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The adenoviral vector AdexCAG-I7-IRES-GFP (Ad-I7) was constructed in the following manner. Adenoviruses lacking the E1 region of their genome are replication-incompetent and are grown in the complementary human embryonic kidney (HEK) 293 cell line, which provides the E1 genes in trans. The entire coding sequence for the rat odorant receptor I7 (2) was amplified by the polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene) from the I7 clone plasmid with the upstream primer 5′-CCCTCGAGTATGGAGCGAAGGAACCAC-3′ and the downstream primer 5′-GCTCTAGACTAACCAATTTTGCTGCCT-3′. The 0.6-kb IRES (9) fragment was cut with Eco RI and Bam HI from plasmid p1162. The fragments of I7, IRES, and the S65T mutant of GFP were first conjugated in the multicloning sites of the expression vector pCA4 (Microbix, Ontario, Canada) and tested by Northern blot with a I7 probe for transcription of mRNA, and by green fluorescence for IRES-driven GFP expression in HEK 293 cells (ATCC, CRL-1573). The I7-IRES-GFP sequence was then subcloned into the Swa I site of the cosmid vector pAdex1pCAw (26) [Y. Kanegae et al., Nucleic Acids Res. 23, 3816 (1995)] to create the cosmid vector pAdexI7-IRES-GFP. The pAdex1pCAw cosmid was created from the human adenovirus type 5 genome from which the E1a, E1b, and E3 regions were deleted and replaced with an expression unit containing the CAG promoter, composed of the cytomegalovirus enhancer plus the chicken β-actin promoter [H. Niwa, K. Yamamura, J. Miyazaki, Gene 108, 193 (1991)], a Swa I site, and the rabbit β-globin polyadenylation signal. The I7 sequence was confirmed by sequencing. Nucleotide 104 in the I7 sequence from GenBank (accession number M64386) is undefined. This nucleotide is deoxycytidine according to our sequence analysis; therefore, amino acid 35 in the deduced protein sequence is alanine. The cosmid vector pAdexI7-IRES-GFP and the Eco T22I-digested DNA-terminal protein complex (DNA-TPC) of Ad5-dIX, which is a human type 5 adenovirus lacking the E3 region, were cotransfected into HEK293 cells by calcium phosphate precipitation. The recombinant adenovirus AdexCAG-I7-IRES-GFP was then generated by homologous recombination in the HEK 293 cells. The DNA-TPC method has been described in detail (26) [S. Miyake et al., Proc. Natl. Acad. Sci. U.S.A. 93, 1320 (1996)]. Bam HI and Xba I digestion of the genomic DNA of AdexI7-IRES-GFP produced the appropriate band pattern, and positive PCR amplification of I7 also verified the construct. Because recombinant viruses do not include the E1a genes, PCR amplification of the E1a region was performed with the primers 5′-ATTACCGAAGAAATGGCCGC-3′ and 5′-CCCATTTAACACACGCCATGCA-3′, as a control for contamination by wild-type adenovirus (Ad5-dIX). Negative PCR amplification of the E1a gene was observed in every stock of recombinant adenovirus. The recombinant adenovirus was propagated in HEK 293 cells and purified by cesium gradient centrifugation [Y. Kanegae, M. Makimura, I. Saito, Jpn. J. Med. Sci. Biol. 47, 157 (1994)]. The viral titer was determined by plaque-forming assay on HEK 293 cells.
