Rapid conditional knock-down–knock-in system for mammalian cells

RNA interference (RNAi) is a powerful tool to analyze gene function in mammalian cells. However, the interpretation of RNAi knock-down phenotypes can be hampered by off-target effects or compound phenotypes, as many proteins combine multiple functions within one molecule and coordinate the assembly of multimolecular complexes. Replacing the endogenous protein with ectopic wild-type or mutant forms can exclude off-target effects, preserve complexes and unravel specific roles of domains or modifications. Therefore, we developed a rapid-knock-down–knock-in system for mammalian cells. Stable polyclonal cell lines were generated within 2 weeks by simultaneous selection of two episomal vectors. Together these vectors mediated reconstitution and knock-down in a doxycycline-dependent manner to allow the analysis of essential genes. Depletion was achieved by an artificial miRNA-embedded siRNA targeting the untranslated region of the endogenous, but not the ectopic mRNA. To prove effectiveness, we tested 17 mutants of WDR12, a factor essential for ribosome biogenesis and cell proliferation. Loss-off function phenotypes were rescued by the wild-type and six mutant forms, but not by the remaining mutants. Thus, our system is suitable to exclude off-target effects and to functionally analyze mutants in cells depleted for the endogenous protein.     

RNA interference: implications for cancer treatment

RNA interference (RNAi) has emerged as one of the most important discoveries of the last years in the field of molecular biology. Following clarification of this highly conserved endogenous gene silencing mechanism, RNAi has largely been exploited as a powerful tool to uncover the function of specific genes and to understand the effects of selective gene silencing in mammalian cells both in vitro and in vivo. RNAi can be induced by direct introduction of chemically synthesized siRNAs into the cell or by the use of plasmid and viral vectors encoding for siRNA allowing a more stable RNA knockdown. Potential application of this technique both as a research tool and for therapeutic purposes has led to an extensive effort to overcome some critical constraints which may limit its successful application in vivo, including off-target and non-specific effects, as well as the relatively poor stability of siRNA. This review provides a brief overview of the RNAi mechanism and of its application in preclinical animal models of cancer.     

Reconstitution of CKMT1 expression fails to rescue cells from mitochondrial membrane potential dissipation: Implications for controlling RNAi experiments

RNA interference (RNAi) is an essential method in molecular biology to reduce the expression of target genes and thereby determine their function. Since this tool is known to also have unspecific effects, control experiments are needed, chiefly among them the exclusion of off-target effects and the reconstitution of the genes' expression for the rescue of the cellular RNAi effects. We show here that the knock-down of the mitochondrial creatine kinase-1 (CKMT1) by RNA interference causes the dissipation of the mitochondrial membrane potential ΔΨ_m. This was accomplished with 11 different RNAi constructs designed to target 7 distinct exons as well as exon/intron junctions making unspecific off-target effects unlikely. However, all our attempts failed to rescue human cells from ΔΨ_m dissipation by the expression of CKMT1 alleles not targeted by RNAi. This included the transient and stable expression of the murine CKMT1 homologue, the expression of human codon usage-modified alleles, the transfection of a novel splice-isoform of CKMT1, and even the introduction of a human genomic clone for CKMT1 with codon usage changes. Our results indicate that while off-target effects of RNA interference can easily be addressed, the rescue of the knock-down phenotype is not necessarily achievable.     

A novel class of temperature-sensitive mutants generated by RNAi-mediated knockdown

Temperature-sensitive (ts) mutants are powerful tools with which to investigate gene function, but it has been difficult to generate ts mutants in mammalian cells. Recently, RNA interference (RNAi) has been widely used for loss of function analyses. In addition, in various organisms, hypothermic-temperature-sensitive RNAi has been reported. By using this characteristic of RNAi, we attempted to generate ts mutants in mammalian cells and were able to successfully generate ts mutants of cell cycle regulator cdc2 and ubiquitin-activating enzyme E1. We compared ts mutants previously isolated by mutagenesis with those generated by RNAi knockdown, and observed similar phenotypes. This method enabled us to generate ts mutants (KDts, knockdown temperature-sensitive mutants) of the genes of interest and will be utilized to facilitate understanding of the biological processes regulated by an essential gene in mammalian cells.     

XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination

Homologous recombinational repair preserves chromosomal integrity by removing double-strand breaks, cross-links, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2/3, Rad51B/C/D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 exists in a single complex with Rad51C. To examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a nonconservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF Chinese hamster ovary cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, whereas ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of dysfunction of the mutants, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon co-expression in bacteria, nickel-affinity purification, and Western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, whereas the K113R mutant did not and was predominantly insoluble. The addition of 5 mm ATP but not ADP also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 probably regulates the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis with both processes being essential for the ability of the complex to participate in homologous recombinational repair.     

