Abstract:
Human adenoviruses (Ads), members of the family adenoviridae,
are medium-sized DNA viruses which have been used as valuable
research tools for the study of RNA processing, oncogenic
transformation, and for the development of viral vectors for use in gene
delivery and immunization technology. The left 12% of the linear Ad
genollle codes for products which are necessary for the efficient
replication of the virus, as well as being responsible for the forlllation of
tumors in animallllodels. The establishlllent of the 293 cell line, by
immortalization of human embryonic kidney cells with th~ E1 region of
Ad type S (AdS), has facilitated extensive manipulation of the Ads and
the development of recombinant Ad vectors.
The study of bovine adenoviruses (BAVs), which cause mild
respiratory and gastrointestinal infections in cattle has, on the other
hand, been limited primarily to that of infectivity, immunology and
clinicallllanifestations. As a result, any potential as gene delivery
vehicles has not yet been realized. Continued research into the
molecular biolo~gy of BAVs and the development of recolllbinant vectors
would benefit from the development of a cell line analogous to that of
the 293 cells.
In an attelllpt to establish such a cell line, the recombinant
plaslllid pKC-neo was constructed, containing the left 0-19.7% of the
BAV type 3 (BAV3) genome, and the selectable marker for resistance to
the aminoglycoside G418, a neomycin derivative. The plasmid construct
was then used to transfect both the Madin-Darby bovine kidney (MDBK)
-iicell
line and primary bovine lung cells, after which G418-resistant foci
were selected for analysis. Two cell lines, E61 (MDBK) and E24 (primary
lung), were subsequently selected and analysed for DNA content,
revealing the presence of the pKC-neo sequences in their respective
genomes. In addition, BAV3 RNA transcripts were detected in the E61
cells. Although the presence of E1 products has yet to be confirmed in
both cell lines, the E24 cells exhibit a phenotype characteristic of partial
transformation by E1. The apparent immortalization of the primary
lung cells will permit exploitation of their ability to take up exogenous
DNA at high efficiency.
