Recombinant baculoviruses used to study estrogen receptor function in human osteosarcoma cells.
We report that modified baculoviruses, termed BacMam viruses, can efficiently deliver multiple genes into mammalian cells to generate a heterologous transcription factor/reporter gene system. Using human estrogen receptor (ER) as a model nuclear receptor, we demonstrate how this approach can be successfully applied to assay development in Saos-2 human osteosarcoma cells. BacMam viruses containing full-length cDNAs were constructed for both human ER subtypes, ERalpha and ERbeta, and a third BacMam virus containing an ER-responsive reporter gene cassette. Using these viruses, we found that BacMam-ER expression/reporter constructs could be used to profile the effects of the agonist 17beta-estradiol and the partial agonist raloxifene in human Saos-2 cells. A comparison of assay data obtained with the BacMam-based system with that using standard DNA transfections demonstrates that the two systems are functionally equivalent, giving comparable EC(50) and IC(50) values for estrogen and estrogen plus raloxifene treatments, respectively. Our results indicate that BacMam-mediated gene transfer offers a novel and efficient method for delivery of nuclear receptors and associated genes for mammalian cell-based assay development.
Clay
William C
WC
Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline Discovery Research, Research Triangle Park, NC 27709-3398, USA. william.c.clay@gsk.com
Condreay
J Patrick
JP
Moore
Linda B
LB
Weaver
Susan L
SL
Watson
Michael A
MA
Kost
Thomas A
TA
Lorenz
Jennifer J
JJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001859:Bone Neoplasms / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D012516:Osteosarcoma / Q000235:genetics* / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000502:physiology*; D014161:Transduction, Genetic; D014162:Transfection / Q000379:methods*; D014982:Xenopus laevis
D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins