The genetic screen of retroviral libraries aimed at the identification of cDNAs altering intracellular phosphorylation events in signaling cascades, here the phosphorylation of Stat1 in response to IFN-γ stimulation, was performed as follows: (i) establishment of a suitable selector cell line not responding to IFN-γ, (ii) transduction of a cDNA library into the selector cells, (iii) stimulation with IFN-γ and subsequent staining for pStat1, (iv) selection of responding cells by cell sorting, (v) amplification of cDNAs from the genomic DNA of the selected cells and re-cloning into a retroviral vector for validation and (vi) analysis of the isolated cDNAs for the reconstitution of the selected phenotype upon expression in the selector cells (for schematic illustration of the experimental design see ).
Figure 1 Schematic representation of the experimental design. Selector cells deficient in responsiveness toward a specific stimulation, here IFN-γ-induced phosphorylation of Stat1, are selected (step 1). After transduction of the retroviral cDNA library (more ...)
Establishment of a selector cell non-responsive to IFN-γ stimulation
A successful genetic screen relies on the utilization of a selector cell exerting a minimal endogenous background for the phenotype desired to enrich upon the selection of a certain cDNA causing it. In this study, we were aiming to identify a cDNA involved in the phosphorylation of Stat1 induced by IFN-γ stimulation. Thus, we intended to establish a cell line that shows weak responsiveness toward the stimulus. G. Poli and co-workers (16
) previously reported on a U937 cell clone (clone #10, plus) supporting HIV-1 replication in the presence of IFN-γ. The analysis of the phenomena revealed undetectable phosphorylation of Stat1 after exposure to IFN-γ. The mechanism by which this blockade to phosphorylation was caused is unknown. However, examination of the respective phosphorylation events using pStat-specific antibodies and FACS analysis demonstrated that ~5% of the cell population responded to stimulation. Thus, the original U937 cell clone #10 we received most probably either contains genetic revertants, which were contaminated with wild-type cells, or possess an effect that was due to epigenetic variation. Therefore, we established subclones from U937 clone #10 by biological cloning. A total of 15 individual clones were analyzed for their induction of pStat1 after exposure to IFN-γ and subjected to a staining procedure using pStat1-specific antibodies conjugated with Alexa 647 as described previously (10
). As shown in , clone #10.13 was demonstrated to respond very weakly in comparison with wt U937 cells indicating its suitability as a selector cell for the intended genetic screen. Revertants were not observed at high frequency; hence, we deemed that this subclone would be appropriate for use in the screen.
Figure 2 Responsiveness of selector cells and selective amplification of cDNAs. (A) FACS analysis of IFN-γ-induced Stat1 phosphorylation of wt U937 cells and cell clone #10.13. Unstimulated cells (gray histogram) or after exposure to IFN-γ (open (more ...)
Detection limit of cDNA-specific amplification using PCR and genomic template DNA of formaldehyde- and methanol-treated cells
Since the screen outlined requires the fixation and permeabilization of cDNA-expressing cells to enable specific staining for pStat1 rather than the sorting of living cells, we first investigated the minimal amount of genomic DNA that allowed for the amplification of cDNAs using PCR. Therefore, U937 cells were transduced three times with one transfer vector from the retroviral cDNA (clone #3) harboring a cDNA of ~2 kb spiked with 2% of an enhanced green fluorescence protein (EGFP)-encoding transfer vector (LibEGFP). Two days after the last transduction round, ~1% of the targets cells were EGFP-positive indicating that about half of the cells were successfully transduced with the Lib cDNA#3. Subsequently, genomic DNA of untreated, formaldehyde- and formaldehyde/methanol-treated cells were isolated and used as the template in a PCR employing oligonucleotides allowing for the specific amplification of the integrated retroviral cDNA. Genomic DNA of untreated transduced and untransduced U937 cells served as positive and negative controls, respectively. As shown in , the PCR protocol described in Material and Methods allowed for the amplification of a 2 kb fragment from 100 ng of untreated and transduced cells but not from untransduced cells indicating the selective amplification of cDNA #3. Moreover, using decreasing amounts of template DNA revealed that >0.01 and >10 ng of genomic DNA of formaldehyde- and formaldehyde/methanol-treated cells were necessary to allow the successful amplification of integrated cDNA, respectively. Thus, the genomic DNA of ~1 and 1000 treated cells, respectively, were sufficient for the amplification. However, when we first amplified 1 ng of genomic DNA with a non-specific amplification method using random hexamer oligonucleotides (Genomi-Phi kit; Amersham), we were able to detect the desired 2 kb band. This demonstrated that >100 cells with a respective integrated cDNA were sufficient to amplify, isolate and clone it, and thus, it was feasible to assume that the screen of a retroviral cDNA library could be performed with fixed and permeabilized cells and their selection by cell sorting.
