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Over the past decades, tremendous efforts have been made to establish pancreatic islet transplantation as a standard therapy for type 1 diabetes. Recent advances in islet transplantation have resulted in steady improvements in the 5-year insulin independence rates for diabetic patients. Here we review the key challenges encountered in the islet transplantation field which include islet source limitation, sub-optimal engraftment of islets, lack of oxygen and blood supply for transplanted islets, and immune rejection of islets. Additionally, we discuss possible solutions for these challenges. [ABSTRACT FROM AUTHOR]

Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid. Materials and Methods: In this experimental study, single homology arm donor was applied along with a single guide RNA (sgRNA) specific to the homology region, and either Cas9 or its mutant nickase variant (Cas9n). Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells (ESCs). Results: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus. Conclusion: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies. [ABSTRACT FROM AUTHOR]

Introduction: Measurement of pancreatic beta cell mass in animal models is a common assay in diabetes researches. Novel whole-organ clearance methods in conjunction with transgenic mouse models hold tremendous promise to improve beta cell mass measurement methods. Here, we proposed a refined method to estimate the beta cell mass using a new transgenic Tg( Pdx1-GFP ) mouse model and a recently developed free-of-acrylamide clearing tissue (FACT) protocol. Methods: First, we generated and evaluated a Tg( Pdx1-GFP ) transgenic mouse model. Using the FACT protocol in our model, we could quantify the beta cell mass and alloxan-induced beta cell destruction in whole pancreas specimens. Results: Compiled fluorescent images of pancreas resulted in enhanced beta cell mass characterization in FACT-cleared sections (2928869±120215 AU) compared to No-FACT cleared sections (1292372±325632 AU). Additionally, the total number of detected islets with this method was significantly higher than the other clearance methods (155.7 and 109, respectively). Using this method, we showed green fluorescent protein (GFP) expression confined to beta cells in Tg( Pdx1-GFP ) transgenic. This enhanced GFP expression enabled us to accurately measure beta cell loss in a beta cell destruction model. The results suggest that our proposed method can be used as a simple, and rapid assay for beta cell mass measurement in islet biology and diabetes studies. Conclusion: The Tg( Pdx1-GFP ) transgenic mouse in conjunction with the FACT protocol can enhance large-scale screening studies in the field of diabetes., (© 2022 The Author(s).)

Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid., Materials and Methods: In this experimental study, single homology arm donor was applied along with a single guide RNA (sgRNA) specific to the homology region, and either Cas9 or its mutant nickase variant (Cas9n). Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells (ESCs)., Results: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1 GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus., Conclusion: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies.