Development of a transgenic mouse model using rat insulin promoter to drive the expression of CRE recombinase in a tissue-specific manner.

Abstract

Background: Tissue-specific ablation of a gene using the Cre-loxP system has been used as an important tool to define its role, in addition to the total ablation, to avoid the embryonic lethality in case of wide expression of the target gene.

Methods: The RIP-Cre genetic construct was generated by standard subcloning techniques and microinjected into one cell embryo to develop the transgenic mouse line. Transgenic mice were screened by polymerase chain reaction (PCR) using DNA isolated from tell digestion. Tissue specificity of RIP was demonstrated by transient transfection of RIP-1acZ construct to NIT-1 cells (mouse insulinoma cell line) in vitro.

Results: The 448 nucleotides of RIP were sufficient for beta-cell specific expression of the reporter gene as evidenced by the presence of blue color in the nucleus of NIT-1 cells. Isolated RIP-Cre transgene was microinjected, and PCR screening identified two independent lines of transgenic mice. Tissue specificity of RIP was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) using the islet RNA from the transgenic mice.

Conclusion: We have established a tissue-specific transgenic mouse model using Cre recombinase linked to rat insulin promoter (RIP) to drive the expression of the reporter gene specifically in the beta-cells. The RIP-Cre transgenic mice will allow beta-cell specific ablation of target gene(s) to define its role in the regulation of islet physiology.