Differential display combined with a regulated transient expression system.

Differential display or RNA fingerprinting (6,9) is widely used to identify altered cellular gene expression at the mRNA level. We describe the differential display of RNAs synthesized from transiently transfected DNA—a technique not previously reported. Although we focus on the application of this methodology to analysis of a pool of RNAs in which cytoplasmic stability can be altered because of its mutant 3′untranslated regions (UTRs), this methodology also allows the selection of RNAs from a pool of mutants having increased levels of abundance due to more efficient RNA processing or polyadenylation, or to enhanced nuclear export. Analysis of RNAs with altered stabilities is greatly facilitated by synthesizing the RNAs in a pulse under the control of a transiently transfected, regulated promoter. Synthesizing the RNAs as a pulse allows them to decay for an appropriate period of time before their analysis using differential display. We used the tetracycline (Tet)-regulated promoter system (4), which is extremely powerful. Expression of a luciferase reporter gene after transient transfection of HepG2, human hepatoma, cells with a Tet-luciferase plasmid (pUHC13-3; Reference 4) was 38fold higher than luciferase expression from the strong cytomegalovirus promoter. Even in transient transfections, the Tet system maintained tight regulation. Expression from the Tet-regulated promoter was repressed 400-fold upon addition of Tet to the medium. To test the use of differential display to analyze RNAs transcribed from transiently transfected DNAs, we constructed pools of random 3′-UTRs fused to the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the Tet-regulated promoter (Figure 1). Since it was not feasible to synthesize a library of oligonucleotides >160 nucleotides (nt) long with approximately 130 nt of randomized sequences, we constructed a library of oligonucleotides with randomized sequences 64 and 65 nt in length (N64 and N65, respectively). The randomized sequences were flanked by both specific sequences that contained a StyI site, so that they could be ligated together, and XbaI and BamHI sites that allowed for insertion into the Tet-CAT plasmid (Figure 1). From this large library of mutants with different 3′UTRs, two pools of mutants containing 1000 and 2000 independent clones, respectively (P1 and P2), were prepared and used in transfections. HepG2 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), and transfected (with a calcium phosphate method) with 10 μg of one of the tetCAT-RM pools (P1 or P2), 5 μg of the Tet-regulated transactivator expressing plasmid (pUHD15-1; Reference 4) and 5 μg of pTZ18U carrier DNA per 100-mm plate. A 5 μg amount of pUHD15-1 and 15 μg of pTZ18U were used for mock-transfections. After a 4-h incubation, the medium was replaced and the cells were harvested, and RNA was extracted after 48 h. Total cellular RNAs were prepared using guanidinium thiocyanate extraction followed by centrifugation in cesium chloride (8). The extracted RNA was treated for 30 min at 37°C with 5 U of RQ1 DNase I in the presence of 40 U of RNasin (both from Promega, Madison, WI, USA), (in 10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2) in a 100-μL reaction mixture, extracted with phenol/ chloroform and ethanol-precipitated in the presence of 0.3 M NaOAc (pH 5.3). Total RNA (8 μg) and 50 pmol of 3′end-specific Tet I primer (5′-TTGTTGTTAACTTGTTTA-3′) were used for reverse transcription (RT) in a 25μL reaction volume in the presence or absence (mock RT) of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Life Technologies, Gaithersburg, MD, USA) used according to the supplier’s instruction. After RT, Tet I primer was removed by using the PrepA-Gene DNA Purification System (Bio-Rad, Hercules, CA, USA). The Benchmarks