Efficient tissue-type specific expression of target genes in a tetracycline-controlled manner from the ubiquitously active Eef1a1 locus

Kazuhito Sakamoto,Hridaya Shrestha,Apostolos Klinakis,Vincenzo Coppola,Argiris Efstratiadis,Barbara L Wehde,Rosa-Maria Ferraiuolo,Foued Amari,Aleata A Triplett,Kay-Uwe Wagner,Patrick D Rädler

doi:10.1038/s41598-019-57052-z

Abstract

Using an efficient gene targeting approach, we developed a novel mouse line that expresses the tetracycline-controlled transactivator (tTA) from the constitutively active Eef1a1 locus in a Cre recombinase-inducible manner. The temporally and spatially controlled expression of the EF1-LSL-tTA knockin and activation of tTA-driven responder transgenes was tested using four transgenic lines that express Cre under tissue-specific promoters of the pancreas, mammary gland and other secretory tissues, as well as an interferon-inducible promoter. In all models, the endogenous Eef1a1 promoter facilitated a cell-type-specific activation of target genes at high levels without exogenous enhancer elements. The applicability of the EF1-LSL-tTA strain for biological experiments was tested in two studies related to mammary gland development and tumorigenesis. First, we validated the crucial role of active STAT5 as a survival factor for functionally differentiated epithelial cells by expressing a hyperactive STAT5 mutant in the mammary gland during postlactational remodeling. In a second experiment, we assessed the ability of the EF1-tTA to initiate tumor formation through upregulation of mutant KRAS. The collective results show that the EF1-LSL-tTA knockin line is a versatile genetic tool that can be applied to constitutively express transgenes in specific cell types to examine their biological functions at defined developmental stages.

Highlights

Using an efficient gene targeting approach, we developed a novel mouse line that expresses the tetracycline-controlled transactivator from the constitutively active eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) locus in a Cre recombinase-inducible manner
To target transgenes exclusively to the mammary gland in a ligand-inducible manner, our team placed the reverse transactivator (rtTA) under control of the endogenous whey acidic protein (Wap) gene[8], and we developed new mammary tumor virus (MMTV)-tTA lines that permit a targeted expression of genes to the epithelium of the prenatal and postnatal mammary gland[9]
To generate EF1-LSL-tTA knockin mice, a conventional homologous recombination approach in mouse embryonic stem (ES) cells was used for the targeted insertion of the tetracycline-controlled transactivator into the endogenous eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) locus

Summary

Introduction

Using an efficient gene targeting approach, we developed a novel mouse line that expresses the tetracycline-controlled transactivator (tTA) from the constitutively active Eef1a1 locus in a Cre recombinase-inducible manner. Soon after Furth and coworkers[3] established the binary tet-controlled expression system as a genetic tool in mice, they developed tTA transgenic lines under regulation of the long terminal repeat (LTR) of the mouse mammary tumor virus (MMTV) to target the expression of oncogenes to the mammary gland and other secretory tissues[4] They were first to apply this model to study divergent roles of the SV40 large T (TAg) oncoprotein in cancer initiation and progression[5]. Despite extensive Cre recombination in selected tissues such as the skin in the MMTV-Cre-based model, none of the females exhibited an expression of the TetO-driven target genes in the mammary gland at levels that were comparable to those observed in crosses with the Wap-rtTA or MMTV-tTA lines (unpublished) In another attempt, we generated embryonic stem (ES) cell-based transgenic lines that express the tTA under the CMV-enhanced chicken β-actin (CAG) promoter in a Cre-inducible manner[13]. The superiority of the constitutively active Eef1a1 locus to drive the expression of oncogenes and to examine their biological significance in diverse cellular subtypes, including their progressively transforming descendants that undergo de- or trans-differentiation, is discussed

Methods

Results

Conclusion