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Kanegae, Y.1
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0025884056
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The adenoviral vector AdexCAG-I7-IRES-GFP (Ad-I7) was constructed in the following manner. Adenoviruses lacking the E1 region of their genome are replication-incompetent and are grown in the complementary human embryonic kidney (HEK) 293 cell line, which provides the E1 genes in trans. The entire coding sequence for the rat odorant receptor I7 (2) was amplified by the polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene) from the I7 clone plasmid with the upstream primer 5′-CCCTCGAGTATGGAGCGAAGGAACCAC-3′ and the downstream primer 5′-GCTCTAGACTAACCAATTTTGCTGCCT-3′. The 0.6-kb IRES (9) fragment was cut with Eco RI and Bam HI from plasmid p1162. The fragments of I7, IRES, and the S65T mutant of GFP were first conjugated in the multicloning sites of the expression vector pCA4 (Microbix, Ontario, Canada) and tested by Northern blot with a I7 probe for transcription of mRNA, and by green fluorescence for IRES-driven GFP expression in HEK 293 cells (ATCC, CRL-1573). The I7-IRES-GFP sequence was then subcloned into the Swa I site of the cosmid vector pAdex1pCAw (26) [Y. Kanegae et al., Nucleic Acids Res. 23, 3816 (1995)] to create the cosmid vector pAdexI7-IRES-GFP. The pAdex1pCAw cosmid was created from the human adenovirus type 5 genome from which the E1a, E1b, and E3 regions were deleted and replaced with an expression unit containing the CAG promoter, composed of the cytomegalovirus enhancer plus the chicken β-actin promoter [H. Niwa, K. Yamamura, J. Miyazaki, Gene 108, 193 (1991)], a Swa I site, and the rabbit β-globin polyadenylation signal. The I7 sequence was confirmed by sequencing. Nucleotide 104 in the I7 sequence from GenBank (accession number M64386) is undefined. This nucleotide is deoxycytidine according to our sequence analysis; therefore, amino acid 35 in the deduced protein sequence is alanine. The cosmid vector pAdexI7-IRES-GFP and the Eco T22I-digested DNA-terminal protein complex (DNA-TPC) of Ad5-dIX, which is a human type 5 adenovirus lacking the E3 region, were cotransfected into HEK293 cells by calcium phosphate precipitation. The recombinant adenovirus AdexCAG-I7-IRES-GFP was then generated by homologous recombination in the HEK 293 cells. The DNA-TPC method has been described in detail (26) [S. Miyake et al., Proc. Natl. Acad. Sci. U.S.A. 93, 1320 (1996)]. Bam HI and Xba I digestion of the genomic DNA of AdexI7-IRES-GFP produced the appropriate band pattern, and positive PCR amplification of I7 also verified the construct. Because recombinant viruses do not include the E1a genes, PCR amplification of the E1a region was performed with the primers 5′-ATTACCGAAGAAATGGCCGC-3′ and 5′-CCCATTTAACACACGCCATGCA-3′, as a control for contamination by wild-type adenovirus (Ad5-dIX). Negative PCR amplification of the E1a gene was observed in every stock of recombinant adenovirus. The recombinant adenovirus was propagated in HEK 293 cells and purified by cesium gradient centrifugation [Y. Kanegae, M. Makimura, I. Saito, Jpn. J. Med. Sci. Biol. 47, 157 (1994)]. The viral titer was determined by plaque-forming assay on HEK 293 cells.
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Niwa, H.1
Yamamura, K.2
Miyazaki, J.3
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19
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0030070724
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The adenoviral vector AdexCAG-I7-IRES-GFP (Ad-I7) was constructed in the following manner. Adenoviruses lacking the E1 region of their genome are replication-incompetent and are grown in the complementary human embryonic kidney (HEK) 293 cell line, which provides the E1 genes in trans. The entire coding sequence for the rat odorant receptor I7 (2) was amplified by the polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene) from the I7 clone plasmid with the upstream primer 5′-CCCTCGAGTATGGAGCGAAGGAACCAC-3′ and the downstream primer 5′-GCTCTAGACTAACCAATTTTGCTGCCT-3′. The 0.6-kb IRES (9) fragment was cut with Eco RI and