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Disease-oriented Genome Networks Cancer
Combating Cancer through Integrated Functional Genomic Research

RNA Interference as a Tool for Functional Gene Analysis and Therapeutic
Gene Silencing in Cancer Research

Matthias Truss, Reinhold Schäfer, Christian Hagemeier
- Charité - Berlin University Medicine -

cellular systems are hard or almost impossible to transfect. RNA interference (RNAi) is an evolutionary well conserved In collaboration with Miletnyi, Bergisch-Gladbach, we have mechanism for post-transcriptional gene silencing. Once constructed vectors that combine expression units for cell incorporated into a protein complex with nuclease activity surface markers and shRNAs (short hairpin RNAs). This (RNA induced silencing complex, RISC), short double strategy allows the selective enrichment of transfected stranded RNA molecules (Small interfering RNAs, siRNAs) shRNA-expressing cells and the subsequent analysis of can serve as a guide to recognize and cleave RNA molecules harbouring a homologous sequence. During the last years, RNAi has turned to be an indispensable tool not only for the functional analysis of cancer related genes but has also been successfully applied for the inhibition of pMACS-KK2-H1
tumour cell proliferation in vitro and in vivo. These findings suggest that, due to the specificity and efficacy of siRNA ,mediated gene silencing, such molecules may hold great therapeutic potential. However, high costs for chemically synthesized siRNA molecules and delivery problems still hamper the broad applicability of RNA interference Fig 1: Human K562 cells have been transfected with either
pMACS-KK2-H1 without insert (EV) or with a gene specific short hairpin insert directed against E2F4 (E2F4). Tranfected To facilitate the applicability of RNAi within the CancerNet, and untransfected K562 cells were mixed in a 1:20 ratio to we have established new RNA interference related tools and mimic a transfection efficiency of 5%. 20 h post transfection, technologies. These include a procedure for the reliable, transfected cells were enriched using the MACS technology cost effective semi automated synthesis of siRNA molecules and bound (b; transfected cells) and flow through (ft; (patent pending) as well as the construction of sets of short untransfected cells) cell populations were cultured separately hairpin (shRNA) expression vectors and a human siRNA for additional 52h. Expression of E2F4 and ß-actin were Enzymatic synthesis of siRNA molecules
This example demonstrates the potential of the pMACS-KK2- We have established a procedure for the cost effective semi- H1 vector system. Magnetic enrichment of transfected cells automated enzymatic synthesis of siRNA molecules in is so efficient that an almost complete knock down of E2F4 96well scale. Such siRNA molecules transferred into human can be observed in the enriched cell population (E2F4 b), leukaemia and colon cancer cells exhibited equally good although the transfected (cell surface marker and shRNA gene silencing activity and specificity as chemically expressing) subpopulation represented only 5% of the total synthesized siRNA molecules at significantly reduced costs. cell population. Therefore, this plasmid system enables A proof of principle experiment with 69 siRNA molecules gene knock down experiments in cells that are hard to synthesized in parallel demonstrated the high, and more transfect without demanding optimization of transfection importantly, comparable yields, underlining the robustness of conditions or purchasing expensive electroporation our method. This technology has already been used to synthesize a set of mutants of a potent siRNA sequence for This strategy can yield sufficient material for subsequent a systematic study of the effects of position dependent point mRNA preparations for Affymetrix chip hybridization experiments and even for extensive biochemical analyses. The availability of pMACS-KK2-H1 vectors harboring positive Human siRNA Database
and negative control inserts and detailed magnetic selection Despite the strong progress made in the siRNA field during protocols as well as the user friendly designed shRNA insert the last two years, gene knock down experiments still require cloning strategy that is compatible with the popular pSuper a labour and cost intensive identification of potent siRNA vectors and their derivatives are additional advantages of sequences and the development of protocols for efficient this plasmid based RNA interference system. siRNA delivery into the cell lines of interest that are frequently refractory towards siRNA incorporation. This pRep-H1: long term gene silencing with
prompted us to establish the human siRNA database replicating vectors
HuSiDa (1). It provides sequences of published functional Silencing of essential genes will result in obvious siRNA molecules targeting human genes and important phenotypes, easily detectable in standard culture conditions technical details of the corresponding gene silencing after transient transfection of siRNA molecules. In contrast, experiments, including the mode of siRNA generation, functional analysis of modulating genes (modifiers) requires recipient cell lines, transfection reagents and procedures and more sophisticated assays monitoring growth factor direct links to published references (PubMed), thereby dependence, responses to genotoxic stress etc. Such supporting the setup and actual procedure of specific RNAi assays are often incompatible with transient transfection of siRNA molecules that often requires high cell densities that can result in cell confluency 48 to 72 hours after transfection. pMACS-KK-H1: Magnetic enrichment shRNA-
Even worse, signal transduction pathways activated by expressing cells.
transient transfection of siRNAs can act synergistically with High transfection efficiencies are an absolute prerequisite for genotoxic stress induced signal transduction pathways. successful knock down experiments. Unfortunately many Disease-oriented Genome Networks Cancer
