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PubMed2004

Signaling and transcriptional changes critical for transformation of human cells by simian virus 40 small tumor antigen or protein phosphatase 2A B56gamma knockdown.

One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein phosphatase 2A (PP2A). Here we have identified changes in gene expression induced by ST and show that ST mediates these changes through both PP2A-dependent and PP2A-independent mechanisms. Knockdown of PP2A B56gamma subunit can substitute for ST expression to fully transform cells expressing LT, hTERT, and Ras-V12. We also identify those genes affected similarly in two cell lines that have been fully transformed from a common parental line by two alternative mechanisms, namely ST expression or PP2A B56gamma subunit knockdown. ST altered expression of genes involved in proliferation, apoptosis, integrin signaling, development, immune responses, and transcriptional regulation. ST reduced surface expression of MHC class I molecules, consistent with a need for SV40 to evade immune detection. ST expression enabled cell cycle progression in reduced serum and src phosphorylation in anchorage-independent media, whereas B56gamma knockdown required normal serum levels for these phenotypes. Inhibitors of integrin and src signaling prevented anchorage-independent growth of transformed cells, suggesting that integrin and src activation are key ST-mediated events in transformation. Our data support a model in which ST promotes survival through constitutive integrin signaling, src phosphorylation, and nuclear factor kappaB activation, while inhibiting cell-cell adhesion pathways.

Antigens, Polyomavirus Transforming↗
PubMed2006

Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells.

We evaluated the contribution of three genetic alterations (p53 knockdown, K-RAS(V12), and mutant EGFR) to lung tumorigenesis using human bronchial epithelial cells (HBEC) immortalized with telomerase and Cdk4-mediated p16 bypass. RNA interference p53 knockdown or oncogenic K-RAS(V12) resulted in enhanced anchorage-independent growth and increased saturation density of HBECs. The combination of p53 knockdown and K-RAS(V12) further enhanced the tumorigenic phenotype with increased growth in soft agar and an invasive phenotype in three-dimensional organotypic cultures but failed to cause HBECs to form tumors in nude mice. Growth of HBECs was highly dependent on epidermal growth factor (EGF) and completely inhibited by EGF receptor (EGFR) tyrosine kinase inhibitors, which induced G1 arrest. Introduction of EGFR mutations E746-A750 del and L858R progressed HBECs toward malignancy as measured by soft agar growth, including EGF-independent growth, but failed to induce tumor formation. Mutant EGFRs were associated with higher levels of phospho-Akt, phospho-signal transducers and activators of transcription 3 [but not phospho-extracellular signal-regulated kinase (ERK) 1/2], and increased expression of DUSP6/MKP-3 phosphatase (an inhibitor of phospho-ERK1/2). These results indicate that (a) the HBEC model system is a powerful new approach to assess the contribution of individual and combinations of genetic alterations to lung cancer pathogenesis; (b) a combination of four genetic alterations, including human telomerase reverse transcriptase overexpression, bypass of p16/RB and p53 pathways, and mutant K-RAS(V12) or mutant EGFR, is still not sufficient for HBECs to completely transform to cancer; and (c) EGFR tyrosine kinase inhibitors inhibit the growth of preneoplastic HBEC cells, suggesting their potential for chemoprevention.

Aged↗
PubMed2006

Cyclin E1 knockdown induces apoptosis in cancer cells.

OBJECTIVES: Cyclin E1 is expressed during the late G1 phase of the cell cycle and mediates the initiation of DNA synthesis by activating cyclin-dependent kinases 2 (CDK2). Abnormally high levels of cyclin E1 expression have frequently been found in cancer cells. Here, we investigate the effect of cyclin E1 knockdown on cancer cells. METHODS: RNA interference, expressed from a DNA-based retroviral vector, was used to knockdown cyclin E1 in adenocarcinoma (HeLa), breast (MDA-MB-31) and glioblastoma (U-373-MG) cell lines and an explant from one glioma patient (GB-LP-2). RESULTS: We have obtained very efficient depletion of cyclin E1 protein (over 80%) and considerable apoptotic induction (50-70%) after 96 hours post-infection. The ability of U-373-MG cells to induce tumor growth in nude mice was also abolished after cyclin E1 knockdown. DISCUSSION: Our results indicate that retrovirus carrying the DNA to be transcribed into a short hairpin RNA (shRNA) against cyclin E1 could be used as a therapeutic agent for cancer therapy.

