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J. Biol. Chem., Vol. 279, Issue 35, 36660-36669, August 27, 2004

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Regulated Tissue-specific Alternative Splicing of Enhanced Green Fluorescent Protein Transgenes Conferred by -Tropomyosin Regulatory Elements in Transgenic Mice^*

Peter D. Ellis, Christopher W. J. Smith, and Paul Kemp

From the Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1QW, United Kingdom

The mutually exclusive exons 2 and 3 of -tropomyosin (TM) have been used as a model system for strictly regulated alternative splicing. Exon 2 inclusion is only observed at high levels in smooth muscle (SM) tissues, whereas striated muscle and non-muscle cells use predominantly exon 3. Experiments in cell culture have shown that exon 2 selection results from repression of exon 3 and that this repression is mediated by regulatory elements flanking exon 3. We have now tested the cell culture-derived model in transgenic mice. We show that by harnessing the intronic splicing regulatory elements, expression of an enhanced green fluorescent protein transgene with a constitutively active promoter can be restricted to SM cells. Splicing of both endogenous TM and a series of transgenes carrying regulatory element mutations was analyzed by reverse transcriptasePCR. These studies indicated that although SM-rich tissues are equipped to regulate splicing of high levels of endogenous or transgene TM RNA, other non-SM tissues such as spleen, which express lower amounts of TM, also splice significant proportions of exon 2, and this splicing pattern can be recapitulated by transgenes expressed at low levels. We confirm the importance in vivo of the negatively acting regulatory elements for regulated skipping of exon 3. Moreover, we provide evidence that some of the regulatory factors responsible for exon 3 skipping appear to be titratable, with loss of regulated splicing sometimes being associated with high transgene expression levels.

Received for publication, May 14, 2004

^* This work was supported by Project Grant PG/2001031 from the British Heart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed. Tel.: 44-1223-333655; Fax: 44-1223-766002; E-mail: cwjs1{at}cam.ac.uk. To whom correspondence may be addressed. Tel.: 44-1223-333627; Fax: 44-1223-766002; E-mail: pk{at}mole.bio.cam.ac.uk.

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