. Within 1 hr, larvae exposed to BODIPY FR-PC showed a bright green fluorescent gallbladder and orange fluorescence within the apical cytoplasm of posterior intestinal cells (Fig. 9C). These cells are known to absorb luminal macromolecules through pinocytosis (107). Orange fluorescence indicative of uncleaved substrate also was observed after 4 hr of incubation in the anterior intestine epithelium, the site of lipid absorption in fish (82, 95). FRET emission was never detected in the liver even after prolonged incubation (Fig. 9D), implying that gallbladder fluorescence following ingestion of labeled lipids is due to cleavage by intestinal PLA 2 s.
  • the evidence of the instant invention is consistent with the predicted pathway of lipid processing observed in mammals since uncleaved substrate (orange) was seen only in the intestinal epithelium. Over time, continuous exposure to BODIPY FR-PC either overwhelms intestinal lipase activity or leads to the integration of BODIPY FR-PC into the cellular phospholipid pool. Regardless, the data of the present invention provide further evidence that in zebrafish, as in mammals, lipids are absorbed and modified in the intestine prior to transport to the liver.
  • mlt mutants retain PED6 processing in the anterior intestine and show normal levels of gall bladder fluorescence.
  • pie (Figs. 10D & 10E) and slj (Fig. 10F) mutants display greatly reduced gall bladder labeling.
  • gall bladder fluorescence was absent in pie larvae but visible in slj larvae, albeit at a reduced level compared with wild-type or mlt larvae (Figs. 10B & 10C).
  • fluorescent lipids can be used to identify mutants with abnormal digestive organ morphology.
  • Lipid reporters also are effective tools for identifying mutations that perturb lipid metabolism without causing obvious morphological defects.
  • larval progeny of individual F2 families derived from an ENU mutagenesis protocol were bathed in PED6 and screened for digestive organ morphology and phospholipid processing.
  • PED6 fluorescent metabolites dramatically enhanced digestive organ structure, facilitating scoring of gallbladder development, intestinal folding, and bile duct morphology.
  • From 190 genomes screened two mutations were identified and confirmed in the subsequent generation. These mutations were recovered based on their pattern of gallbladder fluorescence, not morphological criteria, thereby supporting the use of the fluorescent lipids of the instant invention in large- scale mutagenesis screens.
  • Lipid processing was examined further in the identified mutants using a fluorescent cholesterol analog (Fig. 1).
  • Example 1 Characterize the biochemical pathways responsible for the metabolism of the fluorescent PLA? substrates in zebrafish
  • Larvae are soaked in the identical BODIPY fatty acids injected into the adults, and the rate of gall bladder fluorescence is assayed.
  • the labeling rate is determined by placing a fish in water containing the fluorescent substrate and capturing a digital image of a lateral view (Axiovision, Zeiss and ImageQuant, Molecular Dynamics Inc.).
  • the gall bladder fluorescence is quantified at various time intervals to compute the rate of change over time. Delayed appearance of gallbladder fluorescence after exposure to long chain fatty acids and phospholipids lends further support to the hypothesis that acyl chain length plays a role in lipid transport in zebrafish.
  • PLAp isoform mediation of PED6 processing It is well established that cPLA 2 and some iPLA 2 isoforms exhibit acyl chain specificity and favor phospholipids that contain arachidonic acid, the precursor of eicosanoids (41 , 44). Despite the fact that the fluorescent phospholipids do not contain arachidonic acid, the placement of the BODIPY moiety on the sn2 position results in significant cleavage by cPLA 2 (36, 94). The ability of the BODIPY moiety to disrupt the membrane and its hydrophobicity are both properties similar to arachidonic acid that enable cleavage by cPLA 2 (100). Secreted PLA 2 isoforms exhibit no acyl chain specificity (34). Fluorescent phospholipids that contain a saturated acyl chain on the sn2 position are fine sPLA 2 substrates but poor cPLA 2 ones.
  • the rate of gall bladder fluorescence is compared in larvae exposed to two types of substrates.
  • Larvae are labeled with two phospholipid substrates that differ in their specificity for cPLA 2 : a PL with an sn1 BODIPY and an sn2 saturated acyl chain - a poor PLA 2 substrate, or BODIPY FR-PC - a good cPLA substrate.
  • the critical lipase important for the processing of these lipids is a secreted PLA 2 isoform, a promiscuous enzyme that exhibits little acyl chain preference (96).
