Characterization of 911: A New Helper Cell Line for the Titration and Propagation of Early Region 1-Deleted Adenoviral Vectors

Abstract

Currently, the preferred host for the production of early region-1 (E1)-deleted recombinant adenoviruses (rAdV) is cell line 293, which was generated by transformation of human embryonic kidney cells by sheared adenovirus 5 (Ad5) DNA. To develop alternative hosts for the production of rAdV, we generated adenovirus-transformed human cell lines by transformation of human embryonic retinoblasts (HER) with a plasmid containing base pairs 79-5789 of the Ad5 genome. One of the established HER cell lines, which we called 911, exhibited favorable growth characteristics and was chosen for further study. This cell line is demonstrated to have several characteristics in common with the well-known 293 cell line: The 911 cell line is highly transfectable, and exhibits similar frequencies of homologous recombination. However, it has additional characteristics that make it a useful alternative for 293. The 911 cells perform particularly well in plaque assays. Upon infection with E1-deleted adenoviruses, plaques become apparent in monolayers of 911 cells already after 3-4 days versus 4-10 days in monolayers of 293 cells, thereby reducing the time required for quantitative plaque assays. Furthermore, yields of E1-deleted adenovirus vectors up to three times as high as those achieved with 293 cells can be obtained with 911 cells. Finally, the Ad5-DNA content of the 911 cell line is completely known. These features make the 911 cell line a useful alternative for the construction, propagation, and titration of E1-deleted recombinant adenoviruses.

Profiling of genes which are differentially expressed in mouse liver in response to adenoviral vectors and delivered genes

“…29 Scale up and purification of recombinant adenovirus The scale up and purification of the adenoviral constructs was performed as previously described. 9 Following recombination, each adenoviral construct was amplified in 911 cells 30 (Introgene, Leiden, The Netherlands) from a purified plaque which had been demonstrated to express the transgene. 911 cells, which were 85% confluent, were infected with the recombined adenovirus and 24 to 36 h after infection, cells were harvested and sedimented.…”

Transient cutaneous adenoviral gene therapy with human host defense peptide hCAP-18/LL-37 is effective for the treatment of burn wound infections

“…The Vector Core, Gene Therapy Program, Division of Medical Genetics, Department of Medicine at the University of Pennsylvania, School of Medicine kindly provided the virus. The virus was propagated in 911 cells, 25 purified by two sequential CsCl 2 gradients and dialyzed against 20 mM Tris/HCl pH 8.0 containing 2 mM MgCl 2 . Titer was determined by Adeno-Xt rapid titer kit (Becton Dickinson Bioscience, Heidelberg, Germany).…”

The protective effect of cardiac gene transfer of CuZn–sod in comparison with the cardioprotector monohydroxyethylrutoside against doxorubicin-induced cardiotoxicity in cultured cells

“…The titer of each viral stock was determined on 911 cells. 22 The titers ranged between 4 and 5 Â 10 10 PFU/ml.…”