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* Department Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany; and
Department of Internal Medicine III, Medical School Ulm, Ulm, Germany
1Correspondence: Abteilung "Molekulare Genetik" (B060),Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. E-mail: m.macleod{at}dkfz.de
ABSTRACT
A broad range of malignant diseases, such as mantle cell lymphoma (MCL), is associated with complex genomic alterations, demanding multimodal functional testing of candidate genes. To assess such candidate disease genes, we have developed a bidirectional targeted transgenesis tool, which allows well-controlled modulation of individual gene activities within a cellular MCL system. The engineered versatile transgenesis system permits functional analysis of virtually any candidate gene: for tumor suppressor genes by complementation via integration of respective genomic DNA or for oncogenes by inactivation via integrated shRNA coding plasmids. Complementation by genomic DNA ensures wild-type (WT) regulated gene expression, whereas genomic integration of shRNA coding inserts by an advanced RNAi-strategy mediates specific knock-down of gene expression. Site-specific genomic integration of an unmodified BAC, which contains the CDKN2A/B genes absent in the MCL model system, restored CDKN2A/B expression resulting in the inhibition of cell proliferation. CCND1, strongly overexpressed in the model system, was down-regulated via shRNA expression, again inhibiting proliferation. Notably, the presented site-specific shRNA-strategy circumvents interference by IFN-response induced when using other RNAi gene knock-down methods. In conclusion, we here demonstrate that adequate restoration of a range of different gene activities yields in a desired antiproliferative effect in MCL-derived cells. By antagonizing inactivated tumor suppressor genes or activated oncogenes, the presented approach can be readily used for the functional analysis of a broad range of disease-related genetic defects.— Pscherer, A., Schliwka, J., Wildenberger, K., Mincheva, A., Schwaenen, C., Döhner, H., Stilgenbauer, S., Lichter, P. Antagonizing inactivated tumor suppressor genes and activated oncogenes by a versatile transgenesis system: application in mantle cell lymphoma.
Key Words: functional genomics • RNA • transgenes • oncogenomics • genomic complementation • genetics of disease
MANTLE CELL LYMPHOMA (MCL) represents a non-Hodgkin lymphoma (NHL) (1)
, which is characterized by a block in differentiation, deregulation of cell proliferation as well as insufficient induction of apoptosis in B-cell lymphocytes (2
, 3)
. The characteristic translocation t(11;14)(q31;q32) results in a strong overexpression of the cyclin D1 gene (CCND1) (4)
. However, since transgenic mouse models with t(11;14) do not display a higher incidence of lymphomas (5
, 6)
, additional genomic changes responsible for tumor formation remain to be characterized. Molecular cytogenetics (7
, 8)
revealed a complex karyotype with amplified regions (+18q21, +7p15, +Xq26/28, +3q26, +8q24, +12q13) and deleted or inactivated loci (del 9p21, del 13q14, del 11q23, del 17p13, del 6q21/q27, del 1p22, del 8p22, del 10p15, del Xp22). Quantitative gene expression analyses by real-time PCR and expression-arrays indicated a prevalence of overexpressed proliferation associated genes (9
, 10)
. Mutated tumor suppressor genes were identified on chromosomal bands 9p21 (CDKN2), 13q14 (BCMS), 11q23 (ATM), and 17p13 (TP53), as well as overexpressed oncogenes on 8q24 (MYC) and 18q21 (BCL2) (9
, 11
12
13
14
15)
. Nevertheless, the role of these genes for the disease phenotype is still under investigation.