  • BODIPY FR-PC is used because no other available fluorescent phospholipid contains sn2 arachidonic acid.
  • Example 2 A mutagenesis screen to identify genes that regulate lipid processing in zebrafish
  • a large-scale genetic screen for genes that regulate lipid processing in zebrafish using fluorescent PLA 2 substrates is disclosed.
  • This screen leads to the recovery of mutations that regulate a wide range of developmental and physiological processes.
  • the screen identifies mutations that either resemble or are allelic to previously described intestinal mutations.
  • this screen leads to the recovery of genes that could not be identified using standard screening strategies. These include, but are not limited to, genes that directly regulate PLA 2 ; genes responsible for the development of the liver, biliary system, and the intestinal vasculature and lymphatics; and genes that play a direct role in lipid metabolism and transport.
  • the mutagenesis and screening strategies of the instant invention incorporate several well established methodologies.
  • adult male fish are mutagenized with ENU using established dosing schedules. There exist a number of established dosing schedules (e.g., 110, 111).
  • the F1 progeny of the mutagenized GO fish will be bred to homozygosity using a classical 3 generation protocol (F3 screen), as well as parthenogenetically, using the early-pressure (EP) technique (103).
  • F3 screen classical 3 generation protocol
  • EP early-pressure
  • Progeny refers to any and all future generations derived or descending from a particular organism, whether the organism is heterozygous or homozygous for the mutation.
  • Progeny of any successive generation are included herein, such that the progeny, the F1 , F2, F3 generations, and so on indefinitely are included in this definition.
  • Zebrafish larvae generated in this fashion are soaked in the BODIPY FR-PC, PED6, and /or NBD cholesterol (Fig. 13) lipid reagents at 5 dpf and then screened for defects in lipid processing based upon the timing and pattern of fluorescence in the intestine, liver and gallbladder.
  • Parallel F3 and EP screens are employed because they offer the best opportunity to maximize the use of resources.
  • EP screens are in many ways less efficient than classical F3 screens, they are suited to moderate sized fish facilities and require less manpower because of the reduced number of matings required.
  • ENU mutagenesis For the EP screen, adult male fish from the '*AB' zebrafish line (this line of fish carries the fewest number of haploinsufficient or lethal mutations of common lab strains) are exposed to ENU (3.5 mM) in a secluded fume hood for 30 minutes on 3 consecutive days. After taking the necessary precautions to remove adherent ENU, the fish are returned to the main fish facility and outcrossed to WT fish from the Wik genetic background 3 weeks after mutagenesis, and the F1 progeny are raised to sexual maturity.
  • the mutagenized GO males are mated to double-mutant "tester" female carriers of the alb, spa, and bra mutations and their 32 hpf progeny are analyzed for clones of alb/alb, spa/spa, and bra/bra mutant pigmented cells. Based upon published SLR's for these loci using our ENU dosing schedule there is a need to analyze approximately 1500-2000 progeny from each mutagenized GO fish to accurately determine the efficacy of mutagenesis.
  • 5 dpf larvae derived from pair-wise matings of F2 families or EP treatment of F1 females are soaked fluorescent lipids for 1-10 hrs and analyzed for perturbation of lipid processing using a fluorescent stereo-microscope (Leica MZ FLIII).
  • the BODIPY FR-PC and PED6 reagents used in the screen are purified via TLC, resuspended in ethanol/DMSO and tested in vivo using WT 5 dpf larvae prior to screening.
  • the F3 5 dpf larvae for screening are derived from pair-wise matings of fish from F2 families, while 5 dpf larvae produced parthenogenetically are produced using standard EP protocols: briefly, eggs are collected from anesthetized F1 females (tricaine) and exposed to UV irradiated sperm following standard in vitro fertilization (IVF) protocols. Immediately after IVF (1.4 min.), the fertilized eggs are exposed to 13,000 psi in a French Press for 4-6 min, then slowly returned to ambient atmospheric pressure and allowed to develop until 5 dpf (103).
  • Example 3 Determination of the physiological significance and molecular nature of recovered mutations
