Once the shRNA-expressing lentiviral transfer plasmids were constructed and verified by sequence analysis, they were used to produce a self-inactivating, infectious lentiviral vector to deliver the shRNA-expressing construct to FEK cell lines.
FEK cells were lifted and centrifuged at 300 x g for 3 min at room temperature, the supernatant discarded, and resuspended in 2 ml of viral media containing polybrene.
19]), as was a non-transgenic FEK control cell line.
Transgenic (Pol2-targeting shRNA) and non-transgenic FEK cells were grown in tissue culture conditions as described above.
From these successful cultures, lentiviral particles were harvested and used to generate transgenic FEK cell lines.
Prior to constructing transgenic cells, a primary FEK cell line was established from a male horse fetus.
for transgenic (labels indicate shRNA) and non-transgenic (Non-Tg) FEK cell lines.
The goal of this investigation was to render naive FEK cells resistant to infection by [EIAV.
During the investigation, we attempted to elucidate the mechanisms responsible for resistance to infection in transgenic FEK cell lines.
The naive FEK cells were not resistant to lentiviral infection in general, as evidenced by our ability to transduce them with an HIV-based lentiviral vector.