EP0643768A1 - Sequences d'oligonucleotides et animaux transgeniques transfectes avec celles-ci et presentant une sensibilite reduite aux analgesiques narcotiques - Google Patents
Sequences d'oligonucleotides et animaux transgeniques transfectes avec celles-ci et presentant une sensibilite reduite aux analgesiques narcotiquesInfo
- Publication number
- EP0643768A1 EP0643768A1 EP93912092A EP93912092A EP0643768A1 EP 0643768 A1 EP0643768 A1 EP 0643768A1 EP 93912092 A EP93912092 A EP 93912092A EP 93912092 A EP93912092 A EP 93912092A EP 0643768 A1 EP0643768 A1 EP 0643768A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- animal
- seq
- construct
- bases
- sequence
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
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Classifications
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- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
-
- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
-
- 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
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- 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
Definitions
- This invention generally relates to opioid receptors, and more particularly to oligonucleotide constructs that can be used to produce a transgenic non- human animal, such as rodents, with reduced sensitivity to narcotic analgesics, used as probes or used in therapeutic treatment and diagnoses of narcotic addicts.
- This invention was made with government support under Grant Nos. DA00564, DA02643, DA01583, awarded by the National Institutes of Health. The government has certain rights in this invention.
- a ⁇ -opioid-specific receptor protein was purified to apparent homogeneity with a molecular weight of 58,000 daltons in 1986. Cho et al., Proc. Natl . Acad. Sci . USA, 83 (1986), pp. 4138-4142. However, this protein from rat brain exhibited high binding activity only when it was reconstituted with certain lipids. The role of the lipids is presumably to stabilize a particular conformation of the protein. Hasegawa et al., J. Neurochem. , 49 : 4 (1987), pp. 1007-1012. Poly- clonal antibodies were reported as having been raised against the purified opioid receptor from bovine brain. Roy et al., Biochem. & Biophy. Res.
- the purified opioid-binding protein from bovine brain was characterized by cDNA cloning.
- the primary sequence of the protein was deduced from the cDNA clones, with the sequence being found homologous to various members of the immunoglobulin protein super- family, especially to those molecules involved in cell adhesion. Schofield et al. , The EMBO Journal , 8 : 2 (1989) pp. 489-495.
- a DNA segment corresponds to newly discovered clones.
- This DNA segment is prepared so as to be in the antisense direction.
- the product of DNA construct hybridizes with endogenous mRNA for an opioid binding protein, and is useful within a cell (cultured or in a transgenic animal) in reducing sensitivity to narcotic analgesics.
- Oligonucleotide constructs of the invention can also be RNA. Whether the oligonucleotide constructs are RNA or DNA, they preferably are modified to resist degradation in vivo.
- Uses of the inventive oligonucleotides include as diagnostic or therapeutic agents or as probes. As therapeutic agents they preferably include a means for therapeutically administrating to a patient. Sequences as small as about 12 bases (when single stranded) or about 12 base pairs (when double stranded) can be used, such as in therapeutic treatments of narcotics addicts. Brief Description of the Drawings
- Figure 1 graphically represents the antinoci- ceptive activity of morphine sulfate in the presence of naloxone after control animals (non-transgenic) and inventive animals (transgenic) had received morphine pretreatment, with the inventive transgenic animals showing a marked reduced sensitivity to morphine;
- Figure 2 graphically represents the cDNA of an inventive gene in which the coding region and the region used for antisense generation is shown (where most of the 5' region shown as "antisense fragment" was ligated
- narcotic analgesic is meant the various alkaloids of opium such as morphine, morphine salts (such as morphine hydrobromide, morphine hydrochloride, morphine muscate, morphine oleate, morphine N-oxide, and morphine sulfate) , and morphine analogs and salts such as normorphine, diacetyldihydromorphine, diacetylmorphine hydrochloride, codeine and diacetylmorphine (heroin) .
