Inducible Gene Expression of GFP Reporter Gene in LMH Cell Line Using Inducible Lentivirus Vectors

Aim: By combining Tet-inducible system and lentivirus vectors, we investigated the induction of Enhanced Green Fluorescent Protein (EGFP) reporter gene in poultry cells.
Material and the EGFP gene was placed under Tet-ON system and its induction was studied in liver cell line LMH. First, we transformed competent bacteria separately with an inducible lentivirus vector carrying EGFP and a second vector carrying Tet transactivator rtTA-M2 and prepared a purified maxi-prep stock of either plasmid DNA. Then, we used DNA-calcium phosphate co-precipitation method to co-transfect LMH cells with both plasmid stocks. In the next step, we added different concentrations of Tet analog doxycycline (DOX) to cell growth medium.
Increase of DOX concentration caused elevation of gene expression Within the first 24 hours after post-transfection, the expression of EGFP for 0, 0.01, 0.1 and 1 µg/ml of DOX was 0.4, 6.2, 10.3 and 16% respectively, and in the next 24 hours the expression changed to 6.4, 26, 28.3 and 29.7% respectively. However, treatment with the overdose of DOX caused the cell death.
Combination of Tet-inducible system originating from bacteria and recombinant lentivirus vectors designed for gene transfer to human cells can jointly promote transgene expression in poultry cells. The concentration of the inducer can directly influence the level of transgene induction.