Animals↗
PubMed2003

Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells.

BACKGROUND: Astrocytomas are the most common type of primary central nervous system tumors. They are frequently associated with genetic mutations that deregulate cell cycle and render these tumors resistant to apoptosis. STAT3, signal transducer and activator of transcription 3, participates in several human cancers by inducing cell proliferation and inhibiting apoptosis and is frequently activated in astrocytomas. METHODS: RNA interference was used to knockdown STAT3 expression in human astrocytes and astrocytoma cell lines. The effect of STAT3 knockdown on apoptosis, cell proliferation, and gene expression was then assessed by standard methods. RESULTS: We have found that STAT3 is constitutively activated in several human astrocytoma cell lines. Knockdown of STAT3 expression by siRNA induces morphologic and biochemical changes consistent with apoptosis in several astrocytoma cell lines, but not in primary human astrocytes. Moreover, STAT3 is required for the expression of the antiapoptotic genes survivin and Bcl-xL in the A172 glioblastoma cell line. CONCLUSION: These results show that STAT3 is required for the survival of some astrocytomas. These studies suggest STAT3 siRNA could be a useful therapeutic agent for the treatment of astrocytomas.

Apoptosis↗
PubMed2006

The biological effects of CRP are not attributable to endotoxin contamination: evidence from TLR4 knockdown human aortic endothelial cells.

C-reactive protein (CRP) is the prototypic marker of inflammation and a strong predictor of cardiovascular events in humans. There are questions regarding the validity of the biological effects reported for CRP, in spite of adherence to rigorous control measures minimizing endotoxin [lipopolysaccharide (LPS)] contamination in these in vitro studies. In this study, we addressed the key question of endotoxin contamination in CRP preparations using Toll-like receptor 4 (TLR4) knockdown endothelial cells. Human aortic endothelial cells (HAECs) transfected with prevalidated TLR4 small interfering RNA (siRNA) and scrambled siRNA controls were challenged with pleural fluid-derived CRP or LPS for 12-16 h. Secreted interleukin-6 (IL-6), IL-1beta, IL-8, and plasminogen activator inhibitor-1 (PAI-1) levels and endothelial Nitric oxide synthase (eNOS) activity were determined. TLR4 knockdown in HAECs significantly decreased LPS-induced IL-1beta, IL-6, and IL-8, whereas the stimulatory effects of CRP were similar in both scrambled control and TLR4 knockdown cells. Furthermore, CRP significantly stimulated PAI-1 levels in both control and TLR4-transfected cells and inhibited eNOS activity, whereas LPS effects were negated in TLR4-transfected cells. The data presented cogently demonstrate and further confirm that the biological effects of CRP on HAECs are independent of LPS and thus are attributable to native protein per se. This is the first study to positively authenticate the significance of earlier in vitro reports on CRP biological effects.

Aorta↗
PubMed2006

Lentiviral short hairpin ribonucleic acid-mediated knockdown of GLUT4 in 3T3-L1 adipocytes.

Adipose tissue is an important insulin target organ, and 3T3-L1 cells are a model cell line for adipocytes. In this study, we have used lentivirus-mediated short hairpin RNA (shRNA) for functional gene knockdown in 3T3-L1 adipocytes to assess the molecular mechanisms of insulin signaling. We chose to target GLUT4 to validate this approach. We showed that lentiviruses efficiently delivered transgenes and small interfering RNA (siRNA) into fully differentiated 3T3-L1 adipocytes. We established a strategy for identifying efficient siRNA sequences for gene knockdown by transfecting 293 cells with the target gene fluorescent fusion protein plasmid along with a plasmid that expresses shRNA. Using these methods, we identified highly efficient siGLUT4 sequences. We demonstrated that lentivirus-mediated shRNA against GLUT4 reduced endogenous GLUT4 expression to almost undetectable levels in 3T3-L1 adipocytes. Interestingly, insulin-stimulated glucose uptake was only reduced by 50-60%, suggesting that another glucose transporter mediates part of this effect. When siGLUT1 was introduced into GLUT4-deficient adipocytes, insulin-stimulated glucose uptake was essentially abolished, indicating that both GLUT4 and GLUT1 contribute to insulin-stimulated glucose transport in 3T3-L1 adipocytes. We also found that GLUT4 knockdown led to impaired insulin-responsive aminopeptidase protein expression that was dependent on whether GLUT4 was knocked down in the differentiating or differentiated stage. We further found that GLUT4 expression was not required for adipogenic differentiation but was necessary for full lipogenic capacity of differentiated adipocytes. These studies indicate that lentiviral shRNA constructs provide an excellent approach to deliver functional siRNAs into 3T3-L1 adipocytes for studying insulin signaling and adipocyte biology.