- morphine salts such as morphine hydrobromide, morphine hydrochloride, morphine muscate, morphine oleate, morphine N-oxide, and morphine sulfate
- normorphine diacetyldihydromorphine
- diacetylmorphine hydrochloride codeine and diacetylmorphine (heroin) .
- narcotic analgesics include alpha- prodine, methadone, merperidine, levorphanol, propoxy- phene, fentanyl, oxymorphone, anileridine and metopon. Also included as narcotic analgesics are the endogenous opioids (e.g. the endorphins) and the synthetic opioid peptides (e.g. D-Ala-2, D-Leu-5 enkephalin) .
- endogenous opioids e.g. the endorphins
- synthetic opioid peptides e.g. D-Ala-2, D-Leu-5 enkephalin
- narcotic analgesics As is well known, continued use of at least many of these narcotic analgesics leads to habituation or addiction. However, despite their abuse potential, these narcotic analgesics have therapeutic uses, for example with patients requiring chronic treatment to ease pain.
- narcotic analgesics can be, and are, studied in various mammalian species besides humans, since practical and governmental considerations frequently require that studies be first done in small rodents and/or monkeys before the analgesic properties of pharmaceuticals are tested with humans.
- all drugs that have morphine-like properties in mammals other than man have been found to be morphine-like in man, and a variety of analgesic assays have been developed with animals which have gained widespread acceptance for predicting properties in humans.
- the present invention generally relates to several clones pertaining to opioid receptor functions.
- a particularly preferred, novel clone of this invention (sometimes hereinafter called ⁇ D ⁇ Zl ⁇ , whose sequence is given by SEQ ID N0:1) codes for a protein with opioid receptor function.
- ⁇ D ⁇ Zl ⁇ whose sequence is given by SEQ ID N0:1
- mice transgenic to a unique region of anti-sense DUZ1 and have demonstrated that these transgenic animals exhibit reduced sensi- tivity to the antinociception effects of morphine.
- These transgenic animals, and the underlying oligo ⁇ nucleotide constructs themselves are useful, for example, in diagnoses of and treatments for addiction to narcotic analgesics.
- applications of the invention include therapeutic methods in treating addicts who can potentially be cured of their addiction (because the invention can be used in ways that do not lead to tolerance development) .
- patients requiring chronic treatment with narcotics to ease pain such as terminal cancer patients, can be treated in accordance with the invention by administrations of antisense fragments to block the expression of the opioid receptor gene.
- the patient would still have the pain killing benefit of the narcotic analgesic, such as morphine, but that development of tolerance would be blocked.
- lower doses of the narcotic analgesic could be used and the various, known side effects (such as respiratory depression and constipa ⁇ tion) , which result form chronic treatment with high dosages of narcotics could be lessened.
- Diagnostic kits in accordance with aspects of the invention can be used to determine if a patient is addicted and/or the degree of addiction. For example, cell samples taken from the patient can be screened with oligonucleotides of the invention. Detection of the opioid receptor gene in cells of a patient, such as, for example, blood cells, permits the screening for persons with tolerance to narcotic analgesics (that is, addicts) and differentiates from persons with a narcotic analgesic naivety.
- single stranded forms tend to be more efficacious; however, double stranded forms tend to be more resistant in vivo (that is, to enzyme degradations and the like) , although the action tends to be not as rapid as when singled stranded forms are used.
- the inventive oligonucleotides can also be used as probes for discovering human genomic sequences and for discovering other subunits of opiate receptors.
- the sequences we have given by SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3 are cDNA.
- DNA or RNA constructs of the invention may have at least a portion of the normal phosphodiester backbone modified in order to limit degradation in vivo or to convey resistance to protease or other enzymes. This is because even though antisense oligonucleotides have been shown to act as specific inhibitors of gene expression in a variety of in vitro systems, one of the major problems encountered is the rapid degradation in cells or culture media of the naturally occurring phosphodiester backbone (see, for example, Hoke et al. , Nucl. Acids Res. , 19 (1991), pp. 5743-5748. Methods to replace all or part of the sugar-phosphate backbone of a stretch of DNA and of RNA are being reported, such as by Nielsen et al. , Science, 254 (1991) pp. 1497-1500.