  • the instant invention discloses methods for characterizing, both phenotypically and molecularly, the generated mutations. Phenotypic analysis is approached first. Mutations that affect a wide range of developmental and physiological processes are recovered in the screen. Careful phenotypic analysis of these mutants is important for several reasons. First, it allows quantitation of the range and frequency of phenotypes actually recovered using the fluorescent lipid reagents. Second, it allows the performance of comprehensive complementation analyses so that hypomorphic alleles of interesting mutations are not overlooked. Third, it allow the selection of those mutations worthy of molecular analysis now versus mutations that may be of more immediate interest to other members of the zebrafish scientific community.
  • Mutant phenotypes recovered using the screen of the instant invention can be categorized into several broad categories.
  • This group encompasses, but is not limited to, mutations affecting the intestinal epithelium, liver, vasculature and lymphatics, enteric neuromusculature, and the tissue specific regulation of PLA 2 .
  • Embryological and transient expression assays also are important studies that can aid phenotypic analyses of zebrafish mutants.
  • mutations affecting development of the zebrafish digestive organs are, in general, less easily analyzed using these techniques than mutations affecting early development.
  • the short half-life of injected RNA transcripts and DNA expression constructs coupled with the mosaic distribution of the micro- injected DNA limits the utility of transient expression assays for mutations that are not recognizable until 4-5 dpf.
  • histological analyses are performed by fixing larvae in 4% paraformaldehyde, embedding the fixed larvae in glycolmethacrylate, and followed by sectioning. Sections are stained using toluene blue/azure II as described and analyzed using a Zeiss Axioplan compound microscope. When needed, selected immunocytochemical and molecular markers are employed to further categorize organ specific defects. If necessary ultrastructural studies are performed as well. For 'physiological' mutations, affected larvae are sequentially soaked in the fluorescent lipids of the instant invention, thereby allowing a more detailed categorization. Molecular analyses
  • the molecular characterization of zebrafish mutations can be performed using techniques widely known to those of skill in the art. These techniques include, but are not limited to, use of an ever expanding array of physical and genetic markers, several large insert genomic DNA libraries, outstanding bioinformatics, and a successful EST program.
  • the molecular characterization of identified mutations is accomplished by introducing mutations into a polymorphic genetic background and assigning a chromosomal location using either bulk segregant analyses and polymorphic markers from the 25 zebrafish linkage groups or using half tetrads and polymorphic centromeric markers.
  • DNA from large numbers of mutant progeny from the 'map cross' are extracted and stored.
  • the genetic map of the region surrounding the locus is refined using standard PCR based techniques and genetic markers from existing maps. The closest known genetic markers flanking the mutant locus are then used to screen large insert genomic libraries to identify more closely linked markers.
  • flanking markers within 0.1 cM of the mutant locus are identified, and a BAC or PAC clone spanning the locus is analyzed for the responsible gene. The latter is accomplished by direct sequencing, using the genomic insert to probe an appropriately staged genomic library and/or exon trapping. Confirmation of mutation is accomplished via expression analyses and mutant rescue, using transient analyses, if possible, or via transgenesis. Identification of tightly linked flanking markers generally involves a BAC, PAC, or YAC chromosomal walk, using markers mapped genetically and physically (radiation hybrid panel).
  • genetic mapping is performed using PCR based techniques.
  • SSR polymorphisms are resolved using denaturing (SSR) polyacrylamide gel electrophoresis while single base- pair polymorphisms are resolved using Conformation Sensitive Gel Electrophoresis (CSGE), a non-denaturing technique related to SSCP.
  • DNA detection is accomplished using a Molecular Dynamics Fluorescent Scanner; PCRs use Cy-5 end-labeled primers. PCR fragments generated using non- fluorescent primers are visualized on polyacrylamide gels after staining with the fluorescent intercalating dye Syto-61 (Molecular Probes).
  • SSR denaturing
  • CSGE Conformation Sensitive Gel Electrophoresis
  • the instant invention discloses methods for screening for mutations that perturb lipid processing and characterizing those mutations, both phenotypically and molecularly.
