Heterologous introns can enhance expression of transgenes in mice.

Abstract

In a previous study we showed that genomic constructs were expressed more efficiently in transgenic mice than constructs that were identical except for the lack of introns. Using the mouse metallothionein promoter-rat growth hormone gene construct as a model, we show that the rrst intron ofthe rat growth hormone gene is essential for high-level expression, whereas the other three introns are less effective. Several heterologous introns placed 3' of the coding region of an intronless rat growth hormone gene are also ineffective. However, insertion of some heterologous introns between the metallothionein promoter and the growth hormone gene improves expression. To determine whether addition of heterologous introns would provide a general strategy for improving expression, we have tested them in conjunction with other intronless genes and with different promoters.In principle, it is possible to direct the expression of any gene to any specific cell type of an animal by using established transgenic methodology. This approach relies upon the fact that it is possible to combine the regulatory region(s) ofa gene that is expressed in a cell-specific manner with any mRNAencoding structural gene. The structural gene should then be transcribed according to the specificity dictated by the regulatory elements. This strategy has been used to explore a wide variety of fascinating developmental and physiological problems (see refs. 1-3 for reviews). However, not all gene constructs work well in transgenic mice. The two most common problems are inappropriate expression patterns and failure to achieve adequate expression levels.Inadequate expression is often associated with attempts to express cDNA constructs. The most baffling aspect of this phenomenon is that cDNA constructs that fail to work in transgenic mice are often expressed efficiently when transfected into tissue culture cells. These observations suggested that introns may be of special importance for achieving gene expression in transgenic mice. Because most of the early experiments that led to this suspicion were not well controlled, we set out to test it directly by comparing constructs that were identical except for the presence or absence of introns. With each of three test genes, mouse metallothionein I (mMT-I), mMT-rat growth hormone (rGH), and human ,3-globin (hf3G), we observed a striking improvement both in the number of transgenic mice that expressed the gene and in the average level of expression when the natural introns were included (4). Furthermore, nuclear run-on assays indicated that the introns had a major effect on transcription rather than mRNA processing or stability. Because the mMT and mMTrGH constructs showed no dependence on introns for expression after stable transfection into BHK (baby hamster kidney) cells, we postulated that introns may contain sequences that are recognized during embryogenesis.Introns could improve expression of transgenes by a number of different mechanisms. For example, some introns may contain enhancers or other ...

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“…The intron 1 sequence was tested in transfectant construct 167 (Figure 1b) to assess if the long (~6 kb) intron closest to the PSP94 promoter region 35 Figure 2a) was constructed using a 3.84 kb mouse PSP94 gene promoter/enhancer region to direct targeting of expression of a full length of SV40 Tag (T and t antigens) gene to the prostate. For enhancing transgenic expression by intron sequences, 36 a second transgene (186, Figure 2b) was constructed linking the promoter/enhancer-exon1-part of intron 1 and exon 2 followed by the SV40 Tag gene with the same reading frame. After breeding with the wild type of (C57BL/6 × CBA) F1 hybrids, three transgenic founder breeding lines (F0 of A, B and C) were characterized and successfully established out of 18 transgenic mice: A from transgene 183 (183-2), B and C from 186 (named F0186-3, F0186-9, respectively).…”

Intravenous administration of an AAV-2 vector for the expression of factor IX in mice and a dog model of hemophilia B

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