Conditional Overexpression and Knockout of miR-214 in Mice

Cre-inducible miR-214 expression construct was generated by cloning a miR-214 expression cassette downstream of a CAGGS promoter and a LoxP-flanked transcription STOP element. This construct was targeted into the mouse Collagen A1 locus using a Flippase (FLP) recombinase-mediated genomic integration. mouse Embryonic Stem cells (mESCs) carrying a single copy of the miR-214^STOP construct were identified by resistance to the antibiotic marker hygromycin and Southern blotting. Selected clones were injected into blastocysts to generate pups. To obtain total body overexpressing miR-214^over, miR-214^STOP mice were bred to a Balancer-Cre transgenic strain [16 (link)], kindly provided by E. Hirsch. To generate PyMT miR-214^over transgenic mice, miR-214^over mice were crossed with Mouse Mammary Tumor Virus Polyoma Middle T antigen MMTV-PyMT transgenic mice [17 (link)], kindly provided by F. Cavallo’s laboratory, University of Torino, Italy. miR-214^ko mice [18 (link)] were kindly provided by Eric Olson’s laboratory, UT Southwestern Medical Center, Dallas, USA. The sources of primers used for genotyping are available upon request. All experiments performed with live animals complied with ethical animal care and were approved by the MBC Animal Care Committee and the Italian Ministry of Health (13/2014-PR to DT; 847/2020-PR to DT).

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Orso F., Virga F., Dettori D., Dalmasso A., Paradzik M., Savino A., Pomatto M.A., Quirico L., Cucinelli S., Coco M., Mareschi K., Fagioli F., Salmena L., Camussi G., Provero P., Poli V., Mazzone M., Pandolfi P.P, & Taverna D. (2023). Stroma-derived miR-214 coordinates tumor dissemination. Journal of Experimental & Clinical Cancer Research : CR, 42, 20.

Publication 2023

A 214 Animal Animal care committee Antibiotic Blastocysts Body Clones Collagen Genomic Hygromycin Mice Mmtv mouse mammary tumor virus Mouse embryonic stem cells Polyoma Primers Recombinase Strain T antigen Transcription Transgenic Transgenic mice

Corresponding Organization : Renown Health

Other organizations : Università degli Studi del Piemonte Orientale “Amedeo Avogadro”, VIB-KU Leuven Center for Cancer Biology, Spanish National Centre for Cardiovascular Research, Princess Margaret Cancer Centre, University Health Network, Istituti di Ricovero e Cura a Carattere Scientifico

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Variable analysis

independent variables

Generation of miR-214 overexpressing (miR-214^over) transgenic mice
Generation of miR-214 knockout (miR-214^ko) mice

dependent variables

Expression levels of miR-214
Phenotypic outcomes of miR-214 overexpression or knockout

control variables

Collagen A1 locus used for targeted integration of miR-214 expression construct
Selection of mESC clones carrying a single copy of the miR-214^STOP construct using hygromycin resistance and Southern blotting
Balancer-Cre transgenic strain used to obtain total body overexpression of miR-214
MMTV-PyMT transgenic mice used to generate PyMT miR-214^over transgenic mice

controls

Positive control: Balancer-Cre transgenic strain used to obtain total body overexpression of miR-214
Negative control: miR-214^ko mice

Annotations

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