  • a classic two-generation breeding scheme 110, 111 was employed in the screen.
  • Adult male zebrafish were mutagenized with 3mM ENU by placing them into an aqueous solution for three (3) 1 hr periods within 1 week.
  • the GO males were bred to homozygosity using a classical 3 generation protocol (F3 screen) (110, 1 11).
  • F3 screen classical 3 generation protocol
  • Larval progeny of individual F2 families were bathed in PED6 and screened for digestive and organ morphology and phospholipid processing. Of the 190 genomes screened, two mutations were identified and confirmed in the subsequent generation.
  • the recessive lethal mutation canola produced a phenotype closely resembling that of slj and pie, with degeneration of the intestinal epithelium and reduced gall bladder and intestinal fluorescence.
  • the fat free recessive lethal mutation causes a more pronounce reduction in fluorescence (Fig. 14B).
  • the fat free digestive tract has a normal appearance at 5 dpf (Fig. 14A). Therefore, fat free would have escaped detection in a screen based solely on morphological criteria.
  • the fat free fish will be useful in assessing phospholipid and/or cholesterol processing and will aid in identifying treatments for diseases and disorders associated with phospholipid and/or cholesterol metabolism.
  • mutant zebrafish represent a preferred embodiment of the invention
  • mutant organisms including without limitation mutant amphibia, mutant teleosts, and mutant mammals (for example, rodents, pigs, cattle, and sheep) can be constructed by standard techniques are are included within the scope of this invention.
  • the invention provides methods for identifying compounds or agents that can be used to treat disorders characterized by (or associated with) aberrant or abnormal lipid and/or cholesterol metabolism. These methods are also referred to herein as drug screening assays and typically include the step of screening a candidate/test compound or agent for the ability to modulate (e.g., stimulate or inhibit) lipid and/or cholesterol metabolism.
  • Candidate/test compounds or agents that have one or more of these abilities can be used as drugs to treat disorders characterized by aberrant or abnormal lipid and/or cholesterol metabolism.
  • Candidate/test compounds or agents include, for example, (1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see e.g., Lam, K.S.
  • antibodies e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab') 2 , Fab expression library fragments, and epitope-binding fragments of antibodies
  • small organic and inorganic molecules e.g., molecules obtained from combinatorial and natural product libraries.
  • the invention provides a method for identifying a compound capable of use in the treatment of a disorder characterized by (or associated with) aberrant or abnormal lipid and/or cholesterol metabolism.
  • This method typically includes the step of assaying the ability of the compound or agent to modulate lipid and/or cholesterol metabolism, thereby identifying a compound for treating a disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism.
  • Disorders characterized by aberrant or abnormal lipid and/or cholesterol metabolism are described herein.
  • the invention provides screening assays to identify candidate/test compounds or agents that modulate lipid and/or cholesterol metabolism.
  • the assays include the steps of identifying at least one phenotypic perturbation of lipid and/or cholesterol metabolism in an organism, administering at least one quenched or fluorescently-tagged phospholipid, cholesterol or other lipid analogue to the organism having the phenotypic perturbation, administering a candidate/test compound or agent to the organism under conditions that allow for the uptake of the candidate/test compound or agent by the organism and wherein but for the presence of the candidate/test compound or agent the pattern of fluorescence would be unchanged, and detecting a change in the pattern of fluorescence by comparing the pattern of fluorescence prior to candidate/test compound or agent administration with that seen following administration of the candidate/test compound or agent.