We developed a cellular tool for the functional analysis of candidate genes in a cellular disease system. Both oncogenic gene deregulations, gene inactivation and gene activation, are antagonized in the novel presented cellular system by targeted integration of transgenes via recombinase-mediated cassette exchange (RMCE) (18)
. The method of choice for functional tests of tumor suppressor candidate genes has been transient or stable expression derived from respective cDNAs within a wild-type genetic system. While this expression is driven by ectopic gene promoters, reconstitution of gene defects regulated via endogenous promoters, enhancers, alternative transcription start sites, and splice variants of a specific gene is of distinct advantage (16)
and was recently used to rescue phenotypes in RNAi-experiments (17)
. Therefore, our strategy to compensate for genomic deletion or inactivation of genes is based on the utilization of BACs containing the entire genomic information, including regulatory elements. These BACs became widely available through the Human Genome Project (http://www.ensembl.org). Gene copy number gains, which result in the up-regulation of gene expression, represent another pathogenic mechanism. Many functional studies of such candidate genes analyzed the phenotypic effect of the genomic changes characteristic for a human tumor on introduction in a WT cellular system, like in animal models. In contrast, our system of antagonizing activated oncogenes is based on a cellular disease system. To counteract overexpression of activated genes within the same genetic system used for the complementation studies, we applied RNAi-technology (19)
by stably integrating a modified RNAi-plasmid coding for gene-specific shRNA (20)
. Our system of delivering shRNA coding inserts successfully circumvents the problem of other RNAi strategies inducing IFN response and thereby masking a specific disease gene effect.
MATERIALS AND METHODS
Cell culture and transfections
Granta-519 cells (DSMZ, Braunschwarg, Germany No: ACC 342) were cultured in DMEM medium (Invitrogen, Karlsruhe, Germany), HEK-293 cells (ACC 305) and Jeko-1 cells (ACC 553) were cultured in RPMI medium (Invitrogen). Both media were supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. Transient transfections of Granta-519 cells were performed using the Nucleofector device (Amaxa Biosystems, Koelm, Germany) according to the manufacturer’s instructions with 5 x 106 cells and 5 µg DNA and applying following parameters: cell line solution T and program O-17. Cells were harvested 12 h after transfection for RNA isolation. Stable transfections of Granta-519 cells to introduce the selection cassette were performed by electroporation with an exponential pulse (Bio-Rad GenePulser Xcell, 960 µF, 250 V) and 10 µg ScaI-linearized pGKP511TkneoP plasmid for 1 x 107 cells. Neomycin-resistant cells were selected using a limited dilution with 2 mg/ml G-418 (Invitrogen) in 5% conditioned medium, and monoclonality was tested using Southern-blot assays. Mediating the RMCE reaction for BAC-clone DNA, stable transfections of Granta-519 cells were performed by using the transfection reagent Geneporter (GenLantis, San Diego, CA) according to the manufacturer’s protocol (GTS, previously published for a BAC clone transfection (21)
, 10 µg BAC-plasmid). For stable pSUPERdL (5 µg) transfection, we used the described exponential pulse electroporation protocol. The stable transfections for RMCE were both done by cotransfetion of 2.5 µg Cre-expression plasmid (pCMXhCre) in 1 x 107 neomycin-resistant monoclonal cells. Selection was performed using limited dilution and 2 µM Ganciclovir (GCV, Sigma, Munich, Germany) in 5% conditioned medium with repeated cycles of four days with selection medium and four days without selection medium to minimize the bystander effect.
Plasmids and cloning
Plasmids pCMXhCre, ploxPFRTpGKTkneoFRTloxP, and pGKneo were obtained from E. Greiner (Heidelberg) with permission of F. Stewart (Dresden); pSUPER was obtained from O. Heidenreich (Tübingen) with permission of R. Agami (Amsterdam). All RP11-library BAC-clones, including RP11–149I2 used for genomic complementation, were obtained from the RZPD (library RPCIB753), German Resource Center for Genome Research (Berlin). pGKneo was modified to obtain pGKP511TkneoP by introducing a loxP511 site into the NotI-restriction site and a loxP site into the Asc I-restriction site, followed by the subcloning of the BglII/NcoI digested HSV-Tkneo fragment of ploxPFRTpGKTkneoFRTloxP into the BglII/NcoI- restriction sites of pGKneo. pSUPER was modified to obtain pSUPERdL (dL for "double Lox") by introducing a loxP511 site into the NotI-restriction site and a loxP site into the AflIII-restriction site. Prior to the RMCE mediated knock-down of CCND1, three different synthetic siRNA sequences were tested by transient transfection into Granta-519 cells (data not shown), and the sequence of the most effective one was cloned into the pSUPERdL vector. Short hairpin sequences specific for CCND1 (5'-GATCCCCGATCGTCGCCACCTGGATGttcaagagaCATCCAGGTGGCGACGATCTTTTTGGAAA-3') and, as a control, a shRNA directed against firefly luciferase (Luc, 5'-GATCCCCCTTACGCTGAGTACTTCGAttcaagagaTCGAAGTACTCAGCGTAAGTTTTTGGAAA -3') were inserted as 64 base-pair nucleotides into the BglII/HindIII-restriction sites of pSUPERdL. Plasmids and BAC-clone were isolated and prepared for transfection using High Purity Plasmid Purification System (Marligen, Ijamsville, MD).
