Adenoviral Vector Production Protocol

pAdTrack-CMV (Addgene plasmid #16405) and AdEasier-1 cells (Addgene #16399) were gifts from Bert Vogelstein (52 (link)). The pAdTrack plasmids were linearized by digesting with restriction endonuclease Pme I and subsequently recombined into E. coli BJ5183 cells containing the adenoviral backbone plasmid pAdEasy-1 (AdEasier-1 cells). Recombinants were selected for kanamycin resistance, and the recombination confirmed by restriction endonuclease analyses. Finally, the recombinant plasmids were linearized with PacI before transfection with Lipofectamine 2000 (Thermo Fisher) into the adenovirus packaging cell lines HEK293 cells. The control vector Ad-GFP, Ad-HiBiTUL7, Ad-HiBiTUL8-V5, and Ad-HiBiTUL8-mutDTEL-V5 were produced, purified, and titered in HEK293, as previously described (48 (link)).

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Dirck A., Diggins N.L., Crawford L.B., Perez W.D., Parkins C.J., Struthers H.H., Turner R., Pham A.H., Mitchell J., Papen C.R., Malouli D., Hancock M.H, & Caposio P. (2023). HCMV UL8 interaction with β-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells. Journal of Virology, 97(10), e01241-23.

Publication 2023

AdEasier-1 cells Lipofectamine 2000

Adenoviral Adenovirus Backbone Cell lines Cells E coli Endonuclease i Gifts Hek293 cells Kanamycin resistance Lipofectamine 2000 Plasmids Recombination Restriction endonuclease analyses Transfection

Corresponding Organization :

Other organizations : Children's Cancer Therapy Development Institute

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Variable analysis

independent variables

PAdTrack-CMV (Addgene plasmid #16405) and AdEasier-1 cells (Addgene #16399)
Restriction endonuclease Pme I used to linearize the pAdTrack plasmids
Recombination of linearized pAdTrack plasmids into E. coli BJ5183 cells containing the adenoviral backbone plasmid pAdEasy-1
Linearization of recombinant plasmids with PacI before transfection into HEK293 cells

dependent variables

Recombinants selected for kanamycin resistance
Recombination confirmed by restriction endonuclease analyses
Production, purification, and titration of the control vector Ad-GFP, Ad-HiBiTUL7, Ad-HiBiTUL8-V5, and Ad-HiBiTUL8-mutDTEL-V5 in HEK293 cells

control variables

Lipofectamine 2000 (Thermo Fisher) used for transfection into HEK293 cells

positive controls

Ad-GFP

negative controls

None specified

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