  • a screening procedure for identifying therapeutic compounds that affect phospholipid and/or cholesterol metabolism in vivo involves screening any number of compounds for therapeutically active agents by employing a mutant organism, for example but not limited to fish having the fat free mutation, described herein. Based upon the above results, it will be readily understood that a compound that affects phospholipid and/or cholesterol metabolism in an organism (e.g., the mutant fish described herein) can provide an effective therapeutic agent in a mammal (e.g., a human patient). Since the screening procedures of the invention are performed in vivo, it can be ascertained whether candidate compounds will be highly toxic to a mammalian host organism (e.g., a human patient). In addition, the invention also makes available high throughput in vitro screening methods for screening of large quantities of candidate compounds using the cells and cell lines of the invention.
  • the methods, materials, and organisms of the instant invention simplify the evaluation, identification, and development of active agents, such as drugs, for the treatment of a variety of diseases associated with phospholipid and/or cholesterol metabolism.
  • active agents such as drugs
  • the screening methods of the invention provide a facile means for selecting any number of compounds of interest from a large population that are further evaluated and condensed to a few active and selective materials. Constituents of this pool can then be evaluated in the methods of the invention to determine their activity.
  • Administration of the candidate compound to an organism of the invention can be by any known route, and at a range of concentrations. Following an appropriate period of time, the animal can be assessed for the effect of the compound compared to control animals. Cells from the mutant organisms of the invention are also useful as a source of cells for cell culture.
  • Mutagenesis screens using fluorescent lipids exploit the advantages of combining genetic analyses with imagining of enzymatic function.
  • the evidence of the instant invention demonstrates that lipid metabolism in mammals and fish can be monitored with fluorescent lipids, and that such organisms metabolize ingested fluorescent lipids in an analogous manner.
  • the methods for using the fluorescent lipids of the instant invention to study lipid metabolism, identify diseases of lipid metabolism, and/or to identify agents to treat therapeutically or prophylatically diseases or disorders of lipid metabolism and genetic screening are applicable to all vertebrate model organisms, including, but not limited to, rodents, amphibia, and fish.
  • Fluorescent reporters are predicted to identify genes, including but not limited to, genes involved in diseases of lipid metabolism, such as atherosclerosis; in disorders of biliary secretion, such as biliary atresia; and in cancer, a disease in which lipid signaling plays an important role. Identification of these genes has important implications for cancer research and research related to diseases of the liver, intestine and cardiovascular system.
  • Gata5 is required for the development of the heart and endoderm in zebrafish. Genes & Development "13, 2983-2995.
  • a novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Ce/765, 1043-1051.
  • the secretory phospholipase A2 gene is a candidate for the Moml locus, a major modifier of ApcMin-induced intestinal neoplasia. Ce// 81 , 957-966.
  • Circulating phospholipase A2 activity associated with sepsis and septic shock is indistinguishable from that associated with rheumatoid arthritis. Inflammation 15, 355-367.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Pathology (AREA)
  • Veterinary Medicine (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention utilise des lipides fluorescents, en particulier des phospholipides figés ou des analogues de cholestérol, pour faciliter la recherche de phénotypes représentant des perturbations de la transformation des lipides, la recherche de mutations génétiques qui conduisent à des troubles du métabolisme des phospholipides et du cholestérol et la recherche de composés conçus pour traiter des troubles du métabolisme des phospholipides et/ou du cholestérol. La présente invention concerne un poisson mutant présentant un défaut métabolique affectant la transformation des phospholipides et du cholestérol. Ce poisson mutant convient pour étudier le métabolisme des phospholipides et du cholestérol et pour rechercher des composés conçus pour traiter des troubles du métabolisme des phospholipides et/ou du cholestérol.
PCT/US2002/002766 2001-01-30 2002-01-30 Recherche genetique a haut rendement de lipide et traitement du cholesterol a l'aide de composes fluorescents WO2002061073A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US60/235,226 2000-09-25
US23522601P 2001-01-30 2001-01-30
US09/974,550 2001-10-10
US09/974,550 US20020049986A1 (en) 2000-10-10 2001-10-10 High throughput genetic screening of lipid and cholesterol processing using fluorescent compounds

