Interestingly, although an increase in transgene activity in whole islets was detected in response to glucose, the proportion of -cells that express YFP remained unchanged. ex vivo to high glucose (16. 7 mM) or in vivo infusion of glucose for 24 hours increased luciferase activity in islets, whereas the fraction of YFP-positive -cells after glucose infusion was unchanged. Finally, we showed that fluorescence-activated cell sorting of YFP-positive islet cells can be used to enrich for -cells. Overall, this transgenic line will enable for the first time the application of both fluorescence and bioluminescence/luminescence-based approaches for the study of rat -cells. Diabetes research Rabbit Polyclonal to RPS2 has greatly benefited in recent years from the development of transgenic reporter mice expressing either green fluorescent protein (GFP) (1) or Firefly luciferase (2, 3) under the control Istradefylline (KW-6002) of the mouseins1or ratins2promoter. The sensitivity and broad dynamic range of luciferase assays and their in vivo compatibility enable noninvasive optical measurements of functional -cell mass in situ and after islet transplantation (3, 4). GFP reporter mice have been used for monitoring islet transplants (5), -cell sorting for transcriptomics (6), and time-lapse live imaging, intravitally (7) or in explants (8). Due to its larger size and surgical accessibility, the rat has traditionally been used for physiological studies. As an example, we recently developed a model of prolonged glucose and lipid infusions (911) to examine the -cell response to nutrient excess. This model requires both venous and arterial catheterization of the animals and is technically challenging in mice. We reasoned that a transgenic rat coexpressing luciferase and a fluorescent reporter specifically in -cells would be a useful tool Istradefylline (KW-6002) for this and other existing rat models. In this report we describe the development and characterization of a transgenic rat that expresses a Renilla luciferase (RLuc)-enhanced yellow fluorescent protein (YFP) fusion under the control of the ratins2promoter. We show that islet luciferase activity is responsive to glucose providing a rapid measure of -cell functional mass. Furthermore, we show that YFP is restricted to -cells, facilitating their enrichment using fluorescence-activated cell sorting (FACS). == Materials and Methods == == Generation of RIP7-RLuc-YFP transgenic rats == A plasmid containing rat genomic DNA from 9728 to +167 relative to the start site of transcription of theins2gene followed by an Istradefylline (KW-6002) MHC class I polyA sequences in pBluescript was digested withClaI and a polylinker containingAscI andSbfI sites was inserted (designated RIP7-pBS). In parallel, a 1795-bp RLuc-YFP fusion gene was amplified by PCR using primers with overhangingAscI andSbfI restriction sites. Both the RIP7-pBS plasmid and the RLuc-YFP PCR fragment were digested withAscI andSbfI and ligated to generate RIP7-RLuc-YFP-pBS. Transgenic Sprague Dawley rats were produced by pronuclear injection ofSalI linearized RIP7-RLuc-YFP-pBS (Figure 1). PCR analysis of genomic DNA isolated from tail biopsies was used for the identification Istradefylline (KW-6002) of Tg(RIP7-RLuc-YFP) rats. PCR primers are listed inSupplemental Table 1 . All procedures using animals were approved by the Institutional Committee for the Protection of Animals at the Centre de Recherche du Centre Hospitalier de l’Universit de Montral (CRCHUM). To simplify the analyses and reduce variability, only male transgenic and control littermates were used in this study == Figure 1 . == Reporter expression in adult RIP7-RLuc-YFP rats. A, Schematic showing organization of the RIP7-RLuc-YFP transgene. A 9890-bp fragment of the ratins2gene (RIP7) extending from 9728 to +167 relative to start site of transcription was joined to an RLuc-YFP fusion gene followed by a polyA signal. Primer sets used for PCR.