- chimeric oligonucleotides can form stable heteroduplexes with complementary single-stranded DNA.
- Constructs of the invention thus usefully can have all or a portion of the normal phosphodiester backbone replaced with a variety of synthetic or semi- synthetic analogues capable of mimicking the distance and orientation of the native nucleobase substituents.
- modifications may be by means of a methylphos- phonate, a phosoramidate, a phosphorothioate, a 2'-o- alkyl, a polya ide (2-aminoethylgylcine) or a modification of ribose to a cycloalkane or partially unsaturated cycloalkane.
- Clones of the invention have some coding regions common to the previously published work of Schofield et al., supra , but the inventive clones have unique regions. These unique regions are preferred in preparing the probes, DNA or RNA constructs, the therapeutic and/or diagnostic agents, and in providing transgenic animals with an altered response to narcotic analgesics.
- Example 2 describes our isolation of three clones. The largest clone we sometimes call ⁇ DUZ1 ⁇ (the sequence of which is given by SEQ ID NO:l) with a reading frame comparable to OBCAM in its C-terminal 318 amino acids, but differing in its N-terminal amino acids, differing in all of its 5'-noncoding region, and differing in some of the 3'-noncoding region.
- Example 3 Most of this unique 5'-noncoding region was then used as described by Example 3 (in the antisense orientation) in raising transgenic mice having a dramatically altered response to morphine (taking about three times as much morphine to kill pain as with control animals) . These transgenic animals apparently do not develop tolerance to morphine.
- the clone we sometimes call "SG13" (the sequence of which is given by SEQ ID NO:2) and the third clone we sometimes call “SG8” (the sequence of which is given by SEQ ID NO:3) are also useful in antisense constructs and applications.
- the SEQ ID NO:3 clone also has a unique 5'-noncoding region.
- the inventive transgenic animals for example, can also be used to provide cell cultures where somatic cells from the animals are cultured, and then can be used in a variety of diagnostic techniques and assays.
- Transgenes introduced into animals in order to make transgenic, non-human animals of the invention contain an oligonucleotide sequence introduced into the animals, or an ancestor of the animal, at an embryonic stage.
- the oligonucleotide sequence is expressible in the animals and is effective to alter the animal's response to a narcotic analgesic, as will be exemplified by Example 3.
- the oligonucleotide sequence introduced into the animals forms a transcriptional unit expressible in the animals as a messenger RNA compound capable of hybridizing to endogenous mRNA for an opioid binding protein.
- An altered response by the animal to a narcotic analgesic is, for example, a reduced sensi ⁇ tivity to narcotic analgesic (e.g., such as morphine, morphine analogues, endogenous opioids, and synthetic opioid peptides) .
- the oligonucleotide sequence introduced into the animals is preferably selected from all or part of the sequences illustrated by SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:3, but wherein the sequence selected (or its portion) is read in the antisense direction.
- Example 3 illustrates such a use where the first 500 base pairs of SEQ ID NO:l (a novel portion of SEQ ID NO:l) was used, but used in the antisense direction in constructing the transgene.
- aspect of the invention is the use of DNA or RNA constructs comprising a segment corresponding to all or part of SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3, but reversed with respect to the direction shown
- the segment preferably is sufficient in size to hybridize with endogenous mRNA for an opioid binding protein. We believe a sufficient size is as short as 12 bases (when single stranded) or 12 base pairs (when double stranded) . Shorter sequences than this lose specificities.
- inventive constructs may be operatively linked to an expression vector, such as a mammalian expression vector or a retroviral expression vector, or not. That is, in many therapeutic applications an expression vector is believed not necessary, and the construct can be, for example, directly administered (in single or double stranded form) to a patient by a variety of administration means.