3T3-L1 Cells↗
PubMed2005

Knockdown of the complete Hox paralogous group 1 leads to dramatic hindbrain and neural crest defects.

The Hox paralogous group 1 (PG1) genes are the first and initially most anterior Hox genes expressed in the embryo. In Xenopus, the three PG1 genes, Hoxa1, Hoxb1 and Hoxd1, are expressed in a widely overlapping domain, which includes the region of the future hindbrain and its associated neural crest. We used morpholinos to achieve a complete knockdown of PG1 function. When Hoxa1, Hoxb1 and Hoxd1 are knocked down in combination, the hindbrain patterning phenotype is more severe than in the single or double knockdowns, indicating a degree of redundancy for these genes. In the triple PG1 knockdown embryos the hindbrain is reduced and lacks segmentation. The patterning of rhombomeres 2 to 7 is lost, with a concurrent posterior expansion of the rhombomere 1 marker, Gbx2. This effect could be via the downregulation of other Hox genes, as we show that PG1 function is necessary for the hindbrain expression of Hox genes from paralogous groups 2 to 4. Furthermore, in the absence of PG1 function, the cranial neural crest is correctly specified but does not migrate into the pharyngeal arches. Embryos with no active PG1 genes have defects in derivatives of the pharyngeal arches and, most strikingly, the gill cartilages are completely missing. These results show that the complete abrogation of PG1 function in Xenopus has a much wider scope of effect than would be predicted from the single and double PG1 knockouts in other organisms.

Animals↗
PubMed2006

Stable knockdown of polycystin-1 confers integrin-alpha2beta1-mediated anoikis resistance.

The mechanisms of action of polycystin-1 (PC1) have been difficult to dissect because of its interaction with multiple factors, the heterogeneity of the genetic mutations, and the complexity of the experimental animal models. Here, stable knockdown of PC1 in MDCK epithelial cells was achieved by lentiviral-mediated delivery of a specific small interfering RNA for PKD1. The reduction of PC1 expression prevented tubulogenesis in three-dimensional collagen type I culture in response to hepatocyte growth factor and induced formation of cysts. PC1 knockdown created a condition of haploinsufficiency that led to hyperproliferation, increased adhesion to collagen type I, and increased apoptosis. It was shown that the suppression of PC1 was associated with the increased expression of integrin-alpha2beta1 and reduced apoptosis in cells grown on collagen type I. The engagement of integrin-alpha2beta1 seemed to be essential for the survival because PC1 knockdown cells were significantly less susceptible to anoikis by a mechanism that was reversible by anti-integrin-alpha2beta1 blocking antibodies. Overall, these data link integrin-alpha2beta1 to some of the biologic functions that are ascribed to PC1 and establish the potential of this approach for the direct study of PC1 functions in a genetically defined background. Furthermore, these findings indicate that reduction of PC1 expression levels, rather than the loss of heterozygosity, may be sufficient to induce cystogenesis.

Animals↗
PubMed2006

siRNA mediated knockdown of fibroblast growth factor receptors 1 or 3 inhibits FGF-induced anchorage-independent clonogenicity but does not affect MAPK activation.