An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells

RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.     

The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness.

To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.     

Disturbance of the microRNA pathway by commonly used lentiviral shRNA libraries limits the application for screening host factors involved in hepatitis C virus infection

RNA interference (RNAi) is widely used as a screening tool for the identification of host genes involved in viral infection. Due to the limitation of raw small interfering RNA (siRNA), we tested two commonly used short hairpin RNA (shRNA) lentiviral libraries to identify host factors involved in hepatitis C virus (HCV) infection. It was found that these shRNA library vectors caused non-specific disturbance of HCV replication that was not due to toxicity or interferon response, but related to the high shRNA levels disturbing the endogenous microRNA biogenesis. The high shRNA levels achieved with these vectors reduced the levels of mature microRNAs, including miR-122 known to promote HCV replication. Our findings extend the caution of potential off-target effects of lentiviral shRNA libraries which appear unsuitable to screen microRNA regulated phenotypes, such as HCV replication.     

RISC assembly defects in the Drosophila RNAi mutant armitage

The putative RNA helicase, Armitage (Armi), is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. Here, we show that armi is required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi. Our results suggest that armi is required for RISC maturation.     

The Chinese hamster dihydrofolate reductase replication origin beta is active at multiple ectopic chromosomal locations and requires specific DNA sequence elements for activity

To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb fragment from the Chinese hamster dihydrofolate reductase (DHFR) locus containing the origin beta (ori-beta) initiation region was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-beta in uncloned pools of transfected cells was measured using a competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-beta region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-beta deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-beta region is sufficient to direct initiation and that specific DNA sequences in the ori-beta region are required for efficient initiation activity.     

A relatively low level of ribosome depurination by mutant forms of ricin toxin A chain can trigger protein synthesis inhibition,cell signaling and apoptosis in mammalian cells

The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces ribotoxic stress, which activates stress-induced cell signaling cascades and apoptosis. However, the mechanistic relationship between depurination, protein synthesis inhibition and apoptosis remains an open question. We previously identified two RTA mutants that suggested partial independence of these processes in a yeast model. The goals of this study were to establish an endogenous RTA expression system in mammalian cells and utilize RTA mutants to examine the relationship between depurination, protein synthesis inhibition, cell signaling and apoptosis in mammalian cells. The non-transformed epithelial cell line MAC-T was transiently transfected with plasmid vectors encoding precursor (pre) or mature forms of wild-type (WT) RTA or mutants. PreRTA was glycosylated indicating that the native signal peptide targeted RTA to the ER in mammalian cells. Mature RTA was not glycosylated and thus served as a control to detect changes in catalytic activity. Both pre- and mature WT RTA induced ribosome depurination, protein synthesis inhibition, activation of cell signaling and apoptosis. Analysis of RTA mutants showed for the first time that depurination can be reduced by 40% in mammalian cells with minimal effects on inhibition of protein synthesis, activation of cell signaling and apoptosis. We further show that protein synthesis inhibition by RTA correlates more linearly with apoptosis than ribosome depurination.     

RNAi knock-down mice: an emerging technology for post-genomic functional genetics

RNA interference (RNAi) has been extensively used for sequence-specific silencing of gene function in mammalian cells. The latest major breakthrough in the application of RNAi technology came from experiments demonstrating RNAi-mediated gene repression in mice and rats. After more than two decades of functional mouse research aimed at developing and continuously improving transgenic and knock-out technology, the advent of RNAi knock-down mice represents a valuable new alternative for studying gene function in vivo. In this review we provide some basic insight as to how RNAi can induce gene silencing to then focus on recent findings concerning the applicability of RNAi for regulating gene function in the mouse. Reviewed topics will include delivery methods for RNAi-mediating molecules, a comparison between traditional knock-out and innovative transgenic RNAi technology and the generation of graded RNAi knock-down phenotypes. Apart from the exciting possibilities RNAi provides for studying gene function in mice, we discuss several caveats and limitations to be considered. Finally, we present prospective strategies as to how RNAi technology might be applied for generating conditional and tissue-restricted knock-down mice.     