Fluroescence in situ hybridization (FISH) analysis
For fluorescence in situ hybridization (FISH), the following DNA probes were used: plasmid pGKP511TkneoP (selection cassette), L1.84 (alphaoide, centromer 18 specific probe), BAC-clones RP11–149I2 (CDKN2A/B-specific probe), RP11–536K7 (10p-specific probe), and RP11–56J3 (11q-specific probe) from the library RPCIB753 of the RZPD (German Resource Center Primary Database). Probe labeling by nick translation, preparation of chromosomes and hybridization were performed as described previously (22)
.
DNA and RNA isolation
Whole genomic DNA was isolated by incubating cells from confluent 6-well plates in lysis buffer (50 mM Tris/Cl, pH 8.0; 100 mM EDTA; 100 mM NaCl; 1% SDS), followed by an overnight treatment with proteinase K, mixing with saturated NaCl solution and an isopropanol precipitation of the saturated supernatant. Total RNA was extracted using the TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions.
Synthesis of cDNA
To avoid contamination by genomic DNA, 2 µg total RNA was subjected to a DNase I-digestion (5 U; Roche, Mannheim, Germany) in a buffer containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 5 mM MgCl2, 1 mM DTT for 10 min at 25°C, and 10 min at 37°C. The DNase I-treated total RNA was subsequently subjected to a reverse-transcription reaction obtaining cDNA. First-strand cDNA-synthesis of 2 µg total RNA was performed using the SuperScriptTMII RNase H-Reverse Transcriptase (Invitrogen), according to the manufacturer’s protocol, except reaction buffer concentration was 18.75 mM Tris-HCl and 0.25 mM DTT, including pd(N)6-primers (2.5 µM, Amersham) and T4 Gene 32 Protein (50 µg/ml, Roche).
Genomic and reverse transcription PCR
Genomic PCR was performed with 80 ng genomic DNA as template using the Expand High-Fidelity PCR System (Roche) according to the manufacturer’s instructions. Briefly, primer concentration was 300 nM, and concentration of each dNTP was 350 µM. The PCR profile consisted of specified genomic steps (2 min at 92°C, 35 cycles of 10 s at 92°C, 30 s at 62°C, 3 min at 68°C; 5 min at 68°C) using the following vector-specific PCR oligonucleotides: 5'-PCR- primer for 5'-GTGCCACCTGACGTCTAAGAAAC-3', 3'-PCR-primer rev 5'-GTGTGGAATTGTGAGCGGATAAC-3', 3'-PCR-primer pS rev 5'-GCGCAATTAACCCTCACTAAAGG-3'. Derived cDNA samples (aliquots of 1 µl each) were subjected to RT-PCR analyzes using the AdvantageTM-GC 2 PCR system (BD Biosciences, Heidelberg, Germany) following the manufacturer’s instructions. Briefly, primer and dNTP concentrations were 300 nM and 200 µM, respectively. The PCR profile consisted of 3 min at 94°C, 35 cycles of 20 s at 94°C, 30 s at 60°C, 30 s at 72°C, and 5 min at 72°C, and the following gene specific primers were used: CDKN2Be1 for 5'-CGGAATGCGCGAGGAGAA-3', CDKN2Be1 rev 5'-GCCTCCCGAAACGGTTGA-3', CDKN2Ae3 for 5'-TCCCCGATTGAAAGAACCAGAG-3', CDKN2Ae3 rev 5'- ACGGTAGTGGGGGAAGGCTTAT-3', CDKN2Ae1 for 5'-CAACGCACCGAATAGTTACGG-3', CDKN2Ae1 rev 5'-AACTTCGTCCTCCAGAGTCGC-3', ACTB for 5'-CCTGGGCATGGAGTCCTGT- 3', ACTB rev 5'- ACACGGAGTACTTGCGCTCA-3'. The RT-PCR primers, specific for CDKN2A transcript variant 3 (CDKN2A e1) message, were previously used in a MCL-study (23)
.
