Recombinant baculoviruses used to study estrogen receptor function in human osteosarcoma cells

Area of Science:

• Molecular Biology
• Cell Biology
• Virology

Background:

• Mammalian cell-based assays are crucial for drug discovery and understanding gene regulation.
• Efficient gene delivery into mammalian cells is essential for creating these assays.
• Nuclear receptors, like the human estrogen receptor (ER), play vital roles in cellular processes and disease.

Purpose of the Study:

• To evaluate the efficacy of modified baculoviruses (BacMam) for delivering multiple genes into mammalian cells.
• To develop a heterologous transcription factor/reporter gene system using BacMam for assay development.
• To apply this system for profiling the activity of nuclear receptors, specifically the human estrogen receptor (ER).

Main Methods:

• Construction of BacMam viruses encoding human ER subtypes (ERalpha, ERbeta) and an ER-responsive reporter gene.
• Transduction of Saos-2 human osteosarcoma cells with BacMam viruses.
• Assaying the effects of 17beta-estradiol and raloxifene on ER activity.
• Comparison of BacMam-mediated gene transfer with standard DNA transfection methods.

Main Results:

• BacMam viruses efficiently delivered multiple genes, enabling the creation of a functional ER transcription factor/reporter system.
• The BacMam-based system successfully profiled the effects of estrogenic compounds (17beta-estradiol, raloxifene) in Saos-2 cells.
• Assay data from the BacMam system were functionally equivalent to standard DNA transfection methods, yielding comparable EC(50) and IC(50) values.

Conclusions:

• BacMam-mediated gene transfer is an efficient and novel method for delivering nuclear receptors and associated genes into mammalian cells.
• This approach facilitates the development of robust mammalian cell-based assays for studying nuclear receptor function and screening compounds.
• The BacMam system offers a viable alternative to traditional transfection methods for assay development.