Summary auto-generated
This study developed a tetracycline-inducible expression system for the mouse prion protein (PrP) in murine N2a neuroblastoma cells. Researchers performed two rounds of stable transfection: first introducing either a tetracycline-controlled transactivator (tTA) or reverse transactivator (rtTA), then introducing the prion protein gene (Prnp) under control of a tetracycline-responsive promoter. The reverse transactivator system (rtTA) proved superior, allowing tight control of Prnp expression with minimal background activity when uninduced and robust expression upon induction with doxycycline. Quantitative analysis showed approximately fourfold increases in PrP expression upon induction, with expression levels comparable to transgenic mice overexpressing PrP. The system demonstrated dose-dependent regulation, with detectable increases at 5 ng/ml doxycycline and saturation around 50-150 ng/ml. Expression kinetics showed induction beginning 3-5 hours after doxycycline addition and reaching plateau at 20 hours. Critically, the system proved fully reversible, with expression returning to basal levels within four days of doxycycline withdrawal. These inducible N2a cell clones provide valuable tools for studying PrP cellular function and prion disease mechanisms.
Key findings
- A tetracycline-inducible expression system using reverse transactivator (rtTA) enables tight, dose-dependent control of prion protein expression in N2a neuroblastoma cells with minimal background activity
- Induced PrP expression reaches approximately fourfold higher levels than endogenous levels and is comparable to levels in transgenic mice overexpressing PrP
- The system is fully reversible, with expression returning to basal levels within 96 hours of removing doxycycline, allowing on-off switching of PrP production
- Expression is quantitatively controllable across doxycycline concentrations from 1-50 ng/ml, with induction kinetics showing increases detectable within 3-5 hours and plateau by 20 hours
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Abstract
A tetracycline-inducible expression system has been established for the prion protein (PrP) in murine neuroblastoma cells (N2a). For this purpose, N2a cells were first stably transfected with either the tetracycline-controlled transactivator or the reverse transactivator. After selection of N2a clones which carried one of these transactivators, the murine PrP gene (Prnp) was introduced under the control of the transactivator-responsive promoter in a second round of stable transfection. Stably double-transfected N2a clones carrying the reverse type but not the normal transactivator were found to be fully inducible, giving a low background of Prnp expression before induction and high expression after induction. Stably double-transfected N2a cells were at least as productive as N2a cells over-expressing Prnp permanently under the control of a strong viral promoter. Furthermore, the selected N2a clones allowed the Prnp expression level to be quantitatively controlled by varying the level of the effector substance, the tetracycline-derivative doxycycline. The clones were fully controllable, as over-expression could be switched on and off as desired. These N2a clones may become an important tool for elucidation of the cellular function of PrP and may pave the way for the tetracycline-inducible expression of many genes in this neuroblastoma cell line.