Real-time quantitative reverse transcription PCR (RQ-PCR)
Each cDNA sample was analyzed in triplicate (aliquot of 1 µl each), using the ABI PRISM 7700 Sequence Detector (PE Applied Biosystems, Foster City, CA). Quantitative assessment of DNA amplification was detected via the dye SYBR Green as described previously (9)
. To obtain a standard calibration graph, Universal Human Reference RNA (Stratagene, La Jolla, CA) was DNase I-treated and reverse-transcribed, the cDNA was serially diluted 8 times in H2O at a ratio of 2:1, and measured in every single RQ-PCR run. The resulting calibration graph (Ct vs. log unit of the standard template) correlates Ct values with the amount of template in the PCR reaction. This was performed for every amplicon separately. To standardize the amount of sample cDNA, three endogenous control amplicons were used: the housekeeping genes coding for PGK1, LMNB1 and PPIA. The average value of all three amplicons served as a calibrator for the relative expression value. The oligonucleotides used for RQ-PCR were: CCND1 for 5'-GAAGGAGGTCCTGCCGTCC-3', CCND1 rev 5'-TTTTTCACGGGCTCCAGC-3', OAS1 for 5'-CAAGAGCCTCATCCGCCTAG-3', OAS1 rev 5'-TGCTCCCTCGCTCCCAAG-3', PGK1 for 5'-AAGTGAAGCTCGGAAAGCTTCTAT-3', PGK1 rev 5'-AGGGAAAAGATGCTTCTGGG-3', LMNB1 for 5'-GATTGCCCAGTTGGAAGCCT-3', LMNB1 rev 5'-TGGTCTCGTTAATCTCCTCTTCATACA-3', PPIA for 5'-GCTCGTGCCGTTTGCA-3', PPIA rev 5'-GCAAACAGCTCAAAGGAGACG-3'.
Western-blot analysis
Proteins were isolated by lysing cells from confluent 6-well plates in 10 volumes prechilled RIPA buffer (150 mM NaCl; 50 mM Tris/Cl, pH 8.0; 1% Nonidet P-40; 0.5% Na-DOC; 0.1% SDS including Complete Mini protease inhibitor (Roche) for 30 min at 4°C. After centrifugation, the supernatant was subjected to protein concentration measurement using BCA Protein Assay Kit (Pierce, Rockford, IL), and equal amounts of protein were separated by a 12% SDS-PAGE. Following protein immunoblotting, PVDF membranes were probed with antibodies specific for cyclin D1 (CCND1, DCS-6, BD Biosciences, Cambridge, UK) and Beta-Actin (ACTB, AC-15, Abcam). Signals were visualized by enhanced chemiluminescence (ECL, Amersham, Buckinghamshire, UK).
