Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein

An expression cassette designed for high-level production of rabies virus g lycoprotein (RG) could not be rescued into a replication-defective, adenovi rus-based vector using standard procedures. To overcome this difficulty, a 293-based cell line, designated 293LAP13, was constructed that contained an d expressed a derivative of the lac repressor protein. The lac operator seq uence, to which the repressor binds, was incorporated into an expression ca ssette, containing a promoter and intron, designed for high-level productio n of RG, Insertion of a single operator sequence immediately downstream of the transcription start site and the use of the 293LAP13 cell line allowed recombinant viruses that could not be isolated with 293 cells to be rescued efficiently. The operator-containing virus reached higher titres in 293LAP 13 than in parental 293 cells and also produced plaques more efficiently in 293LAP13 cells. Moreover, in non-complementing human and canine cell lines , adenovirus vectors with a promoter-intron expression cassette expressed R G at much higher levels than vectors lacking the intron, These observations , together with the demonstration that expression of RG by operator-contain ing vectors was repressed markedly in 293LAP13 cells and that this inhibiti on was relieved at least partly by IPTG, suggest that the 293LAP13 cell lin e may be useful for the rescue and propagation of many vectors in which hig h expression of the desired protein prevents vector rescue in 293 cells.