Efficient Generation of Recombinant Adenoviruses Using Adenovirus DNA-Terminal Protein Complex and a Cosmid Bearing the Full-Length Virus Genome
An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1996-02, Vol.93 (3), p.1320-1324 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Miyake, Sanae Makimura, Miho Kanegae, Yumi Harada, Shizuko Sato, Yumi Takamori, Koichi Tokuda, Chikashi Saito, Izumu |
| description | An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with EcoT22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors. |
| doi_str_mv | 10.1073/pnas.93.3.1320 |
| format | Article |
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The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with EcoT22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.93.3.1320</identifier><identifier>PMID: 8577762</identifier><language>eng</language><publisher>United States: National Academy of Sciences of the United States of America</publisher><subject>adenovirus ; Adenovirus E4 Proteins - biosynthesis ; Adenoviruses ; Adenoviruses, Human - genetics ; Adenoviruses, Human - metabolism ; Base Sequence ; Cell Line ; Complementary DNA ; Cosmids ; Deoxyribonucleic acid ; DNA ; DNA, Viral - metabolism ; Embryo, Mammalian ; Gene Expression ; Genetic Vectors ; Genome, Viral ; Genomes ; Homologous recombination ; Humans ; Kidney ; Kidney cells ; Medical research ; Medical sciences ; Molecular Sequence Data ; Mutagenesis, Insertional ; Oligodeoxyribonucleotides ; Proteins ; Recombination, Genetic ; Restriction Mapping ; Viral Proteins - metabolism ; Viruses</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1996-02, Vol.93 (3), p.1320-1324</ispartof><rights>Copyright 1996 National Academy of Sciences</rights><rights>Copyright National Academy of Sciences Feb 6, 1996</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c579t-8e11d6aa28d9e30c55e1d316cbbb718cd32ccea5d15f775e46e955e4a54ea8ba3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/93/3.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/38795$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/38795$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27923,27924,53790,53792,58016,58249</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8577762$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Miyake, Sanae</creatorcontrib><creatorcontrib>Makimura, Miho</creatorcontrib><creatorcontrib>Kanegae, Yumi</creatorcontrib><creatorcontrib>Harada, Shizuko</creatorcontrib><creatorcontrib>Sato, Yumi</creatorcontrib><creatorcontrib>Takamori, Koichi</creatorcontrib><creatorcontrib>Tokuda, Chikashi</creatorcontrib><creatorcontrib>Saito, Izumu</creatorcontrib><title>Efficient Generation of Recombinant Adenoviruses Using Adenovirus DNA-Terminal Protein Complex and a Cosmid Bearing the Full-Length Virus Genome</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with EcoT22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.</description><subject>adenovirus</subject><subject>Adenovirus E4 Proteins - biosynthesis</subject><subject>Adenoviruses</subject><subject>Adenoviruses, Human - genetics</subject><subject>Adenoviruses, Human - metabolism</subject><subject>Base Sequence</subject><subject>Cell Line</subject><subject>Complementary DNA</subject><subject>Cosmids</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA, Viral - metabolism</subject><subject>Embryo, Mammalian</subject><subject>Gene Expression</subject><subject>Genetic Vectors</subject><subject>Genome, Viral</subject><subject>Genomes</subject><subject>Homologous recombination</subject><subject>Humans</subject><subject>Kidney</subject><subject>Kidney cells</subject><subject>Medical research</subject><subject>Medical sciences</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Insertional</subject><subject>Oligodeoxyribonucleotides</subject><subject>Proteins</subject><subject>Recombination, Genetic</subject><subject>Restriction Mapping</subject><subject>Viral Proteins - metabolism</subject><subject>Viruses</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1996</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkUGP0zAQhS0EWsrClQMSksWBW4IdJ3UscSlld1mpAoR2uVqOM2ldJXaxndXyL_jJOLRUXcTJst_3ZsbzEHpJSU4JZ-92VoVcsJzllBXkEZpRImg2LwV5jGaEFDyry6J8ip6FsCWEiKomZ-isrjjn82KGfl10ndEGbMRXYMGraJzFrsPfQLuhMVYlZdGCdXfGjwECvg3Grk-e8MfPi-wG_JDYHn_1LoKxeOmGXQ_3WNkWq3QLg2nxB1B-MscN4Mux77MV2HXc4O9_6qT-boDn6Emn-gAvDuc5ur28uFl-ylZfrq6Xi1WmKy5iVgOl7Vypom4FMKKrCmjL6Fw3TcNprVtWaA2qamnVcV5BOQeRmFJVJai6Uewcvd_X3Y3NAK1OG_CqlztvBuV_SqeMfKhYs5FrdydLQnid7G8Pdu9-jBCiHEzQ0PfKghuDpJxQUYgqgW_-Abdu9GlVQRaEFoJxyhOU7yHtXQgeuuMclMgpZjnFLAWTTE4xJ8Pr0-mP-CHXk_Em31_16Jdd2n6E-3hS6L9g0l_t9W2Izh8BVvP0t9_7ZseR</recordid><startdate>19960206</startdate><enddate>19960206</enddate><creator>Miyake, Sanae</creator><creator>Makimura, Miho</creator><creator>Kanegae, Yumi</creator><creator>Harada, Shizuko</creator><creator>Sato, Yumi</creator><creator>Takamori, Koichi</creator><creator>Tokuda, Chikashi</creator><creator>Saito, Izumu</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><general>National Academy of Sciences</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7QO</scope><scope>5PM</scope></search><sort><creationdate>19960206</creationdate><title>Efficient Generation of Recombinant Adenoviruses Using Adenovirus DNA-Terminal Protein Complex and a Cosmid Bearing the Full-Length Virus Genome</title><author>Miyake, Sanae ; 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The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with EcoT22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.</abstract><cop>United States</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>8577762</pmid><doi>10.1073/pnas.93.3.1320</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Proceedings of the National Academy of Sciences - PNAS, 1996-02, Vol.93 (3), p.1320-1324 |
| issn | 0027-8424 1091-6490 |
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| source | MEDLINE; JSTOR Archive Collection A-Z Listing; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | adenovirus Adenovirus E4 Proteins - biosynthesis Adenoviruses Adenoviruses, Human - genetics Adenoviruses, Human - metabolism Base Sequence Cell Line Complementary DNA Cosmids Deoxyribonucleic acid DNA DNA, Viral - metabolism Embryo, Mammalian Gene Expression Genetic Vectors Genome, Viral Genomes Homologous recombination Humans Kidney Kidney cells Medical research Medical sciences Molecular Sequence Data Mutagenesis, Insertional Oligodeoxyribonucleotides Proteins Recombination, Genetic Restriction Mapping Viral Proteins - metabolism Viruses |
| title | Efficient Generation of Recombinant Adenoviruses Using Adenovirus DNA-Terminal Protein Complex and a Cosmid Bearing the Full-Length Virus Genome |
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