Supplementation with exogenous growth factors such as fibroblast growth factors (FGFs) is essential for anchorage-independent growth of the SW-13 human adrenal adenocarcinoma cell line. We have found that SW-13 cells express mRNAs for FGFRs 1, 3, and 4, but not FGFR2. To assess the roles of individual FGFRs, in anchorage-independent growth, we determined the effects of down-regulation of each FGFR on FGF2- and FGF4-mediated soft agar colony formation in these cells. Using RNAi strategies we found that knockdown of either FGFR1 or FGFR3 leads to inhibition of FGF2- or FGF4-induced soft agar clonogenicity without affecting that induced by heregulin beta1. However, this inhibition is independent of ERK1/2 activation as levels of FGF-induced phospho-ERK 1/2 remain unchanged upon knockdown of either FGFR1 or FGFR3. Conversely, RNAi-mediated knockdown of FGFR4 appeared to have no significant effect on either FGF2- or FGF4-induced anchorage-independent colony formation, or ERK1/2 phosphorylation. These results suggest that constitutive levels of both FGFR1 and FGFR3, but not FGFR4 are essential for FGF-stimulated anchorage-independent growth of SW-13 cells.

Adrenal Cortex Neoplasms↗
PubMed2025

Proliferation Analyses of Conditional Knockdown Strains Using CRISPR Interference in Fission Yeast.

CRISPR interference is a method to conditionally inhibit transcription of an arbitrary target gene. This is useful to study the functions of essential genes, which are required for cellular viability. Although many conditional gene perturbation techniques are available for Schizosaccharomyces pombe, CRISPRi facilitates construction of a large number of knockdown strains because of its systematic, simple procedure. Here, we describe a method to construct and characterize knockdown strains using dCas9-mediated CRISPRi in S. pombe, including a variation of CRISPRi induction technique in a 96-well format for high-throughput studies.

Schizosaccharomyces↗
PubMed2025

CCDC137 knockdown suppresses bladder cancer progression by downregulating SCD.

BACKGROUND: The Coiled-coil domain-containing (CCDC) family, due to its unique protein structural domain and broad involvement in diverse biological processes, has emerged as a focus in oncology research. Nevertheless, its clinical significance and function in bladder cancer (BLCA) remain poorly defined. METHODS: Machine learning algorithms were employed to identify pivotal CCDC genes in the cancer genome atlas (TCGA), and a prognostic model was subsequently constructed. Multi-omics data encompassing pan-cancer cohorts, single-cell sequencing, and spatial transcriptomics were integrated to characterize the expression patterns and prognostic significance of Coiled-coil domain-containing 137 (CCDC137), a previously uncharacterized CCDC family member in BLCA. Tissue microarray confirmed CCDC137 abnormal expression in bladder carcinoma specimens. The effect of CCDC137 knockdown on BLCA progression was evaluated through CCK8 assay, clonogenic formation, wound healing, Transwell, and subcutaneous xenograft models. RNA sequencing, quantitative RT-PCR, and western blot were utilized to delineate its regulatory network. RESULTS: A prognostic model incorporating 10 CCDC genes was successfully established in the TCGA-BLCA cohort. Then, we found that CCDC137 exhibited pan-cancer overexpression and usually correlation with poor clinical outcomes. Immunohistochemistry further substantiated its dysregulation in bladder carcinoma. Integrated multi-omics analyses suggested associations between CCDC137 expression and a tumor immunosuppressive microenvironment. CCDC137 knockdown significantly suppressed bladder cancer cell proliferation and migratory capacity in vitro. Correspondingly, subcutaneous xenograft tumor growth was inhibited in vivo. Moreover, decreased expression of stearoyl-CoA desaturase (SCD), a key lipid metabolic enzyme, accompanied CCDC137 depletion. These findings collectively suggest a cancer-promoting role for CCDC137 in bladder carcinoma. CONCLUSIONS: This systematic investigation combining multi-omics bioinformatics analyses and experimental validation demonstrates the role of CCDC137 in bladder carcinoma progression, providing novel mechanistic insights into the pathogenesis of BLCA and offering a theoretical foundation for therapeutic targeting of CCDC137 in urothelial malignancies.

Urinary Bladder Neoplasms↗
PubMed2006

[Selective knockdown of Angiotensin II receptor subtype 1a in rat vascular smooth muscle cells by RNA interference].

