Generation of GFP-Tagged Recombinant Respiratory Syncytial Virus Using a T7 Expression System

Begin with a plasmid mixture containing helper plasmids encoding RSV replication and transcription proteins and a separate plasmid carrying a full-length, GFP-expressing RSV antigenome cDNA.

Each plasmid is placed under the control of a T7 promoter.

Add a liposome-based transfection reagent and incubate to allow complex formation.

Introduce these complexes into genetically engineered hamster kidney cells that express T7 RNA polymerase and incubate.

The plasmid complexes enter the cells, where T7 RNA polymerase transcribes the RSV plasmids.

This produces viral mRNAs encoding replication and transcription proteins and generates positive-sense RSV antigenomic RNA.

Using host machinery, these mRNAs produce viral replication and transcription proteins that form a functional RSV polymerase complex.

The RSV polymerase complex then converts the positive-sense RNA into viral genomic RNA.

This genomic RNA directs the synthesis of viral mRNAs, which encode structural proteins and GFP.

Under a fluorescence microscope, green fluorescence confirms viral gene expression and the initiation of recombinant RSV production.

The day before transfection, suspend BSR-T7/5 cells in complete medium at 0.5 million ce