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Recombinant Virus Database

Adenovirus Information Site

  Recombinant Adenovirus   Shuttle Vectors for Adenovirus   PCR Primers   Link & References

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RIKEN DNA Bank Adenovirus

Living modified organisms (LMO)
Laboratory use only.
Not intended for intentional introduction into the environment.

Transportation of recombinant adenovirus

    For US transfer, "Veterinary Permission" (VS Forms 16-3 and 16-7) might be required for shipment of the biological materials. Please refer to our website at http://dna.brc.riken.jp/en/furnish.html.

    RIKEN DNA Bank provides recombinant adenoviruses as crude lysates of infected cells with 106-8 pfu/ml virus particle. The volume of lysate provided is 0.1 ml.
    Based on results of our research (Ugai et al., 2002), recombinant adenoviruses are provided at room temperature by air mail.
    After received a sample, amplify the adenovirus immediately by infection of 9x106 cells of HEK293 in 10cm diameter dish with the 100 ul virus solution. Otherwise you save it at -80oC.

Refer a protocol for purification or our published paper (Ugai et al., 2005).

Replication Competent Adenovirus (RCA)

    Recombinant adenoviruses provided by RIKEN DNA Bank are prepared by the 'COS-TPC method' (Miyake et al., 1996) and, in some cases, they might be suspected of contamination by Replication Competent Adenovirus (RCA) even though they were inspected by the "RCA-checking" test (Suzuki et al., 2004) before shipment. In addition, it was reported that RCAs harboring the E1 gene (E1A and/or E1B) derived from HEK293 cells were found in solutions of virus during the preparation of high-titer stocks (Ugai et al., 2003). For these reasons, tests for contamination by RCAs should be performed prior to use of recombinant adenoviruses.

    Moreover, recombinant adenoviruses, as well as recombinant DNA clones, can potentially mutate in some cases. Inserted genes (and the fiber gene, in the case of fiber-substituted recombinant adenoviruses) should be examined prior to your experiments by PCR, nucleotide sequencing and/or Western blotting.

You can find recombinant adenoviruses at
http://dna.brc.riken.jp/search/

Entire list of viruses and shuttle vectors are avairable.

  Cloned DNA Shuttle vector Reconbinant adenovirus
Human c_hum s_hum v_hum
Mouse c_mou s_mou v_mou
Entire list c_all s_all v_all

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  Recombinant Adenovirus   Shuttle Vectors for Adenovirus   PCR Primers   Link & References

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RIKEN DNA Bank Shuttle Vectors for Recombinant Viruses

Living modified organisms (LMO)
Laboratory use only.
Not intended for intentional introduction into the environment.

The viral supernatants produced by transfecting host cells with these vectors under nonstandard conditions could contain hazardous recombinant viruses in some cases. Due caution must be exercised in the production and handling of recombinant adenoviruses.

You can obtain recombinant viruses by transfecting appropriate cells with these shuttle vectors under appropriate conditions. In some cases, however, it is difficult to obtain certain recombinant viruses because of the toxicity of products of inserted genes.

These vectors may also be used as mammalian expression vectors after conversion to plasmid DNA by the self-ligation method.

Recombinant adenoviruses produced by transfecting HEK293 cells with these shuttle vectors are available from the RIKEN DNA Bank, while retrovirus-producing cells are available from the RIKEN Cell Bank (http://www.brc.riken.jp/lab/cell/).

How to obtain virul particle from the shuttle vectors?

  • Intact-genome transfection method
    • In the case shuttle vectors are constructed from the following vector, direct transfection HEK293 with the shuttle vectors yields virul particle (Fukuda et al., 2006).
      • RDB3120 pAxcwit
      • RDB3121 pAxCAwtit
      • RDB5212 pAxcwit2
      • RDB5213 pAxCAwtit2
      • RDB5214 pAxCALNLwtit2
      • RDB5215 pAxEFwtit2
  • COS-TPC method
    • The other case, recombinant adenoviruses are obtained from shuttle vectors by 'COS-TPC method' (Miyake et al., 1996) using DNA-TPC. DNA-TPC is prepared by yourself otherwise purchased from Takara Bio Inc.
      www.takara-bio.com/research.htm

