RRC ID 29128
Author Miyake S, Makimura M, Kanegae Y, Harada S, Sato Y, Takamori K, Tokuda C, Saito I.
Title Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.
Journal Proc Natl Acad Sci U S A
Abstract 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 Eco T22I 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.
Volume 93(3)
Pages 1320-4
Published 1996-2-6
DOI 10.1073/pnas.93.3.1320
PMID 8577762
PMC PMC40078
MeSH Adenovirus E4 Proteins / biosynthesis Adenoviruses, Human / genetics* Adenoviruses, Human / metabolism Base Sequence Cell Line Cosmids* DNA, Viral / metabolism* Embryo, Mammalian Gene Expression Genetic Vectors* Genome, Viral* Humans Kidney Molecular Sequence Data Mutagenesis, Insertional Oligodeoxyribonucleotides Recombination, Genetic* Restriction Mapping Viral Proteins / metabolism*
IF 9.412
Times Cited 780
DNA material pAxcw (RDB00917) pAxc (RDB00918) pAxCAwt (RDB01678) Ax1w1 (RDB01746) Ad5-dlX (RDB01747)