In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters.

The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4(-/-) mice or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter constructs in somatotransgenic mice, respectively

Luciferase expression allows bioluminescence imaging but imposes limitations on the orthotopic mouse (4T1) model of breast cancer

Funding Information: Experiments on the 4T1 and 4Tluc2D6 mouse models of breast cancer were supported by the Russian Scientific Foundation, grant 14-14-00882. MRI measurements were carried out on ClinScan 7T located at Center for Collective Usage (CKP) “Medical nanobiotechologies”, located in Russian National Research Medical University. Experiments on the optimization of protocols for DNA immunization were supported by the Russian Scientific Foundation grant 15-15-30039. Optimization of tumor challenge after DNA immunization was supported by the Russian Fund for Basic Research grant 17-04-00583. Participants were trained in the immunization and tumor challenge experiments in the frame of the European Union Twinning project VACTRAIN, grant agreement #692293, and Swedish Institute PI project 19806/2016. Maria Isaguliants and Stefan Petkov were supported by VACTRAIN, and Maria Isaguliants, also by BALTINFECT, grant agreement #316275. Athina Kilpeläinen was supported by the individual study grant of the Swedish Institute #19061/2014. Patrik Hort is gratefully acknowledged for the language editing. Natalia Belikova is gratefully acknowledged for help with the quantification of protein expression based on the exponential calibration curves. Publisher Copyright: © 2017 Nature Publishing Group. All rights reserved.Implantation of reporter-labeled tumor cells in an immunocompetent host involves a risk of their immune elimination. We have studied this effect in a mouse model of breast cancer after the orthotopic implantation of mammary gland adenocarcinoma 4T1 cells genetically labelled with luciferase (Luc). Mice were implanted with 4T1 cells and two derivative Luc-expressing clones 4T1luc2 and 4T1luc2D6 exhibiting equal in vitro growth rates. In vivo, the daughter 4T1luc2 clone exhibited nearly the same, and 4T1luc2D6, a lower growth rate than the parental cells. The metastatic potential of 4T1 variants was assessed by magnetic resonance, bioluminescent imaging, micro-computed tomography, and densitometry which detected 100-μm metastases in multiple organs and bones at the early stage of their development. After 3-4 weeks, 4T1 generated 11.4 ? 2.1, 4T1luc2D6, 4.5 ? 0.6; and 4T1luc2, 〈1 metastases per mouse, locations restricted to lungs and regional lymph nodes. Mice bearing Luc-expressing tumors developed IFN-? Response to the dominant CTL epitope of Luc. Induced by intradermal DNA-immunization, such response protected mice from the establishment of 4T1luc2-tumors. Our data show that natural or induced cellular response against the reporter restricts growth and metastatic activity of the reporter-labelled tumor cells. Such cells represent a powerful instrument for improving immunization technique for cancer vaccine applications.publishersversionPeer reviewe

Identification of Rtl1, a Retrotransposon-Derived Imprinted Gene, as a Novel Driver of Hepatocarcinogenesis

2012-2013 > Academic research: refereed > Publication in refereed journalVersion of RecordPublishe