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Recombinant adenovirus Information Site

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Recombinant Adenovirus

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., Jpn. J. Cancer Res. 93 (5): 598-603, 2002), recombinant adenoviruses are provided at ambient temperature.
  • 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.

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Empty vectors for construction of recombinant adenovirus

Catalog num. Clone name
Description		 Cloning site Promotera Cre-loxPb Production of recombinant adenovirus
Intact-genome transfectionc COS-TPCd
RDB00917 pAxcw
A cassette cosmid for construction of recombinant adenovirus		 SwaI       Yes
RDB01678 pAxCAwt
A cassette cosmid for construction of a recombinant adenovirus		 SwaI CAG     Yes
RDB03120 pAxcwit
A dual cassette for constructing recombinant adenovirus		 SwaI     Yes Yes
RDB03121 pAxCAwtit
A dual cassette for constructing recombinant adenovirus containing CAG promoter		 SwaI CAG   Yes Yes
RDB05212 pAxcwit2
A dual cassette for constructing recombinant adenovirus		 SwaI     Yese Yes
RDB05213 pAxCAwtit2
A dual cassette for constructing recombinant adenovirus		 SwaI CAG   Yese Yes
RDB05214 pAxCALNLwtit2
A dual cassette for constructing recombinant adenovirus		 SwaI CAG Yes Yese Yes
RDB05215 pAxEFwtit2
A dual cassette for constructing recombinant adenovirus		 SwaI EF-1 alpha   Yese Yes
RDB06016 pAdex CAG-SwaI-IRES-EGFP
Shuttle vector to generate rAd with CAG expression unit. EGFP marker.		 SwaI CAG     Yes

a, CA, a modified promoter of the chicken gene for beta-actin with a cytomegalovirus-immediate early enhancer (CMV-IE); CEA, carcinoembryonic antigen; EF-1 alpha, elongation factor 1 alpha.

b, Target gene harbored in the recombinant adenovirus can be expressed by co-infection with AxCANCre (RDB no. 1748, Gene 181: 207-212, 1996).

c, Fukuda et al., Microbiol. Immunol. 50: 643-654, 2006.

d, Miyake et al., Proc. Natl. Acad. Sci. USA 93: 1320-1324, 1996.

e, PacI restriction enzyme is also applicable for linearization as well as Csp45I restriction enzyme.

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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
jpz1d 880-899 5′-GGGTCCGGTTTCTATGCCAA-3′ S00026
jpz2d 1099-1077 5′-GCCACAGGTCCTCATATAGCAAA-3′ S00026
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′
jpz3d 2392-2413 5′-CCAGACACCGTCCTGAGTGTAT-3′ S00027
jpz4d 2628-2607 5′-CGTTCCCAGAAATGTAGCAACA-3′ S00027
Detection of pIX
jpz5d 3473-3494 5′-CGCTGAGTTTGGCTCTAGCGAT-3′ S00028
jpz6d 3698-3679 5′-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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Link & References

  1. 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.
  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

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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  • 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.
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  • Israel et al., J. Virol. June, 5215-5221, 2001.
  • Michiels F et al. (2002). Arrayed adenoviral expression libraries for functional screening.Nat. Biotechnol. 20: 1154-1157.

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(GRP0046e 2002.01.01 T.M.)

2023.01.28



 

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