Elsevier

Gene

Volume 282, Issues 1–2, 9 January 2002, Pages 65-74
Gene

Effects of mouse strain, position of integration and tetracycline analogue on the tetracycline conditional system in transgenic mice

https://doi.org/10.1016/S0378-1119(01)00793-4Get rights and content

Abstract

The tetracycline conditional system is a very powerful method for achieving control of gene expression in transgenic mice, allowing one to turn expression both off and on in the same animal. We have used it to make a tissue-specific transgenic mouse model of Charcot–Marie–Tooth disease type 1A. This disease is most commonly caused by overexpression of peripheral myelin protein 22 (PMP22) in Schwann cells of the peripheral nervous system. Here we describe the effects of position of integration of the transgene, tetracycline analogue and mouse strain in this model. The small transgenes used to express tTA, the LacZ reporter and the pmp22 cDNA were all very dependent on the position of integration with few of the transgenic lines working successfully. In contrast, the single transgenic made with the 560 kb yeast artificial chromosome construct containing the tTA open reading frame worked well. Tetracycline was found to be cleared from mice relatively fast in comparison with doxycycline and is thus useful if one wants to switch on gene expression after extended periods of administration. Finally, the initial litters were on a mixed genetic background and the level of LacZ or pmp22 expression was very variable between mice. We found that expression became uniform between mice, and occurred in a higher proportion of cells, when the transgenes were crossed onto the CBA/Ca background in comparison with the C57BL/6J background.

Introduction

Several systems for achieving conditional transcription in transgenic mice have been developed. These include those based on cre-loxP where the DNA is recombined and those based on chimeric transcriptional activators that reversibly bind a target DNA sequence in response to a ligand such as RU486, ecdysone, tetracycline or IPTG (reviewed by Lewandoski, 2001, Ryding et al., 2001). The tetracycline conditional system developed by H. Bujard and colleagues (Gossen and Bujard, 1992, Furth et al., 1994, Kistner et al., 1996) allows reversible changes in gene expression over several orders of magnitude and can be made tissue-specific by targeting the expression of tTA using a tissue-specific promotor. It has been used in transgenic mice to achieve conditional modulation of a number of biological systems including; transformation of pancreatic β-cells (Efrat et al., 1995), hyperplasia caused by SV40 T expression (Ewald et al., 1996), memory formation (Mayford et al., 1996, Mansuy et al., 1998a, Mansuy et al., 1998b), prion disease (Tremblay et al., 1998), milk protein content (Soulier et al., 1999), the temporal requirement for endothelin receptor-B (Shin et al., 1999), development of diabetes (Green and Flavell, 2000), and Duchenne muscular dystrophy (Ahmad et al., 2000).

Charcot–Marie–Tooth disease type 1A (CMT1A) is caused by overexpression of peripheral myelin protein 22 (PMP22) in the Schwann cells of the peripheral nervous system leading to demyelination. We have used the tetracycline system to make a tissue-specific and conditional transgenic mouse model of CMT1A (Perea et al., 2001). We achieved good levels of tissue-specific expression that could be controlled by feeding the mice with tetracycline at 2 mg/ml. Overexpression of PMP22 resulted in demyelination of 20–50% of axons. Demyelination was reversible and axons began to remyelinate within 1 week of switching off of Pmp22. Conversely, the upregulation of Pmp22 resulted in active demyelination. However, a number of problems were encountered with the system. Here we describe our experience of chromatin-mediated position effects on the transgenes, the use of two different mouse strains and the use of doxycycline versus tetracycline as the controlling antibiotic.

Section snippets

Transgenic constructs and generation of transgenic mice

Plasmid pUHD15-1 (Furth et al., 1994) which has tTA driven by the CMV promoter, was digested with XhoI and HindIII and gel-purified to remove unwanted plasmid sequences prior to injection into mouse oocytes. Plasmid pUHG16-3 (Furth et al., 1994) which has lacZ under the control of phCMVh*-1 was linearized with XhoI prior to gel purification and microinjection. The tTA open reading frame (ORF) from pUHD15-1 was inserted into a 560 kb YAC so that it was under the control of the PMP22 promoter as

Generation of transgenic mice

Transgenic lines were made separately with the tTA inducer constructs and the responder constructs and these were then mated to produce double transgenics. One construct had tTA driven by the human CMV promoter, plasmid pUHD15-1 (Furth et al., 1994). Another had tTA driven by the human PMP22 promoter and was made by introducing the tTA ORF into the intact PMP22 gene on a 560 kb YAC (Perea et al., 2001). One responder construct had the lacZ reporter gene under the control of the phCMVh*-1

Discussion

Position effects and variegated expression have been observed by almost all investigators using this tetracycline-conditional system (Furth et al., 1994, Kistner et al., 1996, Schultze et al., 1996, Yu et al., 1996, Redfern et al., 1999, Soulier et al., 1999, Ahmad et al., 2000, Diamond et al., 2000, Böger and Gruss, 1999, #897). In our transgenic animals the lacZ reporter and CMV-driven tTA transgenes are clearly very position-dependent as only one of five of the lacZ reporter lines and none

Acknowledgements

We would like to thank Mark Hay for technical assistance with production of transgenic mice. This work was supported by Action Research.

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