- constructs of the invention preferably have been modified for resistance to degradation, such as where at least a portion of the normal phosphodiester backbone is modified with an analogue that is effective to limit degradation in vivo .
- DNA constructs include administration to patients who are addicted to narcotic analgesics in order to assist these patients in curing their addiction while avoiding development of tolerance.
- inventive antisense constructs can be used to suppress expression of the gene.
- the invention includes diagnostic or therapeutic agents comprising an oligonucleotide with at least about 12 bases when single stranded and about 12 base pairs when double stranded and having a backbone, the oligonucleo- tide substantially corresponding to DNA bases (or two RNA analogous thereto) of all or a portion of SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3, but in the antisense direction thereof, and including a means for therapeu ⁇ tically administrating the oligonucleotide to a patient.
- Such therapeutic administrating means includes patches and physiologically acceptable solutions and carriers.
- OBCAM opioid-binding cell adhesion molecule
- cDNA OBCAM DNA copy of gene encoding OBCAM
- bOBCAM bovine gene encoding opioid- binding cell adhesion molecule
- rOBCAM rat OBCAM
- pBOM cDNA for bovine opioid-binding molecule
- pROM cDNA for rat opioid-binding molecule
- bp base pairs
- PCR polymerase chain reaction
- RT reverse transcriptions
- HEPES 4-(2-hydroxy)ethyl-1-piperazineethanesulfonic acid
- aa amino acid(s)
- DUZ2 clone 2 from UZ library
- kb kilobase(s) or 1000 bp
- nt nucleotide(s)
- UZ cDNA library created using rat brain poly(A) RNA (Clontech) and Stratagene's Uni-Zap XR.
- Plasmids were constructed using standard recombinant techniques.
- the eukaryotic expression vector, pSVL was used.
- the rat OBCAM cDNA was digested with Sma ⁇ , and a 649-bp fragment was isolated on agarose gel. This SmaZ fragment bracketed the AUG codon of rat OBCAM cDNA, containing 272 bp upstream of the AUG and 377 bp downstream. This fragment was ligated to a Smal- cleaved preparation of pSVL, followed by transformation of Escherichia coli DH5 ⁇ competent cells. Plasmid DNAs from twenty of these ampicillin-resistant colonies were isolated and nine out of twenty colonies were shown to contain the 649-bp insert.
- the orientation of the insert was verified by the cleavage of internal Xhol site. Two colonies which carried either sense or antisense orientation were selected and named "pSVL sense” or "pSVL antisense", respectively. To ensure that the orientations of pSVL sense and pSVL antisense were correct, the plasmid DNAs were digested by the enzymes Kpnl and PvuII; the products corresponded in size to those expected from pROM sequences.
- NG108-15 neuroblasto a x glioma cells were grown in tissue cultures.
- Tissue culture medium components were purchased from GIBCO. Fetal calf serum was obtained through Hyclone.
- Calcium phosphate-mediated DNA transfection was carried out as follows. On day 1, cells were plated at a density of approximately 1.2 x 10 cells/10 cm dish. On day 2, 3 hours before transfection, all the medium was removed and replaced with 10 ml of fresh medium. Twenty to 30 ⁇ g of plasmid DNA (in the ratio 1:50 of pSVL sense or pSVL antisense plus selectable plasmid pSVNeo) were transfected/plate. The plate was incubated overnight at 37°C in a 5% C0 2 atmosphere.
- Poly(A) + RNA from these clones was isolated from cultures in 10. cm dishes. Poly(A) + RNA of rat brain was purchased from CLONTECH. Genomic DNA was isolated from rat brain, NG108-15, ST8-4, and ST7-3 cells.
