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Beta cell-specific ablation of target gene using Cre-loxP system in transgenic mice
M K Ray1, S P Fagan, S Moldovan
1Department of Surgery, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.
Abstract:
Tissue-specific inactivation of a gene using the Cre-loxP system has been used as an important tool to define its role in which the inactivation of the gene in every cell type results in an embryonic lethality. The expression of Cre recombinase (Cre) can be regulated by controlling the timing or spatial distribution of Cre expression via tissue-specific promoters, ligand-inducible promoters, and ligand-dependent Cre fusion proteins. The rat insulin promoter (RIP) has been used in this study to drive the expression of Cre, specifically in the beta cells. The Cre coding sequence was ligated with the RIP and the isolated RIP-Cre transgene was microinjected into one cell embryo to establish a transgenic mouse line. Tissue specificity of the rat insulin promoter was demonstrated by reverse transcriptase polymerase chain reaction using total RNA from pancreas and other tissues of the RIP-Cre transgenic mice. In addition, the efficiency and specificity of RIP was further analyzed by crossbreeding the RIP-Cre transgenic mice with reporter mice bearing a beta-actin-loxP-CAT-loxP-lacZ transgene. In these mice, lacZ is expressed only after excision of the floxed-CAT gene by Cre-mediated recombination. Here, we present the data for beta cell-specific expression of lacZ in the bigenic mice, as proof of concept in a mouse model for targeting beta cell-specific gene(s). The RIP-Cre transgenic mice will be used as a potential tool for targeting the excision of beta cell-specific gene(s) to study their role in islet cell physiology.
Insights
Researchers developed a new transgenic mouse model using the rat insulin promoter (RIP) to control Cre recombinase (Cre) expression specifically in beta cells. This tool enables targeted gene inactivation in these cells to study their function in islet cell physiology.
Area of Science:
- * Molecular Biology
- * Genetics
- * Endocrinology
Background:
- * The Cre-loxP system is crucial for conditional gene inactivation, essential for studying genes causing embryonic lethality when globally knocked out.
- * Controlling Cre recombinase (Cre) expression spatially and temporally is key for precise gene targeting.
- * Tissue-specific promoters, ligand-inducible systems, and fusion proteins offer methods to regulate Cre activity.
Purpose of the Study:
- * To establish a transgenic mouse line for beta cell-specific gene targeting using the Cre-loxP system.
- * To validate the tissue specificity and efficiency of the rat insulin promoter (RIP) in driving Cre expression in pancreatic beta cells.
- * To demonstrate the utility of RIP-Cre transgenic mice for studying beta cell-specific gene functions.
Main Methods:
- * Construction of a RIP-Cre transgene by ligating the Cre coding sequence with the rat insulin promoter.
- * Microinjection of the RIP-Cre transgene into one-cell embryos to generate transgenic mice.
- * Validation of RIP-Cre expression specificity using reverse transcriptase polymerase chain reaction (RT-PCR) on various tissues.
- * Crossbreeding RIP-Cre mice with reporter mice (beta-actin-loxP-CAT-loxP-lacZ) to assess Cre-mediated recombination efficiency and specificity.
Main Results:
- * Successful establishment of RIP-Cre transgenic mice exhibiting Cre expression driven by the rat insulin promoter.
- * RT-PCR confirmed the tissue-specific expression of the RIP-Cre transgene, primarily in the pancreas.
- * Reporter gene (lacZ) expression was observed specifically in beta cells of bigenic mice, confirming Cre-mediated recombination in these cells.
- * The study demonstrated proof of concept for a mouse model enabling targeted gene manipulation in beta cells.
Conclusions:
- * The RIP-Cre transgenic mouse line provides a reliable tool for achieving beta cell-specific gene inactivation.
- * This model facilitates the study of gene functions critical to islet cell physiology and the understanding of diabetes.
- * RIP-Cre mice represent a significant advancement for targeted research in beta cell biology and related diseases.