Bam HI from plasmid p1162. The fragments of I7, IRES, and the S65T mutant of GFP were first conjugated in the multicloning sites of the expression vector pCA4 (Microbix, Ontario, Canada) and tested by Northern blot with a I7 probe for transcription of mRNA, and by green fluorescence for IRES-driven GFP expression in HEK 293 cells (ATCC, CRL-1573). The I7-IRES-GFP sequence was then subcloned into the Swa I site of the cosmid vector pAdex1pCAw (26) [Y. Kanegae et al., Nucleic Acids Res. 23, 3816 (1995)] to create the cosmid vector pAdexI7-IRES-GFP. The pAdex1pCAw cosmid was created from the human adenovirus type 5 genome from which the E1a, E1b, and E3 regions were deleted and replaced with an expression unit containing the CAG promoter, composed of the cytomegalovirus enhancer plus the chicken β-actin promoter [H. Niwa, K. Yamamura, J. Miyazaki, Gene 108, 193 (1991)], a Swa I site, and the rabbit β-globin polyadenylation signal. The I7 sequence was confirmed by sequencing. Nucleotide 104 in the I7 sequence from GenBank (accession number M64386) is undefined. This nucleotide is deoxycytidine according to our sequence analysis; therefore, amino acid 35 in the deduced protein sequence is alanine. The cosmid vector pAdexI7-IRES-GFP and the Eco T22I-digested DNA-terminal protein complex (DNA-TPC) of Ad5-dIX, which is a human type 5 adenovirus lacking the E3 region, were cotransfected into HEK293 cells by calcium phosphate precipitation. The recombinant adenovirus AdexCAG-I7-IRES-GFP was then generated by homologous recombination in the HEK 293 cells. The DNA-TPC method has been described in detail (26) [S. Miyake et al., Proc. Natl. Acad. Sci. U.S.A. 93, 1320 (1996)]. Bam HI and Xba I digestion of the genomic DNA of AdexI7-IRES-GFP produced the appropriate band pattern, and positive PCR amplification of I7 also verified the construct. Because recombinant viruses do not include the E1a genes, PCR amplification of the E1a region was performed with the primers 5′-ATTACCGAAGAAATGGCCGC-3′ and 5′-CCCATTTAACACACGCCATGCA-3′, as a control for contamination by wild-type adenovirus (Ad5-dIX). Negative PCR amplification of the E1a gene was observed in every stock of recombinant adenovirus. The recombinant adenovirus was propagated in HEK 293 cells and purified by cesium gradient centrifugation [Y. Kanegae, M. Makimura, I. Saito, Jpn. J. Med. Sci. Biol. 47, 157 (1994)]. The viral titer was determined by plaque-forming assay on HEK 293 cells.
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0028037530
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The adenoviral vector AdexCAG-I7-IRES-GFP (Ad-I7) was constructed in the following manner. Adenoviruses lacking the E1 region of their genome are replication-incompetent and are grown in the complementary human embryonic kidney (HEK) 293 cell line, which provides the E1 genes in trans. The entire coding sequence for the rat odorant receptor I7 (2) was amplified by the polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene) from the I7 clone plasmid with the upstream primer 5′-CCCTCGAGTATGGAGCGAAGGAACCAC-3′ and the downstream primer 5′-GCTCTAGACTAACCAATTTTGCTGCCT-3′. The 0.6-kb IRES (9) fragment was cut with Eco RI and Bam HI from plasmid p1162. The fragments of I7, IRES, and the S65T mutant of GFP were first conjugated in the multicloning sites of the expression vector pCA4 (Microbix, Ontario, Canada) and tested by Northern blot with a I7 probe for transcription of mRNA, and by green fluorescence for IRES-driven GFP expression in HEK 293 cells (ATCC, CRL-1573). The I7-IRES-GFP sequence was then subcloned into the Swa I site of the cosmid vector pAdex1pCAw (26) [Y. Kanegae et al., Nucleic Acids Res. 23, 3816 (1995)] to create the cosmid vector pAdexI7-IRES-GFP. The pAdex1pCAw cosmid was created from the human adenovirus type 5 genome from which the E1a, E1b, and E3 regions were deleted and replaced with an expression unit containing the CAG promoter, composed of the cytomegalovirus enhancer plus the chicken β-actin promoter [H. Niwa, K. Yamamura, J. Miyazaki, Gene 108, 193 (1991)], a Swa I site, and the rabbit β-globin polyadenylation signal. The I7 sequence