CREATION OF AN INFECTIOUS RECOMBINANT SENDAI VIRUS EXPRESSING THE FIREFLY LUCIFERASE GENE FROM THE 3'-PROXIMAL FIRST LOCUS

A genetic engineering approach was made to generate a recombinant non- segmented negative-strand RNA virus, Sendai virus (SeV) of the family Paramyxoviridae, that expresses firefly luciferase. The DNA construct containing the entire open reading frame (ORF) of the luciferase gene followed by the SeV transcription stop and restart signals connected w ith the conserved intergenic three nucleotides was inserted immediatel y before the ORF of the viral 3'-proximal nucleocapsid (N) protein gen e in a full-length SeV cDNA copy. After intracellular expression of fu ll-length antigenomic transcripts from the engineered cDNA and of the viral nucleocapsid protein and RNA polymerase from the respective plas mids, a recombinant SeV expressing luciferase activity at a high level was recovered, although the tendency of this particular reporter gene product to aggregate in cells made it difficult to estimate the maxim um level of expression. The increase in genome length brought about by inserting 1728 nucleotides into the 15384 nucleotide parental SeV was associated with reduced plaque size, slightly slower replication kine tics and a severalfold decrease in yield of the virus. The inserted lu ciferase gene was stably maintained after numerous rounds of replicati on by serial passages in chick embryos. These results indicate the pot ential utility of SeV as a novel expression vector.