Methodology

High-efficiency Rosa26 knock-in vector construction for Cre-regulated overexpression and RNAi

Peter Hohenstein , Joan Slight , Derya Deniz Ozdemir , Sally F Burn , Rachel Berry and Nicholas D Hastie

MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK

author email corresponding author email

PathoGenetics 2008, 1:3doi:10.1186/1755-8417-1-3

Published:	3 November 2008

Abstract

Introduction

Rosa26 is a genomic mouse locus commonly used to knock-in cDNA constructs for ubiquitous or conditional gene expression in transgenic mice. However, the vectors generally used to generate Rosa26 knock-in constructs show instability problems, which have a severe impact on the efficiency of the system.

Results

We have optimized the cloning procedure to generate targeting vectors for Cre-regulated expression of constructs within several days with minimal hands-on time, thereby enabling high-throughput approaches. We demonstrate that transient expression of Cre still results in expression of the construct, as shown by the expression level and via functional assays. In addition to its well-established possibilities in expressing cDNA constructs, we show that the Rosa26 locus can be used to drive expression of functional miRNA constructs from its endogenous promoter.

Conclusion

We provide a new high-efficiency cloning system for Rosa26 knock-in constructs to express either cDNA or miRNA fragments. Our system will enable high-throughput approaches for controlled expression of cDNA or miRNA constructs, with the latter providing a potential high-speed alternative for conditional knock-out models.