- RPJ1 [nucleotides 482-499]
- RPJ2 [nucleotides 620-603]
- RNA PCR Reverse Transcription Ten ng of poly(A) + RNA were added in 20 ⁇ l of reaction solution containing 50 mM Tris-HCL (pH 8.3), 3 mM MgCl 2 , 75 mM KC1, 2.5 mM dithiothreitol, 1 mM of each dNTP, 20 units of RNase inhibitor (Promega Biotec) , 200 units of Moloney murine leukemia virus reverse transcriptase (GIBCO/Bethesda Research Laboratories) , and 50 pmol of random hexamer, and incubated for 1 hour at 42°C. The reaction was stopped by heating at 99°C for 5 minutes.
- DNA PCR was performed in 100 ⁇ l containing 50 mg of genomic DNA, 15 pmol of each primer, 20 nmol of each dNTP in 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , and 0.001% (w/v) gelatin.
- the thermal cycling condition was as described above.
- PCR products were electrophoresed on the agarose gel and transferred to the MagnaGraph nylon membranes.
- the membranes were subjected to hybridiza- tion using P-labeled Smal fragment.
- Hybridization was carried out as described previously (Thomas, 1980), using P-labeled probes. The nylon membrane was washed with 40 minutes incubations at
- the SSC composition had 0.5 M sodium chloride and 45 mM sodium citrate.
- Membrane Preparation Cells were harvested in phosphate-buffered saline/EDTA (phosphate buffer + 1 mM EDTA) , homogenized. in 25 mM HEPES (pH 7.4) , 0.32 M Sucrose, and centrifuged at 1,000 x g. The resulting supernatant was centrifuged at 100,000 x g, and the pellet resuspended in 25 mM HEPES (pH 7.4) .
- Cell membranes (about 300 ⁇ g) were incubated for 90 minutes at room temperature with labeled ligand (2 nMunless otherwise noted) in the presence or absence of 1 ⁇ M unlabeled ligands, in 25 mM HEPES (pH 7.4) .
- the ligands used for the binding assays were tritiated diprenorphine (opioid) , scopolamine (muscarinic) , rauwolscme ( ⁇ 2 -adrenerg ⁇ c) , and 125I- ⁇ nsul ⁇ n.
- the unlabeled ligands used for opioid, muscarinic, ⁇ 2 - adrenergic, and insulin binding assays were D-Ala -D- Leu 5 -enkephalin (DADLE) or various other opioid ligands, atropine, phentolamine, and insulin, respectively.
- DADLE D-Ala -D- Leu 5 -enkephalin
- the reaction was terminated by filtration on GF/B filters
- NGJ2 and NGJ6 Two clones, NGJ2 and NGJ6, from PCR products between RPJ1 and RPJ2 were sequenced. These sequences differed in only a few nucleotides from pROM or pBOM, and these differences in nucleotide sequence did not affect the derived amino acid sequences, which were identical to bovine and rat OBCAM.
- the slight differences between the two PCR-derived clones might have resulted from either hetero ⁇ geneity in the NG108-15 cells or from errors introduced by the Taq polymerase. This conservation in the derived amino acid sequences suggested that these RT-PCR products were the corresponding cDNAs of NG108-15 cells.
- Genomic DNA was isolated from NG108-15, ST8-4, and ST7-3 cell lines, and the pROM inserts were amplified by PCR an analyzed on 2% agarose gel. An estimate of the number of ST7-3 antisense and ST8-4 sense inserts was obtained by using densitometry scanning to compare the number of antisense sequences in known amounts of clone pSVL antisense DNA to antisense or sense sequence present in 50 ng of either ST7-3 or ST8-4 genomic DNA.
