Controlled megakaryocytic gene expression

Comment on Nguyen et al, page 1559

Megakaryocytic-specific gene expression gets a boost from the Escherichia coli tetracycline-resistance operon. Transgenic mice expressing a tetracycline-controlled transactivator in megakaryocytes permit an antibiotic-dependent control of megakaryocyte and platelet protein expression.

Using the E coli tetracycline-resistance operon is a well-established method (Tet system) to precisely control gene expression in transfected heterologous cells. The power and beauty of this technology reside in the exquisite nature of the tetracycline-controlled transactivator (tTA) that supports transcription of any sequence downstream of a tetracycline-responsive element (TRE). In heterologous systems, the tTA element is typically encoded by one plasmid, while a second plasmid contains a gene of interest driven by a TRE promoter.FIG1 

Nguyen and colleagues have applied this technology to develop an animal model for megakaryocyte and platelet protein expression. In the first step, the authors generate transgenic mice expressing the tTA under the control of the platelet factor 4 promoter, a well-characterized megakaryocytic-specific promoter. The authors demonstrate megakaryocytic-specific expression for the tTA. As a second step, independently derived transgenic animals were generated containing a bidirectional TRE promoter and coding sequences for β-galactosidase and Aurora (see figure). Transgenic animals containing the TRE construct bred to animals expressing the megakaryocytic-specific tTA produce offspring with the potential for controlled megakaryocytic gene expression.

The attractive property of the Tet system is the ability to precisely control levels of gene expression. In tissue culture, levels of tetracycline or one of its derivatives such as doxycycline (Dox) lead to gene expression in a dose-dependent manner owing to the interaction of Dox with the tTA (see figure). In the case of the transgenic animals, Nguyen et al demonstrate that Dox supplemented in the animals' drinking water represses the TRE-dependent gene expression. Upon removal of Dox from the drinking water, the TRE promoter is active and expression of both β-galactosidase and Aurora is induced in megakaryocytes and platelets. The authors demonstrate that the in vivo expression is both conditional and reversible.

Animals expressing the tTA provide a framework for future in vivo platelet studies. Any gene of interest placed downstream of a TRE promoter can now be conditionally expressed if it is first established in a transgenic colony. Breeding transgenic animals containing the TRE-dependent gene with animals expressing a megakaryocytic-specific tTA will result in double-transgenic offspring with a heritable mechanism to control gene expression. The reported work introduces the possibility of inducing toxic genes for the in vivo analysis of megakaryocyte and platelet biology, work that was heretofore refractory for platelet studies.

How precisely the in vivo levels of protein expression can be controlled remains to be established. However, the possibility that a TRE promoter can be manipulated much like a rheostat is an exciting one. For now, the authors present a detailed characterization of transgenic animals expressing the tTA element and a proof of principle by inducing β-galactosidase and Aurora protein expression. Indeed, a mouse available for conditionally overexpressing any gene in the megakaryocyte and platelet lineage offers new possibilities for studying the anucleate platelet in a variety of experimental situations. ▪