Immunocytochemistry
Fluorescent immunolabeling was performed with 1 x 105 cells of the indicated cell lines, on cells settled down on poly-L-lysine-coated slides for 1 h at RT. After being washedwith PBS, cells were fixed (4% paraformaldehyde, 20 min on ice) and permeabilized (0.2% Triton-X100, 5 min at RT). Following an initial avidin-biotin block (10 min at RT with avidin-solution, 10 min at RT with biotin-solution, DAKO Glastrup, Denmark), slides were additionally blocked with 20% horse-serum (Linaris, Wertheim, Germany S-2000) 30 min at RT. Incubation with the primary Ab (CDKN2B, p15INK4b Ab-6, Clone 15P06, LabVision-NeoMarkers, Fremont, CA) was performed overnight at 4°C. Immunodetection was performed with biotinylated antimouse secondary Ab for 20 min at RT, followed by incubation with Fluorescein-Avidin DCS (Linaris, A-2011) for 30 min at RT. Nuclei were stained and slides were mounted via VectaShield Mounting Medium with DAPI (Linaris, H-1200), and finally slides were examined with a confocal microscopy system. Incubations omitting the specific primary Ab were used as controls.
Proliferation assay
Cell growth was determined by the CellTiter 96TM AQueous Non-Radioactive Cell Proliferation Assay (MTS colorimetric assay, Promega, Mannheim, Germany). The assay was performed in 96-well microtiter plates, according to the manufacturer’s instructions. Cells were seeded in a 48-well plate (1x106 cells per well). For each time point, aliquots of 100 µl were transferred into a 96-well plate. Absorbance was determined at 490 nM using an EL 800 microplate reader (BIO-TEK Instruments Inc., Winooski, VT), and the results were expressed as the mean absorbance of triplicate experiments ± SE.
RESULTS AND DISCUSSION
Construction of a model cell system
The cell line Granta-519, originally established from a MCL patient (24)
, carries most of the MCL typical genomic aberrations, namely t(11;14), del 1p22, del 9p21, del 13q14, del 17p13, and +18q21. This was shown by FISH and comparative genomic hybridization to DNA microarrays (matrix-CGH (25)
, Supplementary Fig. 1
online). To create a cell system for novel bidirectional RMCE approaches, we integrated a selection cassette into Granta-519 (Fig. 1)
. The cassette consists of three main elements: a neomycin gene for positive selection, a Herpes simplex virus thymidine kinase (HSV-Tk) gene for negative selection by ganciclovir, as well as the lox-sites loxP and loxP511 flanking both selection markers and allowing for sequence-specific Cre-mediated recombination. These lox-sites were placed in opposite orientation to allow RMCE directly with BACs from the RP11 library (26)
, where the identical lox-sites are embedded by the vector backbone (Fig. 1)
. Following stable integration of the selection cassette at random genomic sites, homogeneous Granta-519 subclones were selected by neomycin and limited dilution. Subclones were characterized by Southern blot analysis to determine both size and copy number of the selection cassette (data not shown), proliferation assays to compare cell cycling with the parental Granta-519 cells (data not shown), as well as FISH to assess the chromosomal site of integration (Fig. 2
A). Three monoclonal Granta-519 sublines were isolated, each containing a single cassette on chromosome arms 18q, 10p, and 11q, respectively (Fig. 2A
and Supplementary Fig. 2
online). To knock down potential oncogenes by substituting the selection cassette with shRNA coding plasmids, we modified the original RNAi pSUPER-vector. As depicted in Fig. 1
, the new vector pSUPERdL (dL for "double Lox") contains both lox-sites of the selection cassette in the same orientation, in order to use RNAi-plasmid probes directly for RMCE-approaches. Following cotransfection of a Cre-expression plasmid (pCMXhCre) with either a recombinant pBACe3.6 or a recombinant pSUPERdL, the selection markers HSV-Tk and neo are substituted by the cloned insert, and the new subclones are negatively selected via ganciclovir (GCV).