- the OBCAM-transfected cell lines ST8-4 (sense) and ST7-3 (antisense) were tested for the presence of receptors for opioids as well as for several other classes of ligands. Binding of the opioid [ H] diprenorphine was reduced by 80% in the antisense- transfected cells, as compared to non-transfected cells. [ H] Diprenorphine binding to sense-transfected cells was reduced about 10%, which was statistically nonsignificant. In contrast, binding of the ⁇ 2 - adrenergic ligand [ H]rauwolscine, the muscarinic ligand
- the properties of the opioid receptors in the sense- and antisense-transfected cell lines were further tested. Scatchard analysis indicated that essentially all of the 80% reduction of [ 3 H] diprenorphine binding in the antisense-transfected cells was due to a decrease in B mx , while K d was unaffected.
- the opioid receptors in both cell lines retained the ⁇ characteristics of non-transfected NG108-15 cells, as indicated by the rank order of affinity of various opioid ligands: DADLE > ⁇ - endorphin > morphine > U-50,488H.
- the opioid receptors present in either transfected cell retained their stereoselectivity.
- This example illustrates our having conducted a search for cDNAs coding for an OBCAM-like protein, but with one or more additional regions (such as regions coding for a transmembrane and/or intracellular domain, which domains are missing in OBCAM) .
- additional regions such as regions coding for a transmembrane and/or intracellular domain, which domains are missing in OBCAM.
- Genera1 Approach Two cDNA libraries were screened, one purchased from stratagene (SG; amplified one time) , and one created in our laboratory using rat brain poly(A) + RNA (Clontech) and Stratagene's Uni-Zap XR (UZ) . The library was screened with probes repre ⁇ senting two different regions of OBCAM, one from the bovine clone (bOBCAM) and one from the partial rat clone (rOBCAM) . Comparison of these two clones revealed that the coding region was highly conserved, so both probes were generated from within this region.
- bOBCAM bovine clone
- rOBCAM partial rat clone
- D ⁇ Zl (SEQ ID N0:l) contained a slightly shorter putative reading frame, of 338 aa; the C-terminal 318 aa were virtually identical to those of OBCAM, including the same stop codon, but the N-terminal 20 aa were unique.
- Screening of the SG Library Using high stringency conditions, 18 clones were obtained from the SG library, ranging in size from 0.6 kb to 3.5 kb. Only those clones greater than 1.0 kb were further character ⁇ ized, and terminal sequencing quickly identified which clones most likely contained an entire coding sequence.
- SG13 (SEQ ID NO:2) was identical to rOBCAM in the area where the two clones overlapped; however, unlike rOBCAM, SG13 (SEQ ID NO:2) contained a complete reading frame, a short 3' non-coding region, and a 5* non-coding region that extended upstream further than that of rOBCAM. We believe that SG13 (SEQ ID NO:2) represents a sequence extended with respect to rOBCAM, and which contains an additional unique 5' region.
- SG8 also had a complete reading frame, identical to that of SG13 (SEQ ID NO:2), and was also identical to the latter throughout its somewhat shorter 3' non-coding region and in the 200 or so nucleotides immediately upstream of the putative start codon.
- the 5' non-coding region further upstream had no significant sequence homology with SG13 (SEQ ID NO:2), suggesting that it may involve a region regulating translation.
- Transgenic mice containing antisense oriented region from D ⁇ Zl (SEQ ID NO:l) (prepared in a double- stranded construct) exhibited reduced sensitivity to the antinociceptive eff cts of morphine.
- the transgene used in these studies was the first 500 base pairs as a fragment of the clone DUZ1 (SEQ ID NO:l), but constructed to be in the antisense orientation.
- Transgenicmice (founders) were identified and non-transgenic littermates were used as controls for antinociceptive tests. Of 33 D ⁇ Zl (SEQ ID NO:l) founders, 18 (54%) contained the transgene.
- transgenic males #1355 was mated with females of an inbred strain (C57BL/10) to produce Nl generation mice; of 29 offspring, 8 (28%) contained the transgene.
