Figure 1.
Functional organization of pAIM.
The vector is shown in the form of a provirus collinearly integrated in a host gene. The vector is flanked by promoter-deficient LTRs (“dLTR”), which harbor Cre-recombinase recognition sites. The immediate early promoter and enhancer region from human cytomegalovirus (“CMV”) is preceded by a cluster of tet-operators (“TO”), which permit suppression of this promoter in the presence of a tetracycline-controlled transcription silencer (tTS). The coding region of copGFP (“copGFP”) ends with the 2A sequence and an unpaired splice donor (“2A” and “SD” respectively). In the scenario shown above the vector, CMV-driven transcript is polyadenylated on the LTR-derived sequence and encodes only copGFP. In the scenario shown below the vector, transcription continues into the host DNA, and a mature fusion product is produced by splicing, which removes the LTR sequences. If the open reading frames of copGFP and the host gene coincide, both copGFP and a host protein could be produced when the RNA is translated.
Figure 2.
Identification of the fusion transcripts generated by trans-LTR splicing in two independent clones.
The products of nested ligation-mediated PCR on MboI-digested cDNA were sequenced. As predicted, the clones contain host sequence, followed by an MboI site (GATC) and the adapter. The 5′-end of the transcript is derived from the vector. The junction point corresponds to host intron/exon boundary.
Figure 3.
Reversible activation of NF-κB in mutant cells.
(A) Reversible pattern of drug resistance in the mutant clone. Mutant clone 2B2.1 was cultured in Zeo (upper panels) or GCV (lower panels) after infection with Cre (right panels) or the empty vector (left panels). 2.5×105 cells per 6 cm plate were treated for a week, and viable cells were visualized by methanol fixation and methylene blue staining. Similar results were observed in clones 2B1.4 and 2B3. (B) Reversible up-regulation of an NF-κB reporter in the mutant cells. Luciferase reporter assay was used to compare the activity of NF-κB in 2B2.1 cells transduced with either Cre or an empty vector to that in the parental HEK293ZeoTK cell line. The activity of a transiently transfected luciferase reporter was normalized for that of co-transfected constitutive beta-galactosidase expression construct. The data is presented in relative units, with the normalized activity of the reporter in the parental cell line (HEK293ZeoTK) taken as 1. Mutant clones 2B1.4 and 2B3 demonstrated a similar pattern.
Figure 4.
Location of inserts in the three mutant clones.
A fragment of p25 region of chromosome 6 is shown, indicating the relative positions of known genes. The inserts are shown as arrows pointing in the direction of the CMV promoter. Approximate distances from the first annotated exon of RIPK1 are indicated. The drawing in not to scale.
Figure 5.
Examples of regulated expression of RIPK1 in mutant cells.
(A) Elevated expression of RIK1 in the mutant clones. RNA was isolated from mutant clones 2B2.1, 2B1.4 and 2B3, and from the parental HEK293ZeoTK cells, and analyzed by RT-PCR with RIPK1-specific primers (upper panel). The bottom panel presents results of RT-PCR on the same samples with primers for a housekeeping gene (GAPDH). (B) Dependence of RIPK1 expression in the mutant clones on the presence of the insert. Mutant clones 2B1.2 and 2B1.4 were infected with a Cre-expressing construct or the respective empty vector control. The cell lysates were compared by Western blotting with polyclonal anti-RIPK1. (C) Dependence of RIPK1 expression in the mutant clones on the function of the inserted promoter. Mutant clone 2B1.4 infected with a tTS-expressing construct was cultured with or without doxycycline. The cell lysates were analyzed by Western blotting with polyclonal anti-RIPK1 and compared to those of the parental HEK293ZeoTK cells.
Figure 6.
Activity of NF-κB upon overexpression of genes in the vicinity of inserts compared to parental HEK293ZeoTK cells.
NF-êB dependent luciferase reporter activity was measured after cells were co-transfected with respective expression constructs. Only RIPK1 showed elevated NF-κB activity.
Figure 7.
A example of the dependence of NF-κB activity in mutant cells on the function of RIPK1.
Mutant 2B2.1 and parental HEK293ZeoTK cells were assayed for NF-κB activity using transient transfection of a luciferase reporter. A decline in the reporter activity (indicated by the red arrow) resulted from adding an shRNA against RIPK1 to the transfection mix. An shRNA against p53 (a protein that is expressed, but is inactivated in these cells) was used as the control.