Shuttle vector of Sugano full-length human cDNA

    These shuttle vectors were constructed for generation of recombinant adenovirus based on pAxCALNLw cosmid vector. Full-length human cDNA produced by 'oligo-cap method' was inserted into SwaI site of pAxCALNLw.
    Recombinant adenovirus can be obtained by 'COS-TPC method'.
    http://dna.brc.riken.jp/en/sugano.html

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  Recombinant Adenovirus   Shuttle Vectors for Adenovirus   PCR Primers   Link & References

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Examples of PCR Primers for Detection of Adenoviruses

NamePositionaSequenceVID of RVD
Detection of E1A
E1AF1b560-5955'-ATG AGA CAT ATT ATC TGC CAC GGA GGT GTT ATT AC-3'S00797
E1AF2c626-6615'-CTG ATC GAA GAG GTA CTG GCT GAT AAT CTT CCA CC-3' 
E1AR2b1545-15115'-TTA TGG CCT GGG GCG TTT ACA GCT CAA GTC CAA AG-3'S00797
jpz1d880-8995'-GGGTCCGGTTTCTATGCCAA-3'S00026
jpz2d1099-10775'-GCCACAGGTCCTCATATAGCAAA-3'S00026
Detection of E1B
E1BF1c2002-20355'-AGT TTT ATA AAG GAT AAA TGG AGC GAA GAA ACC C-3' 
E1BF2c2097-21315'-ACA CAA GAA TCG CCT GCT ACT GTT GTC TTC CGT CC-3'S00796
E1BR1c2495-24625'-AGT GGT CAG CTG CTC TAT GGA ATA CTT CTG CGC G-3' 
E1BR2c3156-31225'-TGC GAG AGT GGC TGG CTA CGT GAA TGG TCT TCA GC-3'S00796
E1BR3c3285-32515'-TGC TCT CGG GCT CAA GCA ATA TCT TAG TGT GAC TC-3' 
jpz3d2392-24135'-CCAGACACCGTCCTGAGTGTAT-3'S00027
jpz4d2628-26075'-CGTTCCCAGAAATGTAGCAACA-3'S00027
Detection of pIX
jpz5d3473-34945'-CGCTGAGTTTGGCTCTAGCGAT-3'S00028
jpz6d3698-36795'-CATCACATTCTGACGCACCC-3'S00028
Detection of insert cDNA in CAG expression unit
PAXCAF1 5'-GGCTTCTGGCGTGTGACCGGC-3'  
PAXCALNLF1 5'-CACTGCATTCTAGTTGTGGTTTGTCC-3'  
PAXCAR1 5'-CAGAGGGAAAAAGATCTCAGTGG-3'  
aPositions of nucleotide sequences correspond to those of the nucleotide sequence of human adenovirus type 5 (Genbank accession number M73260).
bUgai et al., 2003.
cSuzuki et al., 2004.
dZhu et al., 1999.

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  Recombinant Adenovirus   Shuttle Vectors for Adenovirus   PCR Primers   Link & References

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Link & References

  1. Introduction of Recombinant Virus Bank in RIKEN Gene EngineeringDivision by Takehide Murata, Jianzhi Pan, Megumi Hirose, KumikoInabe, Yukari Kujime, Chitose Kurihara, Yuka Kusa, Satoko Masuzaki,Koji Nakade, Yuri Nakano, Masato Ohkubo, Takahito Yamasaki, YuichiObata, Kazunari K. Yokoyama. Published in the Gene Therapy Review.

  2. Fukuda, H., Terashima, M., Koshikawa, M., Kanegae, Y., Saito, I. (2006) Possible mechanism of adenovirus generation from a cloned viral genome tagged with nucleotides at its ends. Microbiol Immunol. 50: 643-654.

  3. Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Sato, Y., Takamori, K., Tokuda, C., Saito, I. (1996) Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc. Natl. Acad. Sci. USA 93: 1320-1324.

  4. Suzuki, E., Murata, T., Watanabe, S., Kujime, Y., Hirose, M., Pan, J., Yamazaki, T., Ugai, H., Yokoyama, K.K. (2004) A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks. Oncol. Rep. 11: 173-178.