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Site-specific genomic integration
Regarding deleted regions in Granta-519, the deletion of 9p21 is the only one with loss of both copies. Therefore, the respective genes CDKN2A/B were selected for the complementation study. Biallelic loss of CDKN2A/B was confirmed by FISH using BAC-clone RP11–149I2 as probe (data not shown, indirect illustration in Fig. 2B
) and by genomic PCR (data not shown). BAC-clone RP11–149I2 was used for RMCE with all three Granta-519 sublines. Site-specific integration resulted in new sublines with a single insert on chromosome arms 10p, 11q, and 18q, respectively, as determined by FISH (illustrated for 11q in Fig. 2A, B
). Sublines with integrates at 11q and 18q were selected as monoclonal sublines and used for further analysis. Interestingly, CDKN2A/B-BAC clone FISH signals, one per RMCE modulated cell, can be detected only at the transgene integration site (11q); no signals are detected at the endogenous chromosomal site of CDKN2A/B, 9p21. B-cell lymphocytes like Granta-519 are notoriously difficult to transfect, in particular using BAC DNA. The high specificity of the RMCE system shown in the present study overcomes this limitation, since modulation of the cells was obtained even with low transfection efficiencies. CCND1 overexpression arising from the translocation t(11;14) is the genetic hallmark of MCL. Knocking down this gene is expected to have profound effects on MCL pathogenesis. High expression of CCND1 in Granta-519 was confirmed by Western blot and real-time PCR analysis (data not shown, indirectly illustrated in Figs. 3
C and 4
). A recombinant CCND1-shRNA plasmid was stably integrated into the Granta-519 sublines harboring the selection cassette on chromosome 18q and 11q, respectively. This was determined by genomic PCR, including sequence verification of these PCR products (Fig. 2C
). Further sublines were generated by integration of an empty pSUPERdL vector, and an insert coding for unspecific firefly luciferase-shRNA. These clones serve as additional controls for locus or position-dependent effects (27)
or shRNA-effects like IFN response.
|
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Modulation of gene activity
To validate our strategy for the specific modulation of gene activity, we performed expression studies on both RNA and protein concentration. As illustrated in Fig. 3A
, CDNK2A/B transcripts derived from stably integrated BAC DNA were detected by RT-PCR. Three different fragments were amplified, two of which were specific for CDKN2A (one for exon 1, the second for exon 3) and one was specific for CDKN2B (exon 1). Based on the distance of the exons of both genes, this indicates integration of the complete insert of the original BAC clone. Additionally restoration of the expression of protein coded by the CDNK2A/B-BAC clone was detected by immunocytochemical staining of CDKN2B, which was absent in unmodified Granta-519 cells (Fig. 3B
). The wild-type regulated CDKN2B protein expression in the BAC-complemented subclone is documented in relation to the expression concentration of another MCL cell line, Jeko-1, for which overexpression of CDKN2A/B had been reported (28)
. Analyzing the stable CCND1-shRNA cells, we observed significant decrease in cyclin D1 transcript (Fig. 4)
and protein (Fig. 3C
) levels. The decreased protein concentration of CCND1 was not observed in subclones stably transfected with the empty vector pSUPERdL, the selection cassette, or an unspecific shRNA. With inhibition of 72 vs. 61% measured by real-time PCR, the stable integration was superior to transient expression of CCND1-pSUPERdL (Fig. 4)
. Thus, we achieved a highly efficient knock down of an overexpressed target gene from a single pol III-shRNA transcription unit.
Prevention of RNAi induced IFN response
Since RNAi reflects an evolutionary conserved, endogenous defense mechanism against virus infection in mammalian cells, the potentially costimulated IFN-response might be difficult to distinguish from effects by the gene knock down (29
, 30)
. However, the stable CCND1-shRNA modulated cell clones did not show any up-regulation of IFN-stimulated genes like OAS1 (Fig. 4)
and STAT1 (data not shown). In contrast, transient expression of CCND1-shRNA induced OAS1 expression by a factor of 2.56. Since stable integration of the same shRNA coding insert did not induce OAS1 expression, this effect cannot be due to the CCND1 short hairpin sequence. A more potent OAS1 or STAT1 activation has been reported in various studies applying synthetic siRNA-, transient, or lentiviral shRNA-techniques on other cell types (29
, 30)
. The lack of IFN response in our system could be due to at least one of the following reasons. In transient or lentiviral applications the vector sequences, including the original viral LTRs, which are not co-integrated by RMCE, may be responsible for an antivirus response via IFN induction. This could be mediated by an IFN stimulated response element (ISRE), which was found to localize adjacent to the HIV-1 LTR sequences (31)
. Alternatively, transient and lentiviral expression systems overload cells with multiple copies of vector backbone sequences and transcription units, whereas in our RMCE-mediated RNAi-system only one copy of a transcription unit per cell is transferred to the host cells’ genome. The excessive amount of ectopic nucleic acids might be responsible for induction of the IFN pathways. Thus, our system results in a highly specific gene knock-down (Fig. 3C
and 4
) without unspecific effects due to the IFN system.