- AD 50 for morphine as shown in Table 1. These values were similar whether non-transgenic littermates of transgenic animals or age- and sex-matched naive C57BL/10 mice were used as a control group, as illustrated by the data of Table 1. TABLE 1
- AD 50 Antinociceptive Activity
- Fertilized hybrid zygotes were obtained approximately 12 hours postcoitus from the mating of (C57BL/6 female x SJL male) F2 hybrid mice. Zygote isolation, injection, and reimplantation into the oviducts of pseudopregnant Swiss-Webster recipient females. Transgenic offspring were identified by using the polymerase chain reaction on mouse tail DNA samples. Approximately 1 cm of tail was removed and incubated overnight at 55°C in 200 ⁇ l of 50 mM Tris, pH 8, 100 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate, and 2.5 mg/ml proteinase K. The digested solution was precipi- tated with isopropanol, spooled, transferred into 50 ⁇ l water, and boiled for 15 minutes.
- Antinociceptive activity of morphine sulphate was determined by the tail flick method. Morphine sulphate was injected s.c. Because of the small number of animals available (6 in each group) , the up-and-down method was used to determine AD 50 . (Dixon, Amer. Statistical J . , (1965), 967-978.)
- mice Manipulation of zygotes had no effect on morphine sensitivity of the animals as non-transgenic adults.
- founder mice, and first generation containing the antisense construct had a morphine AD 50 of 11.22 ⁇ 1.32, while the Nl generation mice, the second generation, had a morphine AD 50 of 22.4 ⁇ 1.22.
- Nl generationmice derived from founder male 1355 contained the antisense transgene in another inbred strain (DBA/2J) show significantly less sensitivity to morphine. This observation, taken with the difference in sensitivity to morphine in founders and C57BL/10 Nl offspring suggests the expression of the transgene is influenced by the genetic background of the mouse strain harboring it.
- mice were pretreated with a fixed dose of morphine given s.c. in 0.1 ml physiological saline (see below) , and tail flick latencies monitored again until they had returned to baseline. Morphine antinociception was then determined in the presence of 125 ⁇ g/kg s.c. naloxone, given ipsilateral to the morphine sulfate administration at the same time. As in the initial antinociceptive tests, the morphine AD 50 s were determined by the up-and-down method.
- the AD S0 of morphine was increased in the acute tolerance studies, and the higher the pretreatment dose of morphine, the higher the AD 50 .
- the AD 50 in the nontransgenic animals reached a maximum of 67.45 mg/kg, a nearly 10-fold increase over the control mice not pretreated with morphine.
- the AD 50 of morphine in the transgenic animals increased relatively slightly with morphine pretreatment dose, with a maximum value of 35.53 mg/kg in transgenic animals pretreated with 100 mg/kg morphine. Since the control AD 50 in these animals was elevated to 22.44 mg/kg, this represented an increase of only about 50%.
- mice containing the transgene showed a reduced sensitivity to morphine.
- this reduced sensitivity was manifested in less of an effect of morphine pretreatment in raising the AD 50 .
