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AdMax™ Hi-IQ

AdMax™ – Hi IQ for High Protein Expression From Adenoviral Vectors

"My laboratory uses the AdMax-HiIQ Ad system to express viral membrane proteins for studies of protein function. In many cases, these viral proteins are retained in the ER and this causes toxicity in the 293 cells used to produce the vectors unless there is regulation of transgene. The HiIQ system strongly represses expression of these membrane proteins in 293IQ cells allowing us to produce high titer stocks of these Ad vectors. Moreover, the rapid Cre-mediated recombination gives us Ad vectors quickly and efficiently. We have found that this approach is particularly valuable to allow expression of these viral membrane proteins in difficult to transfect cells, such as epithelial cells. Also important is the fact that we can get high-level expression in animal models. For all of these reasons, in my laboratory this system has replaced several other systems for producing Ad vectors." Dr. David C. Johnson Professor, Molecular Microbiology & Immunology Oregon Health & Sciences University

 

The problem:
It is possible to enhance expression of proteins cloned in Ad vectors by introducing an intron between the transcription start and the translation start of the protein. However, high levels of transgene expression can then have inhibitory effects on virus replication, even with proteins that are not cytotoxic when expressed at moderate levels. Further, mild inhibition of virus replication can significantly reduce the efficiency or prevent rescue of vectors, given that rescue from transfected or cotransfected plasmid DNA is inefficient. Consequently, isolation of high expression vectors designed for certain proteins fails. It is difficult to predict which proteins can be driven to high expression, but the problem is common in the expression of glycoproteins and DNA binding proteins or enzymes.

The solution:

The inhibition of protein expression, even if only partial, can significantly improve the rescue efficiency of cassettes containing a cDNA expressed from a promoter-intron combination (DA Matthews et al.; Cummings and Graham, unpublished). In cell lines that express the lac repressor, and expression cassettes that contain the lac operator (repressor binding sequence), transcription from the cassette is inhibited when the vector is rescued or propagated in host cells that express the repressor. Unlike other gene regulation systems, high level expression in normal cells or in vivo does not require a second transactivator.

This “Hi-IQ” system has been combined with the AdMax™ two plasmid vector rescue system for isolation of vectors expressing all but the most toxic proteins at very high levels (see Figure). The system differs from the original AdMax™ in its use of 293 cells expressing lac repressor (293-IQ cells) and shuttle plasmids containing expression cassettes with the MCMV IE gene promoter, an intron, and a lac operator sequence for regulation of expression. The vectors are rescued and propagated in 293-IQ cells and the transgene can then be expressed by transduction of any other cell type where transcription is relieved of repression.

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