Establishment of Cre/LoxP recombination system in transgenic rats

doi:10.1016/j.bbrc.2004.04.204

Biochemical and Biophysical Research Communications

Volume 319, Issue 4, 9 July 2004, Pages 1197-1202

https://doi.org/10.1016/j.bbrc.2004.04.204 Get rights and content

Abstract

The rat has offered an important animal model in biomedical research including surgical procedure. However, advanced genetic manipulation has progressed less far in the rat than in the mouse. Here we report the Cre/LoxP transgenic rat system, demonstrating conditional chromosomal translocation both in the fertilization and adult stage, spatio-temporal gene controlling by catheter-based adenoviral gene transfer, and muscular fusion events in the limb transplant. Taking advantage of the larger body size of the rat than the mouse, this rat system provides a potential value to evaluate biomedical and therapeutic significance for gene therapy and regenerative medicine.

Section snippets

Materials and methods

Experimental animals. Wild male Wistar rats (body weight 180–250 g) were used to establish the DsRed2/GFP double reporter Tg rat and NCre Tg rat. The Wistar rats were originally purchased from Charles River Japan (Yokohama, Japan). All experiments in this study were performed in accordance with the Jichi Medical School Guide for Laboratory Animals.

Generation of Tg rats. The DsRed2 plus poly(A) additional signal DNA fragment flanked with LoxP at both ends was amplified by polymerase chain

Results and discussion

We have established one line of double reporter Tg rats, carrying DsRed2 flanking LoxP sites at both ends as a stuffer and GFP downstream of the DsRed2. This line was generated by microinjection into the egg pronuclei of the Wistar rat. The genes are controlled under a ubiquitous CMV enhancer/chicken β-actin promoter (CAG promoter), and DsRed2 expresses in the rat before Cre/LoxP site-specific excision (Fig. 1A). The skeletal muscle, pancreas, heart myocardium, and bronchus showed relatively

Acknowledgements

We thank Drs. R. Takahashi and S. Ueda (Y.S. Technology Institute, Tochigi, Japan) for their expert transgene injection. We also thank Dr. T. Osumi (Himeji Institute of Technology, Hyogo, Japan) for his generous gift of phGFP-105-C1 plasmid, Dr. I. Saito (Inst. Med. Sci., Tokyo Univ., Tokyo, Japan) for providing pxCANCre and AdV-Cre (AxCANCre), Dr. J.-I. Miyazaki (Univ. Osaka, Osaka, Japan) for providing pCAGGS expression vector, and Drs. M. Sato and M. Ichida (Department of Biochemistry, Jichi

References (10)

J.Z Tsien et al.
Cell
(1996)
Y Hakamata et al.
Biochem. Biophys. Res. Commun.
(2001)
M Takahashi et al.
Biochem. Biophys. Res. Commun.
(2003)
Y Sato et al.
Biochem. Biophys. Res. Commun.
(2003)
Y Zan et al.
Nat. Biotechnol.
(2003)

There are more references available in the full text version of this article.

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¹: The first three authors were equally contributed to the work.

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Establishment of Cre/LoxP recombination system in transgenic rats

Abstract

Section snippets

Materials and methods

Results and discussion

Acknowledgements

Cell

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Nat. Biotechnol.