  5. Ugai, H., Watanabe, S., Suzuki, E., Tsutsui-Nakata, H., Yokoyama, K.K., Murata, T. (2002) Stability of a recombinant adenoviral vector: optimization of conditions for storage, transport and delivery. Jpn. J. Cancer Res. 93: 598-603.

  6. Ugai, H., Suzuki, E., Inabe, K., Murata, T., Hamada, H., Yokoyama, K.K. (2003) Spontaneous mutations in the human gene for p53 in recombinant adenovirus during multiple passages in human embryonic kidney 293 cells. Biochem. Biophys. Res. Commun. 300: 448-456.

  7. Ugai H, Yamasaki T, Hirose M, Inabe K, Kujime Y, Terashima M, Liu B, Tang H, Zhao M, Murata T, Kimura M, Pan J, Obata Y, Hamada H, Yokoyama KK.(2005) Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration. Biochem Biophys Res Commun. 331: 1053-1060.

  8. Zhu, J., Grace, M., Casale, J., Chang, A.T., Musco, M.L., Bordens, R., Greenberg, R., Schaefer, E., Indelicato, S.R. (1999) Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector. Human Gene Ther. 10: 113-121.


Efficiency of infection of adenovirus

  • Wickham et al., Nat. Biothech. 14, 1570-1573, 1996.
  • Mizuguchi and Hayakawa, Gene 285, 69-77, 2002.
  • Li et al., Mol. Cancer Ther. 4, 1850-1859, 2005.
  • Huch et al., Hum. Gene Ther. 17, 1-14, 2006.
  • Yoshida et al., Hum. Gene Ther. 9, 2503-2515, 1998.
  • Nakamura et al., Hum. Gene Ther. 13, 613-626, 2002.
  • Steinwaerder et al., Hum. Gene Ther. 11, 1933-1948, 2000.
  • Dehari et al., Cancer Gene Ther. 10, 75-85, 2003.
  • Bruning and Runnebaum, Gene Ther. 10, 198-205, 2003.
  • Shayakhmetov and Lieber, J. Virol. Nov, 10274-10286, 2000.
  • Israel et al., J. Virol. June, 5215-5221, 2001.
  • Michiels F et al., Nat. Biotechnol. 20, 1154-1157, 2002.
  • Michiels F et al. (2002). Arrayed adenoviral expression libraries for functional screening. Nat. Biotechnol. 20: 1154-1157.
Table of infection efficiency of human cells and cell lines with standard Ad 5 viruses at the indicated Multiplicity of Infection (MOI). (form Michiels F et al. (2002), Nat. Biotechnol. 20: 1154-1157)
Cell type Origin of cells % efficiency MOI
Primary cells
Adipocytes Human liposuction tissue 25 2500
Bronchial epithelial cells Human lung 70 1000
Mast cells Cultured from human CD34+ cord blood cells 25 2500
CD14+ Monocytes Human peripheral blood 10 2500
CD3+ T-lymphocytes Human peripheral blood, naive 3 2500
Human Osteoblasts Differentiated mesenchymal stem cells 75 3000
HUVEC Human umbilical cord vascular endothelium 100 1000
Mesenchymal cells Human pluripotent bone marrow progenitors <10 1000
Pre-adipocytes Human liposuction tissue 40 2500
GM09503 Human foreskin fibroblasts 28 1000
Cell lines
A549 Human lung carcinoma 90 250
BxPC3 Pancreatic cancer 50 1000
HaCat Human keratinocyte 47 1000
HCT116 Human fibroblast 75 100-500
Hela-cells Human cervix tumor 90 1000
HepG2 Human liver carcinoma 88 50
Jurkat Human T lymphoma 81 2500
K562 Human erythroid leukemia 31 1000
MCF-7 Human breast cancer 58 1000
Ramos Human B-cell lymphoma 13 2500
SH-SY5Y Human neuronal tissue 99 250
T47D Human breast cancer 68 375
U2OS Human osteosarcoma 87 100
 

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(2002.01.01 T.M.)

2016.06.08