An approach for controlling the timing and order of engineered mutations in mice

Abstract

Significant advances in our understanding of normal development and disease have been facilitated by engineered mice in which genes can be altered in a spatially, temporally, or cell type restricted manner using site specific recombinase systems like Cre-loxP or Flp-frt. In many circumstances it is important to understand how interactions between multiple genes influence a given phenotype. Robust approaches for precisely controlling multiple genetic alterations independently are limited, however, thus the impact of mutation order and timing on phenotype is generally unknown. Here we describe and validate a novel Gt(ROSA)26Sor targeted transgene allowing precise control over the order and timing of multiple genetic mutations in the mouse. The transgene expresses an optimized, Flp-estrogen receptor fusion protein (Flpo-ERT2) under the control of a loxP-stop-loxP cassette. In this system, genes modified by loxP sites are altered first upon expression of Cre. Cre also eliminates the loxP-stop-loxP cassette, permitting widespread expression of Flpo-ERT2. Because of the estrogen receptor fusion, Flp activity remains inert until administration of tamoxifen, allowing genes modified by frt sites to be modified subsequently with controllable timing. This mouse transgene will be useful in a wide variety of applications where independent control of different mutations in the mouse is desirable.

Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network (EVNet) to fuel tumour progression

“…To further confirm the role of CSC EVs in the progression of PDAC, we developed a GEMM that allows the inducible and conditional knockout of Rab27a mediated by flippase recombination ( Rab27a Frt/Frt ). We crossed Rab27a Frt/Frt and R26 LSL-FLPOERT2/+ alleles24 with the KPC. The final model spontaneously develops PDAC ( LSL-Kras G12D/+ , LSL-Tp53 R172H/+ , Pdx-1-Cre , R26 LSL-FLPoERT2/+ , Rab27a Frt/Frt , hereafter referred to as KPC iRab27a Frt/Frt ).…”

Exosomes define a local and systemic communication network in healthy pancreas and pancreatic ductal adenocarcinoma

“…In the second approach, we included an additional Pdx1-Flp allele, which allows recombination of the ExoBow transgene specifically in pancreas cells when Pdx1 promoter is first active 22 . In the third approach, we included an additional R26 LSL-FLPoERT2/ + allele to create the KPC-iExoBow model, which allows tamoxifen-inducible Flp recombination in PDAC 23 (Fig. 1j, k ).…”

ExBoX: a simple Boolean exclusion strategy to drive expression in neurons

“…These activity-modulating tools, although well characterized in a variety of circuits (see Bando et al, 2016; Burrone et al, 2002; Johns et al, 1999; Lin et al, 2010; Okada and Matsuda, 2008; Priya et al, 2018; Sim and Antolin et al, 2013; Sweeney et al, 1995; Xue et al, 2014 for examples), will need to be validated in each particular setting and/or system. To facilitate studies where the timing of CDS expression is critical, we are currently generating AAV vectors driving expression of a tamoxifen-dependent FlpO (data not shown) (Goodrich et al, 2018). This AAV-FlpOERT2 could be delivered at the same time as the ExBoX AAVs and used to turn Off expression by orally providing tamoxifen at a later time point.…”