Transient expression of two luciferase reporter gene constructs in developing embryos of Macrobrachium lanchesteri (De Man)

Lipoprotein lipase, an enzyme of central importance to lipid metabolism, is most abundant in adipose tissues, cardiac and skeletal muscle, and portions of the brain. The current work examined the murine lipoprotein lipase promoter using transient transfection, gel-retention analyses, and transgenic mice. Maximum expression of the luciferase reporter gene in transfected cells was observed with -101 bp of the promoter. Nuclear extracts from tissues expressing lipoprotein lipase contained DNA binding proteins that recognize the CCAAT box (-64 bp) and an octamer motif (-46 bp); this combination of factors was absent in nonexpressing tissues. Transgenic mice from three of five founders prepared with -1,824-bp promoter constructs expressed the luciferase reporter gene at highest levels in brown adipose tissue and brain. These findings suggest that the -1,824-bp promoter region contains sequence elements responsible for the tissue-specific transcription of lipoprotein lipase in vivo.

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Non-invasive somatotransgenic bioimaging in living animals

F1000Research ◽

10.12688/f1000research.25274.1 ◽

2020 ◽

Vol 9 ◽

pp. 1216 ◽

Cited By ~ 1

Author(s):

Juliette M. Delhove ◽

Rajvinder Karda ◽

Lorna M. FitzPatrick ◽

Suzanne M.K. Buckley ◽

Simon N. Waddington ◽

...

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Viral Vectors ◽

Es Cells ◽

Embryonic Stem ◽

Whole Body ◽

Luciferase Reporter ◽

Gene Promoters ◽

Luciferase Reporter Gene ◽

Endogenous Gene

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.

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