The use of integrating DNA vectors to analyse the molecular defects in ionising radiation-sensitive mutants of mammalian cells including ataxia telangiectasia

Integrating DNA vectors, encoding selectable recombinant genes, were used to assess rejoining and recombination in wild-type mammalian cells and their ionising radiation-sensitive mutants. To provide a simple model of an important radiation-induced lesion - the DNA double-strand break - the vectors were cut with restriction endonucleases at specific single sites. If these breaks were made in the coding sequence of a selectable gene, the fidelity of the rejoin/recombination process could be measured by survival of vector-transformed cells in selective medium. Rejoining was assessed using vectors without internal homologies, while recombination was measured using pairs of fragments or deletion vectors carrying homologous regions. Initial experiments were made with vectors carrying a single selectable gene but, to overcome potential artefacts, 2-gene vectors were then constructed where one gene acts as a linked marker and (unbroken) control for the other (broken) gene. Available data are reviewed to show that, compared to their respective wild-type counterparts: (1) an ataxia telangiectasia (A-T) cell line and the hamster irs1 mutant show a consistent reduction in the fidelity of rejoining double-strand breaks (while the hamster mutants irs2, irs3, xrs series, and EM9 show wild-type fidelity); (2) the hamster EM9 mutant shows a reduction in ability to recombine homologous vector fragments (while the A-T line and probably the xrs mutants show show wild-type abilities); and (3) the xrs mutants show a reduction in overall transformation frequency with vector DNA, whether broken or not, while the other mutants tested show approximately wild-type frequencies. A critical account of the techniques and data is given, together with speculations on the molecular nature of the processes which are defective in these mutants, leading to radiosensitivity.     

Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs

A number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand fundamental cellular mechanisms, they require information and knowledge of existing sequences since the RNAi sequences are generated synthetically. An alternative strategy would be to create an RNAi library from cDNA. Unfortunately, the complexity of such a library of siRNAs would make screening difficult. To reduce the complexity, longer dsRNAs could be used; however, concerns of induction of the interferon response and off-target effects of long dsRNAs have prevented their use. As a first step in creating such libraries, long dsRNA was expressed in mammalian cells. The 250 nt dsRNAs were capable of efficiently silencing a luciferase reporter gene that was stably transfected in MDA-MB-231 cells without inducing the interferon response or off-target effects any more than reported for siRNAs. In addition, a long dsRNA expressed in the same cell line was capable of silencing endogenous c-met expression and inhibited cell migration, whereas the dsRNA against luciferase had no effect on c-met or cell migration. The studies suggest that large dsRNA libraries are feasible and that functional selection of genes will be possible.     

The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division

We identify a mitochondrial E3 ubiquitin ligase, MARCH5, as a critical regulator of mitochondrial fission. MARCH5 RING mutants and MARCH5 RNA interference induce an abnormal elongation and interconnection of mitochondria indicative of an inhibition of mitochondrial division. The aberrant mitochondrial phenotypes in MARCH5 RING mutant-expressing cells are reversed by ectopic expression of Drp1, but not another mitochondrial fission protein Fis1. Moreover, as indicated by abnormal clustering and mitochondrial accumulation of Drp1, as well as decreased cellular mobility of YFP-Drp1 in cells expressing MARCH5 RING mutants, MARCH5 activity regulates the subcellular trafficking of Drp1, likely by impacting the correct assembly at scission sites or the disassembly step of fission complexes. Loss of this activity may account for the observed mitochondrial division defects. Finally, MARCH5 RING mutants and endogenous Drp1, but not wild-type MARCH5 or Fis1, co-assemble into abnormally enlarged clusters in a Drp1 GTPase-dependent manner, suggesting molecular interactions among these proteins. Collectively, our data suggest a model in which mitochondrial division is regulated by a MARCH5 ubiquitin-dependent switch.     

An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase

Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is a neurodegenerative disease characterized by motor neuron degeneration, paralysis and death. One cause of this disease is mutations in the Cu,Zn superoxide dismutase (SOD1) gene. As mutant SOD1 acquires a toxic property that kills motor neurons, by reducing the mutant protein the disease progression may be slowed or prevented. While mutant SOD1 is toxic, the wild-type SOD1 is indispensable for motor neuron health. Therefore, the ideal therapeutic strategy would be to inhibit selectively the mutant protein expression. Previously we have demonstrated that RNA interference (RNAi) can selectively inhibit some mutant SOD1 expression. However, more than 100 SOD1 mutants can cause ALS and all mutants cannot be inhibited selectively by RNAi. To overcome this obstacle, we have designed a replacement RNAi strategy. Using this strategy, all mutants and wild-type genes are inhibited by RNAi. The wild-type SOD1 function is then replaced by designed wild-type SOD1 genes that are resistant to the RNAi. Here we demonstrate the concept of this strategy.