cDNA library transfer and enrichment of cells responding to IFN-γ
To transduce the retroviral cDNA derived from human PBL, 3 × 106 MLV-based Phoenix gp packaging cells were co-transfected with the plasmid library spiked with 5% of the vector pLibEGFP harboring the reporter gene EGFP and a VSV-G encoding construct. Two days post–transfection, 6 ml of cell-free supernatants containing MLV(VSV-G) pseudotype vectors were harvested and subsequently used to transduce 3 × 106 U937 clone #10.13 cells. After 3 days of expansion, ~1–2% of the cells expressed EGFP indicating that 20–40% of the cells were successfully transduced with cDNA-encoding retroviral transfer vectors. A total of 5 × 106 of the transduced cells were stimulated with IFN-γ as described and stained using Alexa 647-conjugated pStat1-specific antibodies. Stimulated and unstimulated wt U937, and untransduced and transduced #10.13 cells served as controls.
As shown in , ~1% of the cDNA library expressing #10.13 cells revealed detectable pStat1 levels. The cells were sorted using FACS and their genomic DNA was isolated to serve as a template for unspecific amplification using the Genomi-Phi protocol. Subsequently, the pre-amplified DNA was subjected to cDNA-specific amplification using PCR. While no signal was obtained using control DNA of untransduced #10.13 cells, the use of genomic DNA of the selected cDNA-transduced cells as a template resulted in a single DNA fragment of ~1.5 kb termed cDNA1.2. Although it is feasible to assume that more than one cDNA was present in the selected cell population, these were possibly not amplified as they were not enriched by the selection scheme applied and, therefore, represented at a much lower frequency in the genomic DNA used as a template. However, in two previous screens using the same experimental design a number of cDNAs were obtained of which one was shown to encode the IFNGR2. The other cDNAs were demonstrated not to reconstitute responsiveness to IFN-γ stimulation (data not shown).
Figure 3 Cell sorting of cDNA expressing cells and amplification of cDNA. (A) pStat1 detected in unstimulated and IFN-γ-stimulated transduced selector cells. Boxes indicate the gates used to select pStat1-positive cells. Numbers within the boxes represent (more ...)
This blunt end fragment was ligated into the vector pCR-Blunt II-TOPO and sequenced demonstrating that the coding region encoded human IFNGR2 (17
). Thus, remarkably, after only one round of selection a single cDNA was enriched encoding a member of the IFN-γ-induced signaling cascade leading to the downstream phosphorylation of Stat1.
The selected cDNA reconstitutes responsiveness of the selector cells to IFN-γ stimulation
Next, we aimed to demonstrate that expression of the selected cDNA1.2 mediated the responsiveness to IFN-γ stimulation of U937 #10.13 cells. By using specific primers that introduced flanking NotI restriction sites and a standard PCR protocol, cDNA1.2 was amplified, digested with NotI and ligated into the retroviral vector BMN-I-GFP, which was previously digested with NotI and dephosphorylated with calf intestine phosphatase. In this construct, the MLV promoter in the 5′ long terminal repeat drives the expression of an inserted cDNA and an IRES-GFP cassette. The accuracy of the coding region and its right orientation of the inserted cDNA were analyzed by sequencing. MLV(VSV-G) vector particles packaging the mRNA transcripts of retroviral transfer vectors, BMN-I-GFP and BMN-I-GFP-cDNA1.2, were harvested from transiently transfected packaging cells as described and used to transduce U937 #10.13 cells. Within 2 weeks after transduction, the transduced cells expressing GFP were sorted twice using FACS analysis and expanded. As shown in , FACS analysis for the abundance of pStat1 of unstimulated and IFN-γ-stimulated cells using phospho-specific antibodies revealed that the cells transduced with the control vector BMN-I-GFP did not respond to IFN-γ. In contrast, cells expressing GFP and cDNA1.2 showed clear phosphorylation of Stat1 in response to IFN-γ. Notably, cells that were negative for GFP, and therefore did not contain the cDNA, did not show appreciable pStat1 levels. To exclude the possibility that this resulted in different amounts of Stat1-substrate proteins in the transduced cells, western-blot analysis of cell lysates was performed employing murine monoclonal antibodies directed against Stat1 and pStat1, respectively, and anti-murine IgG antibodies conjugated with HRP. Wild-type U937 cells expressed ~2–3-fold more Stat1 compared with all #10.13-derived cell lines. Most importantly, besides wt U937 cells, only #10.13 cells transduced with BMN-I-GFP-cDNA1.2 responded toward IFN-γ stimulation by Stat1 phosphorylation in agreement with the observations obtained using FACS analysis. This showed that the recovery of the phenotype was mediated by cDNA1.2-encoded IFNGR2 expression rather than the different amount of Stat1 present in the cytoplasm.
Figure 4 Reconstitution of IFN-γ responsiveness upon the expression of cDNA1.2. (A) Detection of pStat1 in unstimulated and IFN-γ-stimulated cells transduced with BMN-I-GFP and BMN-I-GFP-cDNA1.2, respectively. Numbers represent the percentage of (more ...)