Vectors for high-level expression of cDNAs controlled by tissue-specific promoters in transgenic mice.

The use of transgenic mice has become an important tool for creating novel models of disease by expressing target genes in a tissueor cell typespecific fashion. One highly successful approach for generating transgenic mice has been to use a genomic clone containing the gene of interest. In these genomic clones, the gene is under the control of its own endogenous promoter, so that it is expressed in an appropriate tissue-specific and regulated fashion, and contains introns, exons, and a polyadenylation (polyA) addition site, resulting in efficient transcription, processing, and transport of the resultant mRNA. Although highly reliable, the use of genomic clones is not always feasible because of either size limitations or the need for an experimental strategy whereby a protein is targeted to a specific cell type. In situations like this, the use of a cDNA encoding the gene of interest fused behind a promoter/enhancer with a known cellular expression profile may prove more desirable and convenient. Previous studies have indicated that transgenes consisting of cDNAs are expressed at a much higher level when a spliceable intron is included in the construct (2,3). Indeed, introns are thought to be required for the proper and efficient maturation of mammalian mRNAs. Numerous constructs have been successfully employed that contain a 3′ cassette consisting of a spliceable intron immediately upstream of a polyA addition site. Transgenes generated in this way have large first exons (consisting of the cDNA) and small terminal exons. However, an analysis of the structure of many genes indicates that most mammalian genes contain small first exons and large terminal exons. We therefore considered that placing a spliceable intron between a promoter and cDNA would result in a transgene construct that may look and function more like a bona fide normal mammalian gene and thus be expressed more reliably. Several plasmid vectors are commercially available in which cDNAs can be placed behind an intron and used in vitro for transient transfection analysis. However, since many of these vectors contain a strong viral promoter to drive expression of the construct, they are not good candidates for use in the generation of transgenic mice when a restricted tissue-specific pattern of expression is desired. Therefore, we developed two cloning vectors allowing an investigator to: (i) easily insert a promoter/enhancer sequence designed to specifically target expression of the desired protein in a tissueor cell type-specific fashion upstream of a cDNA; (ii) easily insert a cDNA encoding the protein of interest downstream of a cell-specific promoter and spliceable intron, and upstream of a polyA addition site; and (iii) easily excise the transgene from the prokaryotic vector. Each of these features was considered in the design pSTEC-1 and pSTEC-2, which differ only in the available restriction enzyme sites (Figure 1). Specifically, both plasmids contain unique restriction enzyme sites for the insertion of a promoter/enhancer. Although any combination of the sites can be used, it is important to recognize that one of these unique sites must remain intact to excise the transgene from the plasmid backbone before injection of the fertilized eggs. Each plasmid also contains a chimeric intron composed of the 5′ splice site from the β-globin intron and the 3′ splice site from an IgG intron. A short 30-bp DNA sequence (derived from the original pCI plasmid) between the PstI site and the 5′ splice site becomes exon I. Downstream of the intron lies a polylinker region with a wide variety of unique restriction sites for the insertion of cDNAs. Further downstream lies a simian virus 40 (SV40) polyA addition site required for proper processing of the transgene mRNA. The cloned cDNA/polyA site portion becomes defined as the terminal exon (exon II) of the construct. Lastly, each plasmid contains several unique restriction sites downstream of the polyA addition site allowing for excision of the transgene from the plasmid backbone. Additional terminal unique restriction sites will be generated if their duplicate sites at the cDNA-insertion polylinker are eliminated during cloning. For example, if a cDNA is cloned into the NheI-NotI sites of Benchmarks