- ORGANISM Rattus rattus
- D DEVELOPMENTAL STAGE: Adult
- F TISSUE TYPE: brain
- CTCTCCCTCC CTTTGCAAAC ATTGGATTTA AACCTGCTCA GAATTCAGTA CAGAGGAAGC 540
- CTGTGTGGCC ACAAACAAGC TTGGGAACAC CAATGCCAGC
- ORGANISM Rattus rattus
- D DEVELOPMENTAL STAGE: Adult
- F TISSUE TYPE: brain
- GACGGTCCGG CAGGGAGAGA GCGCCACCCT CAGGTGTACC ATAGATGACC GGGTCACCAG 660
- CTACACTGCG GAGGGGCAAA CCAGTTTGGG CTTTCTTTTG GTTATTTTTT TGTTCTTCTT 1620
- GACTGTTTAG TTTTTTGGTT TGATTTCTGG GATTTTCAAT TTGATTTGTT TTTCTTTT 1680
- ORGANISM Rattus rattus
- D DEVELOPMENTAL STAGE: Adult
- F TISSUE TYPE: brain
- GTAAGACTTA CAGGCTTGAT GAAATATACA TGTATCCAAA GCTACAAATT TAGAAGTCAC 2040
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Abstract
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US86667792A | 1992-04-10 | 1992-04-10 | |
PCT/US1993/002913 WO1993021309A1 (fr) | 1992-04-10 | 1993-03-29 | Sequences d'oligonucleotides et animaux transgeniques transfectes avec celles-ci et presentant une sensibilite reduite aux analgesiques narcotiques |
US866677 | 1997-05-30 |
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JP (1) | JPH08501442A (fr) |
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PT656007E (pt) * | 1992-08-13 | 2003-09-30 | Univ California | Genes de receptores opioides delta |
US20020061554A1 (en) | 1992-08-13 | 2002-05-23 | Evans Christopher J. | Orphan opioid receptor and recombinant materials for its production |
US6258556B1 (en) * | 1993-02-26 | 2001-07-10 | The United States Of America As Represented By The Department Of Health And Human Services | cDNA and genomic clones encoding human μ opiate receptor and the purified gene product |
JP4191796B2 (ja) * | 1995-08-15 | 2008-12-03 | ユーロスクリーン・ソシエテ・アノニム | 拮抗剤または作動剤として、ヒトorl1受容体に結合できることが知られていない化合物を回収する方法 |
US7083932B1 (en) | 1995-08-15 | 2006-08-01 | Euroscreen, S.A. | Nucleic acid molecules encoding peptides having pronociceptive properties |
FR2750825B1 (fr) | 1996-07-15 | 2002-08-23 | Centre Nat Rech Scient | Animal transgenique non humain dans lequel l'expression de l'un au moins des genes codant pour les recepteurs aux opiaces est modifiee |
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- 1993-03-29 EP EP93912092A patent/EP0643768A4/fr not_active Withdrawn
- 1993-03-29 WO PCT/US1993/002913 patent/WO1993021309A1/fr not_active Application Discontinuation
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- 1993-03-29 JP JP5518364A patent/JPH08501442A/ja active Pending
- 1993-03-29 AU AU42769/93A patent/AU677200B2/en not_active Expired - Fee Related
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1994
- 1994-10-07 NO NO943808A patent/NO943808L/no unknown
Non-Patent Citations (6)
Title |
---|
GENE, vol. 117, no. 2, 15 August 1992, AMSTERDAM NL, pages 249-254, XP000602047 LIPPMAN, D.A. ET AL.: "Opioid-binding cell adhesion molecule (OBCAM)-related clones from a rat brain cDNA library" * |
JOURNAL OF BIOLOGICAL CHEMISTRY (MICROFILMS), vol. 267, no. 11, 15 April 1992, MD US, pages 7921-7926, XP000602135 ANN D.K. ET AL.: "Specific reduction of delta opioid receptor binding in transfected NG108-15 cells" * |
JOURNAL OF BIOLOGICAL CHEMISTRY (MICROFILMS), vol. 268, no. 24, 25 August 1993, MD US, pages 18280-18285, XP000602144 GOVITRAPONG, P. ET AL.: "Transfection of NG108-15 cells with antisense opioid-binding cell adhesion molecule cDNA alters opioid receptor G-protein interaction" * |
MAMMALIAN GENOME, vol. 4, no. 3, 1993, pages 179-182, XP000602417 CHAKRABORTI, A. ET AL.: "Genetic mapping of opioid binding protein gene(s) to mouse chromosome 9" * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 89, no. 23, 1 December 1992, WASHINGTON US, pages 11234-11238, XP000602145 LANE, C.M. ET AL.: "Regulation of an opioid-binding protein in NG108-15 cells parallels regulation of delta opioid receptors" * |
See also references of WO9321309A1 * |
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WO1993021309A1 (fr) | 1993-10-28 |
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