was confirmed by sequencing. Nucleotide 104 in the I7 sequence from GenBank (accession number M64386) is undefined. This nucleotide is deoxycytidine according to our sequence analysis; therefore, amino acid 35 in the deduced protein sequence is alanine. The cosmid vector pAdexI7-IRES-GFP and the Eco T22I-digested DNA-terminal protein complex (DNA-TPC) of Ad5-dIX, which is a human type 5 adenovirus lacking the E3 region, were cotransfected into HEK293 cells by calcium phosphate precipitation. The recombinant adenovirus AdexCAG-I7-IRES-GFP was then generated by homologous recombination in the HEK 293 cells. The DNA-TPC method has been described in detail (26) [S. Miyake et al., Proc. Natl. Acad. Sci. U.S.A. 93, 1320 (1996)]. Bam HI and Xba I digestion of the genomic DNA of AdexI7-IRES-GFP produced the appropriate band pattern, and positive PCR amplification of I7 also verified the construct. Because recombinant viruses do not include the E1a genes, PCR amplification of the E1a region was performed with the primers 5′-ATTACCGAAGAAATGGCCGC-3′ and 5′-CCCATTTAACACACGCCATGCA-3′, as a control for contamination by wild-type adenovirus (Ad5-dIX). Negative PCR amplification of the E1a gene was observed in every stock of recombinant adenovirus. The recombinant adenovirus was propagated in HEK 293 cells and purified by cesium gradient centrifugation [Y. Kanegae, M. Makimura, I. Saito, Jpn. J. Med. Sci. Biol. 47, 157 (1994)]. The viral titer was determined by plaque-forming assay on HEK 293 cells.
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The production of other viral proteins is also hindered because the E1 genes provide an essential function as transcriptional activators [T. Shenk, in Fields Virology, B. N. Fields et al., Eds. (Lippincott-Raven, Philadelphia, 1996), vol. 2, pp. 2111-2138]. In an earlier study using an adenovirus vector containing the lacZ marker gene, we observed strong but heterogeneous viral infection and protein expression in rat nasal epithelium, and no virally induced cell loss out to 21 days after infection [H. Zhao, J. M. Otaki, S. Firestein, J. Neurobiol. 30, 521 (1996)].
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24
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6844227448
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-
note
-
9 pfu/ml and 0.3% fast green dye was slowly injected through the nostril into the right side of the nasal cavity with a thin plastic tubing. The solution was allowed to remain in the nasal cavity. After recovery, the animals were maintained at room temperature with no other treatment until they were killed.
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25
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0004136246
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Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
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Northern blot for detection of I7 mRNA was performed using a standard procedure [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)]. Total RNAs were extracted from tissues with TRlozol reagent (Gibco-BRL); 20 μg of total RNA was loaded on each lane of the gel. The I7 probe was synthesized by PCR with primers that covered the entire I7 coding sequence and was labeled with digoxigenin (DIG-11-dUTP, Boehringer Mannheim) according to the manufacturer's protocol. After hybridization, the probe was detected with the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
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(1989)
Molecular Cloning: A Laboratory Manual
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Sambrook, J.1
Fritsch, E.F.2
Maniatis, T.3
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27
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6844238223
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-
note
-
The olfactory turbinates were dissected out, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 2 hours, and cryoprotected in 20% sucrose. Cryostat sections (15 μm) were cut and incubated with polyclonal antibody to GFP (Clontech). Specific staining was then visualized with the Vectastain Elite ABC kit (Vector Lab).
