The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes.

Abstract

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

Localization of an Insulin-like Growth Factor (IGF) Binding Site of Bovine IGF Binding Protein-2 Using Disulfide Mapping and Deletion Mutation Analysis of the C-terminal Domain

“…This introduced a PstI site at the 5Ј end and cDNA encoding the amino acids MQPRLGGPALLLLP of the signal peptide as described by Bourner et al (19). The full-length PstI/EcoRI bIGFBP-2 cDNA fragment was introduced into the pXMT-2 expression vector (20), kindly provided by P. D. Rathjen (Department of Biochemistry, University of Adelaide) resulting in the plasmid pGF-8. Four C-terminal deletion mutants were created by polymerase chain reaction (PCR) mutagenesis as described by Clackson et al (21) using pGF-8 template.…”

gp130-mediated Signal Transduction in Embryonic Stem Cells Involves Activation of Jak and Ras/Mitogen-activated Protein Kinase Pathways

“…The inducible Jak1 antisense construct was obtained by cloning a 2.7-kilobase BglII fragment, derived from pCDHJ5 and encompassing the site of translational initiation of a human Jak1 cDNA (24) in the antisense orientation into the BglII site immediately downstream of the human 6 -16 promoter in the expression plasmid p1OX (25). Expression construct were co-transfected with the resistance marker plasmids PGKneo (26) or PGKhprt (10).…”

An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives

“…As opposed to existing systems (6,8,10) that are designed and tested for inducible expression restricted to undifferentiated ES cells only, we show that inducible expression with GTEV49 ES cells can be achieved both in undifferentiated ES cells and their ectodermal, mesodermal, and endodermal differentiated derivatives in midgestation embryos. Because these embryos are chimeric, we could not determine precisely the fraction of ES-derived cells that actually activates the expression of hAP after injection of 4ЈOHT to foster mothers.…”

“…Whyatt et al (6) developed an interferon-based inducible expression system to drive conditional expression of the chloramphenicol acetyltransferase (CAT) gene. Expression of the reporter gene was shown to be controlled tightly.…”