Transportation of recombinant adenovirus
- 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 9×106 cells of HEK293 in 10cm diameter dish with the 100 uL virus solution. Otherwise you save it at -80oC.
- Refer a protocol for purification in Recombinant virus protocols or our published paper (Ugai et al., 2005).
- 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
Current information relevant to resources [link]
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.
- Living modified organisms (LMO)
Laboratory use only.
Not intended for intentional introduction into the environment.
Shuttle Vectors for Recombinant Viruses
- 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 (https://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.
Examples of PCR Primers for Detection of Adenoviruses
|Name||Positiona||Sequence||Link to data sheet|
|Detection of E1A|
|E1AF1b||560-595||5′-ATG AGA CAT ATT ATC TGC CAC GGA GGT GTT ATT AC-3′||S00797|
|E1AF2c||626-661||5′-CTG ATC GAA GAG GTA CTG GCT GAT AAT CTT CCA CC-3′|
|E1AR2b||1545-1511||5′-TTA TGG CCT GGG GCG TTT ACA GCT CAA GTC CAA AG-3′||S00797|
|Detection of E1B|
|E1BF1c||2002-2035||5′-AGT TTT ATA AAG GAT AAA TGG AGC GAA GAA ACC C-3′|
|E1BF2c||2097-2131||5′-ACA CAA GAA TCG CCT GCT ACT GTT GTC TTC CGT CC-3′||S00796|
|E1BR1c||2495-2462||5′-AGT GGT CAG CTG CTC TAT GGA ATA CTT CTG CGC G-3′|
|E1BR2c||3156-3122||5′-TGC GAG AGT GGC TGG CTA CGT GAA TGG TCT TCA GC-3′||S00796|
|E1BR3c||3285-3251||5′-TGC TCT CGG GCT CAA GCA ATA TCT TAG TGT GAC TC-3′|
|Detection of pIX|
|Detection of insert cDNA in CAG expression unit|
|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.
Link & References
- Introduction of Recombinant Virus Bank in RIKEN Gene Engineering Division by Takehide Murata, Jianzhi Pan, Megumi Hirose, KumikoInabe, Yukari Kujime, Chitose Kurihara, Yuka Kusa, Satoko Masuzaki,Koji Nakade, Yuri Nakano, Masato Ohkubo, Takahito Yamasaki, Yuichi Obata, Kazunari K. Yokoyama. Published in the Gene Therapy Review.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
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|
|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|
|A549||Human lung carcinoma||90||250|
|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|
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(GRP0046e 2002.01.01 T.M.)