OBJECTIVE: To selectively knockdown the expression of Angiotensin II receptor subtype 1a (AT1aR) in rat vascular smooth muscle cells (VSMCs) by RNA interference and the sequential effects on cellular viability and proliferation. METHODS: The primary cultured rat aortic VSMCs were transfected by plasmids pAT1a-shRNA1 and pAT1a-shRNA2, each carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence, or by a control plasmid pGenesil-Control (pCon) carrying a nonspecific shRNA-coding sequence. The mRNA and protein expressions of AT1a, AT2 were analyzed by semi-quantified RT-PCR and Western blot, respectively and normalized to the internal control gene beta-actin. Cellular viability and proliferation were determined with methylthiazoletetrazolium (MTT) assay. RESULTS: AT1a mRNA and protein were reduced by 82% and 69% by pAT1a-shRNA1, 77% and 56% by pAT1a-shRNA2, respectively while no change was found in pCon treated VSMCs. AT2 receptor level in VSMCs remains unchanged after various treatments. The A(490nm) values obtained by MTT measurements were similar among groups in the absence of Ang II but decreased significantly in pAT1a-shRNA1 and pAT1a-shRNA2 treated VSMCs in the presence of Ang II. CONCLUSION: RNA interference can selectively knockdown AT1a expression in cultured VSMCs and attenuate the Ang II induced cell proliferation. Future studies are warranted to explore the potential role of RNA interference on AT1 function and as a new gene therapy tool for cardiovascular diseases.

Animals↗
PubMed2005

RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisation.

TES was originally identified as a candidate tumour suppressor gene and has subsequently been found to encode a novel focal adhesion protein. As well as localising to cell-matrix adhesions, TES localises to cell-cell contacts and to actin stress fibres. TES interacts with a variety of cytoskeletal proteins including zyxin, mena, VASP, talin and actin. There is evidence that TES may function in actin-dependent processes as overexpression of TES results in increased cell spreading and decreased cell motility. Together with TES's interacting partners, these data suggest that TES might be involved in regulation of the actin cytoskeleton. Here, for the first time, we have used RNAi to successfully knockdown TES in HeLa cells and we demonstrate that loss of TES from focal adhesions results in loss of actin stress fibres. Similarly, and as previously reported, RNAi-mediated knockdown of zyxin results in loss of actin stress fibres. TES siRNA treated cells show reduced RhoA activity, suggesting that the Rho GTPase pathway may be involved in the TES RNAi-induced loss of stress fibres. We have also used RNAi to examine the requirement of TES and zyxin for each other's localisation at focal adhesions, and we propose a hierarchy of recruitment, with zyxin being first, followed by VASP and then TES. Cell Motil.

Actins↗
PubMed2006

siRNA gelsolin knockdown induces epithelial-mesenchymal transition with a cadherin switch in human mammary epithelial cells.

Epithelial-mesenchymal transition (EMT) describes a process occurring during development and oncogenesis by which epithelial cells obtain fibroblast-like properties and show reduced cell adhesion and increased motility. In this report, we demonstrated typical EMT in human mammary epithelial MCF10A small interfering (si)RNA gelsolin-knockdown cells. EMT was characterized by fibroblastic morphology, loss of contact inhibition and focus formation in monolayer growth, enhanced motility and invasiveness in vitro, increased actin filaments, overexpression of RAC, activation of both extracellular signal-regulated kinase and AKT, inactivation of glycogen synthase kinase-3, conversion of cadherin from the E- to N-type and induction of the transcription factor Snail. These results suggested that gelsolin functions as a switch that controls E- and N-cadherin conversion via Snail, and demonstrated that its knockdown leads to EMT in human mammary epithelial cells and possibly to the development of human mammary tumors.

Breast Neoplasms↗
PubMed2006

RNAi-mediated knockdown of aldehyde dehydrogenase class-1A1 and class-3A1 is specific and reveals that each contributes equally to the resistance against 4-hydroperoxycyclophosphamide.