Publications (2)

Publication Number Publication Date
WO2002061073A2 true WO2002061073A2 (fr) 2002-08-08
WO2002061073A3 WO2002061073A3 (fr) 2003-07-03

Family

ID=26928698

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/002766 WO2002061073A2 (fr) 2001-01-30 2002-01-30 Recherche genetique a haut rendement de lipide et traitement du cholesterol a l'aide de composes fluorescents

Country Status (1)

Country Link
WO (1) WO2002061073A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1606759A2 (fr) * 2003-03-26 2005-12-21 Synergy Biosystems Ltd. Procede d'identification d'agents biologiquement actifs et combinaisons synergiques
WO2010051288A1 (fr) 2008-10-27 2010-05-06 Revivicor, Inc. Ongulés immunodéprimés
EP2527456A1 (fr) 2004-10-22 2012-11-28 Revivicor Inc. Porcs transgéniques déficients en chaîne légère d'immunoglobuline endogène
CN113234664A (zh) * 2021-05-11 2021-08-10 澳门大学 一种胰腺祖细胞的制备方法及其应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CARNEGIE INSTITUTION: "YEAR BOOK 98-99" CARNEGIE INSTITUTION YEAR BOOK, XX, XX, 1998, page 44,52 XP002193394 *
FARBER S A ET AL: "CHARACTERIZATION OF CA2+-DEPENDENT PHOSPHOLIPASE A2 ACTIVITY DURING ZEBRAFISH EMBROYGENESIS" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 274, no. 27, 2 July 1999 (1999-07-02), pages 19338-19346, XP002193392 ISSN: 0021-9258 cited in the application *
FARBER S A ET AL: "GENETIC ANALYSIS OF DIGESTIVE PHYSIOLOGY USING FLUORESCENT PHOSPHOLIPID REP ORTERS" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, vol. 292, 18 May 2001 (2001-05-18), pages 1385-1388, XP002193393 ISSN: 0036-8075 *
HENDRICKSON H STEWART ET AL: "Intramolecularly quenched BODIPY-labeled phospholipid analogs in phospholipase A2 and platelet-activating factor acetylhydrolase assays and in vivo fluorescence imaging" ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 276, no. 1, 1 December 1999 (1999-12-01), pages 27-35, XP002186412 ISSN: 0003-2697 cited in the application *
MULLINS M C ET AL: "LARGE-SCALE MUTAGENESIS IN THE ZEBRAFISH: IN SEARCH OF GENES CONTROLLING DEVELOPMENT IN A VERTEBRATE" CURRENT BIOLOGY, CURRENT SCIENCE,, GB, vol. 4, no. 3, 3 March 1994 (1994-03-03), pages 189-202, XP002042326 ISSN: 0960-9822 *
PACK MICHAEL ET AL: "Mutations affecting development of zebrafish digestive organs." DEVELOPMENT (CAMBRIDGE), vol. 123, no. 1, 1996, pages 321-328, XP002236851 ISSN: 0950-1991 cited in the application *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1606759A2 (fr) * 2003-03-26 2005-12-21 Synergy Biosystems Ltd. Procede d'identification d'agents biologiquement actifs et combinaisons synergiques
EP1606759A4 (fr) * 2003-03-26 2007-11-28 Synergy Biosystems Ltd Procede d'identification d'agents biologiquement actifs et combinaisons synergiques
EP2527456A1 (fr) 2004-10-22 2012-11-28 Revivicor Inc. Porcs transgéniques déficients en chaîne légère d'immunoglobuline endogène
WO2010051288A1 (fr) 2008-10-27 2010-05-06 Revivicor, Inc. Ongulés immunodéprimés
CN113234664A (zh) * 2021-05-11 2021-08-10 澳门大学 一种胰腺祖细胞的制备方法及其应用
CN113234664B (zh) * 2021-05-11 2024-05-10 澳门大学 一种胰腺祖细胞的制备方法及其应用

Also Published As

Publication number Publication date
WO2002061073A3 (fr) 2003-07-03

Similar Documents

Publication Publication Date Title
US8124056B2 (en) High throughput genetic screening of lipid and cholesterol processing using fluorescent compounds
Pisharath et al. Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase
JP4383533B2 (ja) 真骨魚類を使用する、活性についての薬剤のスクリーニング方法
Yee et al. Exocrine pancreas development in zebrafish
Haldi et al. Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish
Zarbalis et al. A focused and efficient genetic screening strategy in the mouse: identification of mutations that disrupt cortical development
JP6605452B2 (ja) 逐次ハイブリダイゼーションバーコーディングによる分子の多重標識化
US7482507B2 (en) Methods of screening agents for activity using teleosts
US20070130632A1 (en) Methods for introducing heterologous cells into fish
Hama et al. In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters
Cui et al. Disruption of planar cell polarity activity leads to developmental biliary defects
Yasutake et al. Twist functions in vertebral column formation in medaka, Oryzias latipes
Rojas-Muñoz et al. chokh/rx3 specifies the retinal pigment epithelium fate independently of eye morphogenesis
Harish et al. Real-time monitoring of an endogenous Fgf8a gradient attests to its role as a morphogen during zebrafish gastrulation
US20110126300A1 (en) High Through-Put Method of Screening Compounds for Pharmacological Activity
Li et al. Generation of living color transgenic zebrafish to trace somatostatin-expressing cells and endocrine pancreas organization
WO2002061073A2 (fr) Recherche genetique a haut rendement de lipide et traitement du cholesterol a l'aide de composes fluorescents
Yee et al. Zebrafish as a model for pancreatic cancer research
Askjaer et al. Modern tools to study nuclear pore complexes and nucleocytoplasmic transport in Caenorhabditis elegans
Bozzo et al. Whole mount in situ hybridization and immunohistochemistry for studying retinoic acid signaling in developing amphioxus
JP2002531115A (ja) 表現型プロフィールのライブラリーを構築する方法
Ho et al. Analysis of small molecule metabolism in zebrafish
Campanale et al. Methods to label, isolate, and image sea urchin small micromeres, the primordial germ cells (PGCs)
Davison et al. Characterization of Kras‐mediated pancreatic tumorigenesis in zebrafish
Sun et al. Embryological manipulations in zebrafish

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP