Abstract
Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.
Highlights
In the past, the study of gene expression during normal development or induced by disease in rodent models have been assessed largely by end-point assays involving the sacrifice of large cohorts of animals or restricted to invasive serial blood or urine sampling
Light-producing transgenic (LPT) mice, where a luciferase transgene is inserted in the first intron of an endogenous gene, can be subjected to bioluminescence imaging (BLI) to quantify gene activity in living animals[3]
hypoxia-inducible factor (HIF) response element (HRE) is used as a biological sensor of hypoxia, important in cancer and ischemia models of disease
Summary
The study of gene expression during normal development or induced by disease in rodent models have been assessed largely by end-point assays involving the sacrifice of large cohorts of animals or restricted to invasive serial blood or urine sampling. Light-producing transgenic (LPT) mice, where a luciferase transgene is inserted in the first intron of an endogenous gene, can be subjected to BLI to quantify gene activity in living animals[3]. Genome editing technologies have expedited the generation of whole body luciferase reporter transgenics[4], and have massively increased the usage of LPT rodents, but critically do not reduce animal wastage in transgenic colony formation. The numbers of mice used in biomedical research has increased substantially since CRISPR/Cas[9] genome editing has been applied to the production of transgenic mice with the time taken for mouse models to be created taking months instead of years to generate. It was reported that in 2016, 226,000 animals were used to create new lines of genetically altered animals[5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
