An enhanced promoter trap protocol

A. Toyoda, a'2 J. Kusuda, a H. Maeda, 2 K. Hashimoto 1 1Division of Genetic Resources, National Institute of Health, Shinjuku-ku, Tokyo 162, Japan 2Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji-shi, Tokyo 192, Japan Received: 28 November 1994 / Accepted: 20 February 1995 Isolation and identification of functional genes from the mamma- lian genome have become an important task. Analysis of the mo- lecular mechanisms involved in human genetic disorders requires an efficient and simple approach to identification of candidate genes in genomic DNA fragments cloned from a target region within chromosomes. Isolation of genes from cloned DNA frag- ments is attempted mostly by hybridization of cDNA to a given genomic DNA fragment or by the exon-trapping method (Buckler et al. 1991). The exon-trapping method has the disadvantage that the 5'-flanking region and the first exon in genomic DNA can not be isolated directly. In most cases, DNA clones containing pro- moters have been isolated from genomic libraries by hybridization with cDNA as a probe. This approach is problematic because isolation of the full length of cDNA is required, and the 5'-fianking region corresponding to a given cDNA should be distinguished from that of pseudogenes. In the murine embryonic stem cells, various vectors have been constructed with a promoterless reporter gene to trap genes by integration into the genome (Friedrich and Soriano 1991; von Melchner et al. 1992; Niwa et al. 1993). The expression of the trapped gene in these constructs depends on the reading frame of the coding region in the genomic DNA which is fused with the reporter gene. Overcoming the above limitations, we have constructed a pro- moter trap vector named pPT1 to isolate the promoter regions of certain genes from cloned genomic DNA. The vector was con- structed to utilize the functions of a splicing acceptor site, a stop codon linker, and the internal ribosomal entry site (IRES) sequence located in the 5' non-translated region of poliovirus genome. The pPT1 was constructed as follows (Fig. t): first, the se- quence (554 bp) connecting the first intron and the second exon of the human NADH-cytochrome b5 reductase (b5R) gene (Tomatsu et al. 1989) was generated by the polymerase chain reaction (PCR) with genomic DNA as a template. The primer set used comprised the first intron region (nucleotide nos. 12328-12347: 5"-AGCCA- TTTGGTCAGTGTAGG-3'), and the second exon (nucleotide nos. 12 8 62-128 81 : 5'ATCGGTACCTGAGCAGACTGTACAG- GAAC-3'; the underlined part indicates the KpnI site). The PCR product was digested with HinclI/KpnI and subcloned into pUC18. The stop codon linker containing the NotI site (Nippon Gene Ltd, Japan) was ligated downstream from the b5R gene fragment at the blunt-ended SacI site of the above construct. Secondly, with the RNA prepared from the superuatant of the cell cultures infected with poliovirus type 3 (provided by K. Yoshii), the first DNA strand complementary to the RNA was synthesized. The mixture of the RNA and the cDNA was subjected to PCR amplification. The primer set comprised 5"-ACG- Correspondence to: A. Toyoda at Hachioji-shi GCGGCCGCTTAAAACAGCTCTGGGGTTGTT-3' (nucleotide nos. 1-22; the underlined portion indicates NotI site) and 5'- ACGAAGCTTACTGAAATCCTGTCTTCAAACAA-3" (nucle- otide nos. 714-763; the underlined portion indicates the HindlII site) for the IRES region (Cann et al. 1984). The NotI-digested product (736 bp) of the amplified IRES region was inserted be- tween the NotI and the blunt-ended EcoRI site on the pUC18- based construct. The 1.2-kb HinclI-HindIII fragment excised from this construct was cloned into pUC19, to which was introduced a linker containing three cloning sites [5'-CCTCGAGAGATCT- TCTAGAGTC-3' (the underlined portion indicates the XhoI, the BgtII, and the XbaI sites) and 5"-GACTCTAGAAGATCTCTC- GAGGACGT-3' (the complementary strand)] at the AatlI/HinclI sites. Finally, the 3.7-kb blunt-ended HindIII-BamHI fragment cod- ing the E. coil lacZ gene and the SV40 poly A signal sequence derived from pRSV-~Gal (Edlund et al. 1985) was ligated into the blunt-ended HindIII site of the pUC19-based construct. The constructed vector has the following characteristics: (i) it contains the IacZ gene as a reporter whose product can be readily detected by staining with X-gal, (ii) the first exon attached to a promoter region can also be trapped by use of the splice acceptor site of the bSR gene, and some reading frame preceding the lacZ gene is stopped by the stop codon linker so that the correct ex- pression of the lacZ gene can be ensured, and (iii) when the IRES sequence is positioned in an intercistronic space of dicistronic mRNA, it stimulates efficient translation of the downstream gene, that is, its expression depends on the upstream promoter activity (Pelletire and Sonenberg 1988; Kim et al. 1992; Palmer et al. 1993). Thus, if there is a promoter region in an insert fragment, not necessarily containing coding sequences, the lacZ gene expression will be controlled by the IRES sequence. To examine whether the lacZ gene in the constructed vector can be expressed with the known promoter sequences, a 1.9-kb or 1.5-kb genomic DNA fragment coding the 5'-flanking region as well as the first exon of either the human b5R gene or the human dehydrofolate reductase gene was inserted into pPT1 in either the right or the reverse direction. These recombinant plasmids were transfected with a LipofectAMINE reagent (BRL) into HeLa or COS cells. After 2 days, these cells were washed with PBS and fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 min at room temperature. The cells were rinsed with PBS and overlaid with activity staining solution (1 mg/ml X-gal, 5 ms potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgC12 in PBS). The cells were incubated overnight at 37~ The activity staining showed that the lacZ gene was expressed when the given DNA fragment was cloned in the right direction (data not shown). The result indicates that the DNA sequence (about 400 bp) containing the splicing acceptor site functions properly to insert the promoter sequence with the first exon and also that DNA