LinX retroviral expression system

The LinX retroviral expression system created by Greg Hannon¹ at CSHL is an efficient method for retroviral packaging. It produces amphotrophic retrovirus that infects cells from a broad range of mammalian species. This line is derived from human embryonic kidney (HEK 293) cell line. The viral gag, pol and env genes- necessary for particle formation and replication- are stably integrated into the genome of the LinX packaging cell line. The separate introduction and integration of the structural genes minimizes the chances of producing replication-competent virus due to recombination events during cell proliferation^2,3. The retroviral expression vector provides the viral packaging signal (Ψ), the shRNA to the target gene and a puromycin resistance marker. Transfection of the pSM2 self-inactivating retroviral vector into the LinX packaging cell line produces replication-incompetent virus that can efficiently transfer genes into a variety of mammalian cell types.
 

 

Once the packaging cell line is transfected with a retroviral expression vector that contains a packaging signal (1), the viral genomic transcript containing the target gene and selectable marker are packaged into infectious virus within 48–72 hrs (2-4).  Virus produced in this way can infect target cells and transmit target genes; however, it cannot replicate within target cells because the viral structural genes are absent.

Overview of the process of generating packaged virus particles using shRNA and LinX cells, infection of target cells and assaying for mRNA or protein expression (readout).