Proliferation of modulated cells is inhibited
The functional consequences of modified Granta-519 subclones were assessed using proliferation assays. The RMCE modulated cell sublines complementing CDKN2A/B and knocking down CCND1, respectively, were both derived from two parental lines with integration site on chromosome 18q and 11q. As shown in Fig. 5
, all sublines modulated in gene activity displayed a decrease in proliferation after selection to monoclonality. With respect to cell proliferation, knock down of CCND1 was sufficient to antagonize a tumor-specific gene activation. Due to the vital role of CCND1, such a test would be impossible in a knockout system. The inhibition of proliferation by restored CDKN2A/B activity was so efficient that it was difficult to maintain the respective cell clones in culture, highlighting the dramatic effect of CDKN2A/B in the pathogenesis of MCL. Interestingly, a recent study provided evidence for a direct effect of CDKN2A/p14ARF on CCND1, repressing its transcription (32)
. Thus, restoration of CDKN2A/B may affect cell proliferation also via the CCND1 pathway.
|
In conclusion, we report here for the first time a stable modulation and correction strategy for gene expression regardless of whether the gene is inactivated, deleted, or overexpressed. We document the establishment and application of a novel and flexible RMCE targeted transgenesis system, allowing for either knocking down up-regulated genes via RNA interference or complementing inactivated genes via knock-in of genomic DNA within a cellular disease system. In this regard the presented cellular tool is a sophisticated enhancement of formerly reported stable and efficient cassette exchange techniques (33)
. The bidirectional architecture of the tool is completed by the implementation of single-copy shRNA configurations, which are recently reported to study mouse models in a wild-type genetic system (34)
. The versatile transgenesis system presented in this study can be used as a screening tool exploiting BAC or shRNA plasmid libraries. It is a well-suited system to analyze the relevance of a single genomic aberration in the context of multiple genomic alterations. In animal models, genomic changes characteristic for human tumors are often introduced to mimic tumorigenesis and to test for therapeutic strategies. In contrast, the efficient RMCE-based modulation of human cells derived from disease tissue, carrying different genetic alterations, provides a powerful alternative to study pathogenic gene function and to test interfering therapeutic measures. As demonstrated by the modulated MCL-cells, the presented tool will enhance the understanding of diseases, which originates from complex genetic defects.
Online Supplemental Material
Supplementary Figure 1: Matrix-CGH profile of cell line Granta-519. Supplementary Figure 2: Detection of integration site by FISH.
ACKNOWLEDGMENTS
We thank Erich Greiner for critical and fruitful discussions, Sabine Görisch and Holger Kohlhammer for their methodical support, and Francis Stewart for his critical reading of the manuscript. This work was supported by a grant of the Fritz Thyssen Stiftung (10.04.1.169) and a grant of the BMBF (NGFN2:SMP-DNA/01GR0417), and grants of the EU(LSHC-CT-2004-503351) and Krebschilfe (70-3173-Tr3,B4) to S.S. to P.L.
FOOTNOTES
2 Conflict of Interest statement: The authors declare that they have no potential conflicts of interests. 
Received for publication September 13, 2005. Accepted for publication January 20, 2006.
REFERENCES
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  E. Ortega-Paino, J. Fransson, S. Ek, and C. A. K. Borrebaeck Functionally associated targets in mantle cell lymphoma as defined by DNA microarrays and RNA interference Blood, February 1, 2008; 111(3): 1617 - 1624. [Abstract] [Full Text] [PDF]
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