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28
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6844230226
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note
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A panel of 74 odors were screened. They included odors from several classes and groups: Aromatics Alcohols Cinnamyl alcohol, eugenol, guaiacol Aldehydes para-Anisaldehyde, (-)-carvone, cinnamaldehyde, salicylaldehyde, lilial Esters Cinnamylformate, geranyl acetate, isoamyl salicylate, linalyl formate Ethers Anisole, cineole, 2-methylanisole, 4-methylanisole, isoeugenol, methyl eugenol Heterocycles 2-Isobutyl-3-methoxypyrazin Hydrocarbons 2-Ethyltoluene, 3-ethyltoluene, 1,2-diethylbenzene, limonen Ketones Acetophenone, 2-decalone Aliphatics Alcohols n-Propyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-hexyl alcohol, n-heptyl alcohol, n-octyl alcohol, n-nonyl alcohol, n-decyl alcohol, 2-ethylfenchol, geraniol, β-citronellol, linalool Aldehydes Propion aldehyde, isobutyraldehyde, n-valeraldehyde, n-hexaldehyde, n-heptaldehyde, n-octyl aldehyde, n-nonyl aldehyde, n-decyl aldehyde, undecylic aldehyde, dodecyl aldehyde, frans-2-octenal, 2-octynal, trans-2-tridecanal, citral, lyral Acids Propionic acid, n-valeric acid, n-octanoic acid, n-nonanoic acid Alkanes n-Octane, n-nonane, n-decane Amines Isopentylamine, phenethylamine Esters Amyl acetate, ethyl butyrate, ethyl hexanoate, isoamyl acetate, octyl butyrate, octyl isovalerate Ethers Citral diethyl acetal, citral dimethyl acetal Ketones 2,3-Butanedione, 1-fenchone, 2-nonanone Other Heptyl cyanide, 1,1,3,3-tetramethylbutyl isocyanide
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3 airspace was allowed to equilibrate for more than 1 hour. All solutions were used within 8 hours. Two 18-gauge needles provided the input and output ports for the odorant-containing vapor above the solution. For stimulation, a 100-ms pulse of the odorant vapor at 9 psi was injected into the continuous stream of humidified air. The pulse was controlled by a Picospritzer solenoid-controlled valve (General Valve). The odorant stimulus pathway was cleaned by air between each stimulus presentation. The minimum interval between two adjacent stimuli was 1 min.
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The animal was overdosed with anesthetics (ketamine and xylazine) and decapitated. The head was cut open sagitally and the septum was removed to expose the medial surface of the olfactory turbinates [S. G. Shirley, E. H. Polak, D. A. Edwards, M. A. Wood, G. H. Dodd, Biochem. J. 245, 185 (1987)]. The right half of the head was mounted in a wax dish filled with rat Ringer solution. The medial surface of turbinates was face up and exposed to the air. A continuous stream of humidified clean air was gently blown on the turbinates through tubing to prevent tissue from drying. The opening of the tubing was 8 mm in diameter and was placed about 10 mm from the turbinate surface. The EOG recording electrode was an Ag-AgCl wire in a capillary glass pipette filled with rat Ringer solution containing 0.6% agarose. The electrode resistance was 0.5 to 1 megohm. The recording pipette was placed on the surface of the olfactory epithelium and connected to a differential amplifier (DP-301, Warner Instruments). Placement of the electrode was determined by visualizing GFP fluorescence with a modified stereomicroscope (Kramer Scientific). The EOG potential was observed on a chart recorder, recorded with a digital audio tape recorder, and later transferred to computer. For most experiments, two electrodes and two amplifiers were used to record EOGs from two different sites of epithelium simultaneously. All experiments were performed at room temperature (22° to 25°C).
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2, 10 mM Hepes, and 10 mM glucose (pH 7.4). Odor stimuli were delivered with the SF77 Fast Perfusion system (Warner Instruments), allowing precise concentrations to be applied for steps of various durations.
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Five animals were infected with virus containing only the lacZ gene. In each animal the EOG electrode was positioned in several areas over the epithelium. After recording, the epithelia were reacted with X-gal and 18 electrode positions were determined to have been within areas of high infection. Responses to octanal at those positions were not different from those of uninfected animals.
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We thank K. Mikoshiba, E. Falck-Pedersen, M. Chao, and in particular S. O. Yoon for expert advice and assistance; L. Buck and H. Breer for receptor clones; T. Lufkin for the IRES construct; L. Richards and K. Moriyoshi for GFP adenovirus; IFF and Harmon & Reimer Inc. for lyral and lilial; Y. Huang for technical assistance; and R. Axel, D. Kelley, and P. Mombaerts for thoughtful comments. J.M.O. would like to dedicate this paper to the memory of Yoko Sakaki. This work was supported by the Whitehall and McKnight Foundations and the National Institute on Deafness and Other Communication Disorders.
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