Generation and characterization of a Tet-On (rtTA-M2) transgenic rat

Meena Sukhwani,Junming Yue,Yi Sheng,Chih-Cheng Lin,Jennifer J Shuttleworth,Kyle E Orwig,Tianjiao Chu

doi:10.1186/1471-213x-10-17

Abstract

BackgroundThe tetracycline-inducible gene regulation system is a powerful tool that allows temporal and dose-dependent regulation of target transgene expression in vitro and in vivo. Several tetracycline-inducible transgenic mouse models have been described with ubiquitous or tissue-specific expression of tetracycline-transactivator (tTA), reverse tetracycline-transactivator (rtTA) or Tet repressor (TetR). Here we describe a Tet-On transgenic rat that ubiquitously expresses rtTA-M2 driven by the murine ROSA 26 promoter.ResultsThe homozygous rat line (ROSA-rtTA-M2) generated by lentiviral vector injection, has a single integration site and was derived from the offspring of a genetic mosaic founder with multiple transgene integrations. The rtTA-M2 transgene integrated into an intron of a putative gene on chromosome 2 and does not appear to affect the tissue-specificity or expression of that gene. Fibroblasts from the ROSA-rtTA-M2 rats were transduced with a TetO7/CMV-EGFP lentivirus and exhibited doxycycline dose-dependent expression of the EGFP reporter transgene, in vitro. In addition, doxycycline-inducible EGFP expression was observed, in vivo, when the TetO7/CMV-EGFP lentivirus was injected into testis, kidney and muscle tissues of ROSA-rtTA-M2 rats.ConclusionsThis conditional expression rat model may have application for transgenic overexpression or knockdown studies of gene function in development, disease and gene therapy.

Highlights

The tetracycline-inducible gene regulation system is a powerful tool that allows temporal and dosedependent regulation of target transgene expression in vitro and in vivo
Pronuclear injection is generally considered less efficient in rats [11], germline competent embryonic stem (ES) cells were only recently established for this species [12,13] and there are no reports of gene targeting in ES cells to produce knockout rats
These transferred embryos resulted in 35 live progeny and Reverse transcription (RT)-polymerase chain reaction (PCR) results indicated that 12 (34%) of these carried the ROSA26-reverse tetracycline-transactivator (rtTA)-M2 transgene

Summary

Introduction

The tetracycline-inducible gene regulation system is a powerful tool that allows temporal and dosedependent regulation of target transgene expression in vitro and in vivo. There is over a century of phenotypic and physiological data on anatomy, physiology and disease for various inbred rat models [15,16] Combining these data with improved genetic and genomic resources will provide unprecedented opportunities for functional genetic studies in this species. Recognizing this opportunity, NIH has funded the Rat Expressed Sequence Tag (EST) Program, the Rat Genome Program and the Rat Genome Database and established the Rat Resource Research Center (RRRC; http://www.nrrrc.missouri.edu) during the past decade [17]. There is a strong impetus to expand the transgenic tools for this species

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Conclusion