PURPOSE: Aldehyde dehydrogenases class-1A1 (ALDH1A1) and class-3A1 (ALDH3A1) have been associated with resistance to cyclophosphamide (CP) and its derivatives. We have previously reported the downregulation of these enzymes by all-trans retinoic acid (ATRA). METHODS: In this study, we used siRNA duplexes as well as retrovirally expressed siRNA to knockdown one or both enzymes together in A549 lung cancer cell line in order to investigate the role of each one in mediating the resistance and the effect of the addition of ATRA. RESULTS: The results show that significant and specific knockdown of each enzyme can be achieved and that each one contributes similarly to cell resistance to 4-hydroperoxycyclophosphamide (4-HC), an active derivative of CP. Added effects were seen when both enzymes were inhibited. The addition of ATRA also exhibited additional inhibitory effects on ALDH activity and increased 4-HC toxicity when added to single siRNA aimed at one of the enzymes. On the other hand, ATRA had minimal and insignificant additional inhibitory effects on ALDH enzyme activity when added to a combination of siRNAs against both enzymes, but still increased 4-HC toxicity beyond that seen with RNAi-mediated inhibition of both enzymes together. CONCLUSIONS: We conclude that both enzymes, ALDH1A1 and ALDH3A1 will need to be blocked in order to achieve the highest sensitivity to 4-HC. Furthermore, ATRA increases 4-HC toxicity even when added to a combination of siRNAs against both enzymes, thus suggesting additional mechanisms by which ATRA can increase drug toxicity.

Actins↗
PubMed2006

Establishing efficient siRNA knockdown in mouse embryonic stem cells.

FAM-labeled oligo dT (FAMdT) was utilized as a means to gauge efficient transfection of small nucleic acids into mouse embryonic stem cell (mESC) colonies. Using colonies grown overnight, transfection was restricted largely to the periphery of the colonies with only a 40% decrease in Oct-3/4 RNA transcript levels following cognate Oct-3/4, small interfering RNA (siRNA) delivery. However, transfection of mESC 4 h after seeding gave greater than 90% cells being successfully transfected based on quantitative real-time PCR detection of approximately 90% Oct-3/4 RNA transcript knockdown. This method provides an economical and efficient means by which to determine effective transfection conditions, and establish efficient siRNA knockdown of reportedly difficult to transfect cell lines such as mESC.

Animals↗
PubMed2005

Construction of a functional transporter analysis system using MDR1 knockdown Caco-2 cells.

PURPOSE: The efflux transporter, P-glycoprotein (P-gp), located in the brush-border membrane of intestinal absorptive cells, reduces the bioavailability of a wide range of orally administered drugs. Using P-gp inhibitors in transport experiments in Caco-2 cell monolayers is widely accepted as an efficient way to estimate the contribution of P-gp to the intestinal absorption of drugs. However, there still remain some arguments that the inhibitors might affect the function of other proteins. Multidrug resistance 1 gene (MDR1) specifically inhibited Caco-2 cells were constructed, therefore, as a better in vitro evaluation system of intestinal drug absorption. METHODS: The effective sites of RNAi were selected using siRNA libraries and single siRNAs and MDR1 stable knockdown Caco-2 cells were constructed using a tRNA(val)-shRNA expression vector. RESULTS: In siRNA stably expressed Caco-2 cells, the expression level of MDR1 was reduced at mRNA and protein levels. Transcellular transport studies using digoxin revealed that the P-gp function was suppressed completely, similar to that in verapamil-treated cells. CONCLUSIONS: MDR1 stable knockdown Caco-2 cells were successfully constructed by RNAi technology. This will consequently allow the development of a selection system for candidate drugs with improved absorption properties.

Biological Transport, Active↗
PubMed2004

Transposon-based RNAi delivery system for generating knockdown cell lines.

RNA interference is rapidly becoming a powerful tool for genetic analyses in mammalian systems. A potential drawback to transient small inhibitory RNA silencing is the short duration of downregulation it confers, usually only 24-72h. Viral-based vector systems for the long-term delivery of RNA hairpins have been developed, yet they require expertise in viral production and transduction. Here we describe a simple plasmid-based system for the generation of long-term gene knockdown utilizing RNA interference combined with the gene delivery capabilities of the mammalian Tc1-like transposon Sleeping Beauty. Designated Maleficent, this system is shown to downregulate exogenous expression of GFP in a constitutively positive cell line. In addition, targeting of the endogenously expressed lamin A gene results in long-term silencing with significant reduction in protein levels (> 95%). Maleficent therefore provides a relatively easy, efficient, and stable means of delivering RNAi hairpins to generate long-term gene-specific knockdown cell lines.

Animals↗