Dual recombinase action in the normal and neoplastic mammary gland epithelium

Patrick D Rädler,Yong Li,Rayane Dennaoui,Aleata A Triplett,Rosa-Maria Ferraiuolo,Dieter Saur,Kay-Uwe Wagner,Kerry Vistisen,Amalraj Thangasamy,Hridaya Shrestha

doi:10.1038/s41598-021-00231-8

Abstract

We developed a transgenic mouse line that expresses the codon-optimized Flp recombinase under the control of the MMTV promoter in luminal epithelial cells of the mammary gland. In this report, we demonstrate the versatile applicability of the new MMTV-Flp strain to manipulate genes in a temporally and spatially controlled manner in the normal mammary gland, in luminal-type mammary tumors that overexpress ERBB2, and in a new KRAS-associated mammary cancer model. Although the MMTV-Flp is expressed in a mosaic pattern in the luminal epithelium, the Flp-mediated activation of a mutant KrasG12D allele resulted in basal-like mammary tumors that progressively acquired mesenchymal features. Besides its applicability as a tool for gene activation and cell lineage tracing to validate the cellular origin of primary and metastatic tumor cells, we employed the MMTV-Flp transgene together with the tamoxifen-inducible Cre recombinase to demonstrate that the combinatorial action of both recombinases can be used to delete or to activate genes in established tumors. In a proof-of-principle experiment, we conditionally deleted the JAK1 tyrosine kinase in KRAS-transformed mammary cancer cells using the dual recombinase approach and found that lack of JAK1 was sufficient to block the constitutive activation of STAT3. The collective results from the various lines of investigation showed that it is, in principle, feasible to manipulate genes in a ligand-controlled manner in neoplastic mammary epithelial cells, even when cancer cells acquire a state of cellular plasticity that may no longer support the expression of the MMTV-Flp transgene.

Highlights

Metastatic progression[23,24,25]
An optimal expression of transgenes under the control of the extended MMTV promoter is dependent on the FVB/N strain background[2], and the Jak1fl allele was the only suitable Flp reporter strain in our colony at that time
It should be noted that in Jak1fl/wt females from intermediary crosses with the MMTV-Flp, the PCR assay resulted in the co-amplification of a smaller fragment from the Jak[1] wildtype allele (Jak1wt) that frequently obscured the specific PCR band that resulted from the Flp-mediated excision of the Pgk-neomycin cassette (Jak1fl-neo)

Summary

Introduction

Metastatic progression[23,24,25] This genetic tool makes it possible to predict the biological relevance of candidate target genes for breast cancer prevention and treatment. To understand the genetic cues that drive normal epithelial cell differentiation or the etiology of breast cancer, it is necessary to accurately model consecutive genetic events in the order they may occur in vivo. It is, challenging to develop animal models that permit a genetic modification of two or more endogenous loci at different stages of the developmental process or during neoplastic progression. We report the generation of a novel transgenic strain that expresses the codon-optimized Flp recombinase under the control of the mouse mammary tumor virus long terminal repeat (MMTV-Flp) and how it can be applied in combination with Cre recombinase to delete and activate genes in the desired order in normal and neoplastic epithelial cells of the mammary gland

Methods

Results

Conclusion