Increased Specific Labeling of INS-1 Pancreatic Beta-Cell by Using RIP-Driven Cre Mutants with Reduced Activity

Abstract

Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P) in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P) was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.

The use of mice in diabetes research: The impact of physiological characteristics, choice of model and husbandry practices

“…The amount of ectopic expression should be measured and the functional significance of ectopic gene manipulation in a specific tissue or cell population, as well as its influence on the target tissue should be investigated and controlled for through Cre‐positive/ loxP ‐negative controls. Attempts to improve tissue specificity can have a negative impact on recombination efficiency or decrease the population of target cells able to maintain transgenic expression 40 . Levels of gene knock‐in or knockout should, therefore, be measured, preferably in a way that can measure cell heterogeneity of expression prior to interpretation of results.…”

“…Attempts to improve tissue specificity can have a negative impact on recombination efficiency or decrease the population of target cells able to maintain transgenic expression. 40 Levels of gene knock-in or knockout should, therefore, be measured, preferably in a way that can measure cell heterogeneity of expression prior to interpretation of results.…”

Membrane tethering of CreER decreases uninduced cell labeling and cytotoxicity while maintaining recombination efficiency

“…Finally, Cre driver lines with RIP promoter frequently showed non-specific labeling of cells other than islet beta-cells, especially under pathological conditions. 34 , 35 We interrogated this problem by expressing mCreER in Ad293 cells ( Figure 6 G), which are not insulin-producing cells. As expected, transduction with Lenti-RIP-CreER led to 12.4%–24.8% of cell labeling at 24–48 h after induction ( Figure 6 H).…”