Therefore, long term gene silencing with shRNA expressing availability of vectors already containing positive and vectors is the method of choice for the functional negative control shRNA-inserts make it an attractive characterization of nonessential genes. Unfortunately, stable alternative to existing retroviral shRNA expression vectors. transfection of conventional shRNA expression vectors has severe disadvantages. In most cases, only a very small pFlp-H1: FLP-In shRNA expression vector
fraction of transfected cells (>0.1%) will integrate transfected One disadvantage of replicating vectors is that their plasmids into their genome. Even worse, not all antibiotic maintenance requires continuous selection pressure. resistant cells will express shRNAs. Therefore, cell clones Therefore, these vectors are not suited for the generation have to be picked and characterized. In addition to the extra stably transfected cell pools to study gene function effort, the usage of cell clones can also complicate the (metastasizing, response to genotoxic stress etc.) in identification of target genes, because already randomly xenograph mouse models. This prompted us to construct the picket clones from untransfected “homogeneous” cell pFlp-H1 vector. It enables the generation of isogenic cell populations may display severe differences in gene pools by Flp-recombinase mediated, site specific integration of a shRNA expression cassette thatara also especially In an attempt to establish alternatives to retroviral vectors for qualified for Affymetrix array analysis of knock down the use in non S2 laboratories, we have constructed replicating hairpin expression vectors for long-term gene Services and collaborations
The performance of the pRep-H1 vector has been For NGFN2 members, the proprietary shRNA expression extensively tested in several cell lines, including K562, vectors are available on request, including all necessary LN229, HEK293, and U373. In collaborations with several positive and negative control constructs. As a special other CancerNet-members, it was successfully used to service for NGFN2 members, we also offer to generate pools generate numerous pools of stably transfected cells for the of cell lines, stable transfected with our replicating vector functional analysis of cancer related genes. pRep-H1 and its derivatives for the functional analysis of nonessential genes in cell culture. This includes assistance in the design of shRNA sequences, cloning of shRNA coding inserts, sequencing, transfection into the cell line provided by the collaborator, the generation of stable transfected cell pools and the monitoring cell proliferation during the initial antibiotic selection period to detect and to document proliferation associated knock down phenotypes followed by shipping the cell pools to our collaboration partners for detailed characterization. We will continue to support CancerNet RNA interference i
F g 1: Human HEK293 tranfected with either pRep-H1
users by providing informations about functional siRNA vectors targeting EGFP (EGFP); E2F4 (E2F4 21) and E2F6 sequences, suitable recipient cell lines and validated (E2F6A; E2F6B). Four week after transfection, expression transfection protocols. We will also continue to provide our levels of E2F4 and E2F6 were analyzed by western blotting. “siRNA transfection efficiency control kit” that includes positive and negative siRNAs (both, synthetic siRNAsand As shown in Fig. 2, the replicating, shRNA expression vector
shRNA expression vectors), real time PCR primers and pREp-H1 mediates efficient long term knock down of target detailed protocols for siRNA knock down experiments. genes in mammalian cells (>4 weeks in continuous culture We are trying to advance siRNA delivery methodology tested). pRep-H1 transfected cells are unable to separate focusing on alternatives to conventional siRNA transfection antibiotic resistance and shRNA expression, because first, even after 4 weeks the complete pool of antibiotic cells still We are also continuously improving our shRNA expression displays almost quantitative gene silencing and second, all vectors. Derivatives of pRep-H1 with alternative antibiotic our attempts to generate antibiotic resistant K562 cell pools resistance genes and/or co-expressing red fluorescent expressing shRNA directed against BCR-ABL-kinase, protein have already been constructed and are under essential for K562 cell survival, have failed (data not shown). examination. The main focus here is on the development of This eliminates the need for picking and characterization of Cre-lox and tetracycline regulated shRNA expression vectors cell clones with all the aforementioned associated that will enable the functional characterization of any gene in disadvantages. Transcripts of some shRNA expression pools of stable transfected cell populations. vectors, especially such based on the U6 promoter, have been described to induce interferon response. This limits the Lit.: 1. Truss et al., HuSiDa—the human siRNA database: an
applicability of such vectors. The comparative Affymetrix open access database for published functional siRNA array analysis of the expression profiles of HEK293 cell sequences and technical details of efficient transfer into pools stably transfected with pRep-H1 expression with or recipient cells. Nucleic Acids Res. 2005 Jan 1; 33 Database without shRNA-inserts using did show not any evidence for a Issue:D108-11. 2. Wiebusch et al., Inhibition of human
shRNA specific activation of interferon response genes. cytomegalovirus replication by small interfering RNAs. J. Consequently, pRep-H1 seems especially suited for long Gen. Virol. 2004 Jan; 85 (Pt 1): 179-84. 3. Tchernitza, O. et
term gene silencing in mammalian cells. Its user friendly al., Transcriptional basis of KRAS oncogene-mediated designed shRNA insert cloning strategy that is compatible cellular transformation in ovarian epithelial cells. Oncogene with the pSuper vectors and their derivatives and the



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