Single Gene Knockdown Research Articles

Abstract 3869: SOCS3-mediated regulation of inflammatory cytokines in triple-negative breast cancer

Abstract We demonstrated that knock-down of tumor suppressors p53 and PTEN in mammary epithelial MCF10A cells (called MCF10A-p53-PTEN-) results in generation of aggressive tumors. Furthermore MCF10A-p53-PTEN- cells also display an increased EMT phenotype as well as expansion of cancer stem cell (CSC) population. Utilizing Affymetrix gene expression analyses, we confirmed that malignant MCF10Ap53-PTEN- cells displayed a gene expression pattern which closely resembled that of the basal/claudin molecular subtype with the TN. This subtype is previously characterized by the expression of EMT and stem cell genes. The Inflammatory cytokines IL6 and TGF-β have been found to regulate stem cells and EMT. Consistent with these data, we found that MCF10A-p53-PTEN- cells secreted significantly higher levels of IL6 (>1000 fold) and TGF-β (>200 fold) compared to parental or single gene deleted cells. Furthermore combined deletion of p53 and PTEN compared to single gene knockdown resulted in transformation of MCF10A cells generating aggressive metastatic tumors in NOD/SCID mice. IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the SOCS3 mediated Stat3/NF-κB pathway. Consistent with these reports, we demonstrated that SOCS3 is abundantly expressed in MCF10A cells where it negatively regulates the IL6 feedback loop. Unexpectedly, we found that SOCS3 protein was undetectable in MCF10A-p53-PTEN- cells. Basal/claudin-low breast cancer cell lines that display an activated IL6 feedback loop also had low SOCS3 protein levels. Furthermore, we found a similar discordance between SOCS3 protein and mRNA levels in primary basal/claudin-low breast cancer samples as demonstrated by immunochemistry utilizing a primary breast cancer tissue array. Together these findings suggest that loss of SOCS3 protein expression allows for maintenance of the IL6 mediated inflammatory feedback loop in aggressive/metastatic TN breast cancers. We found that MCF10A-p53-PTEN- cells displayed increased proteasome activity as evidenced by SOCS3 protein accumulation upon proteasome inhibition. Proteolytic degradation of SOCS3 protein in turn is controlled by constitutive activation of the IL6 mediated Stat3/NF-κB pathway. We demonstrate that enforced expression of SOCS3 or IL6 pathway inhibition interfering with this loop through IL6R blockade repress the CSC population, reducing the tumor growth and metastasis in mouse xenografts. These studies provide a strong rationale for development of IL6 pathway-targeting agents for the treatment of TNBC, an aggressive disease that currently lacks molecularly targeted therapeutics. Citation Format: Hasan Korkaya, Maria Ouzounova, Gwangil Kim, Ali Quraishi, April Davis, Nader Tawakkol, Sumeyye Korkaya, Max S. Wicha. SOCS3-mediated regulation of inflammatory cytokines in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3869. doi:10.1158/1538-7445.AM2014-3869

Relevance of different prior knowledge sources for inferring gene interaction networks.

When inferring networks from high-throughput genomic data, one of the main challenges is the subsequent validation of these networks. In the best case scenario, the true network is partially known from previous research results published in structured databases or research articles. Traditionally, inferred networks are validated against these known interactions. Whenever the recovery rate is gauged to be high enough, subsequent high scoring but unknown inferred interactions are deemed good candidates for further experimental validation. Therefore such validation framework strongly depends on the quantity and quality of published interactions and presents serious pitfalls: (1) availability of these known interactions for the studied problem might be sparse; (2) quantitatively comparing different inference algorithms is not trivial; and (3) the use of these known interactions for validation prevents their integration in the inference procedure. The latter is particularly relevant as it has recently been showed that integration of priors during network inference significantly improves the quality of inferred networks. To overcome these problems when validating inferred networks, we recently proposed a data-driven validation framework based on single gene knock-down experiments. Using this framework, we were able to demonstrate the benefits of integrating prior knowledge and expression data. In this paper we used this framework to assess the quality of different sources of prior knowledge on their own and in combination with different genomic data sets in colorectal cancer. We observed that most prior sources lead to significant F-scores. Furthermore, their integration with genomic data leads to a significant increase in F-scores, especially for priors extracted from full text PubMed articles, known co-expression modules and genetic interactions. Lastly, we observed that the results are consistent for three different data sets: experimental knock-down data and two human tumor data sets.

MP25-09 HIGH THROUGHPUT GENETIC SCREENING WITH DROSOPHILA MELANOGASTER IDENTIFIES NOVEL GENES ASSOCIATED WITH STONE FORMATION

You have accessJournal of UrologyStone Disease: Basic Research II1 Apr 2014MP25-09 HIGH THROUGHPUT GENETIC SCREENING WITH DROSOPHILA MELANOGASTER IDENTIFIES NOVEL GENES ASSOCIATED WITH STONE FORMATION Thomas Chi, Man Su Kim, Sven Lang, Tiffany Zee, Gulinuer Muteliefu, Sarah Blaschko, David Killilea, Katja Bruckner, Arnold Kahn, Pankaj Kapahi, and Marshall Stoller Thomas ChiThomas Chi More articles by this author , Man Su KimMan Su Kim More articles by this author , Sven LangSven Lang More articles by this author , Tiffany ZeeTiffany Zee More articles by this author , Gulinuer MuteliefuGulinuer Muteliefu More articles by this author , Sarah BlaschkoSarah Blaschko More articles by this author , David KillileaDavid Killilea More articles by this author , Katja BrucknerKatja Bruckner More articles by this author , Arnold KahnArnold Kahn More articles by this author , Pankaj KapahiPankaj Kapahi More articles by this author , and Marshall StollerMarshall Stoller More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.311AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Vertebrate models for nephrolithiasis are limited in their ability to rapidly screen multiple and varied interventions that modulate urinary stone formation. A short lifespan and rapid stone formation makes Drosophila melanogaster an ideal system to screen large numbers of interventions to assess their effect on kidney stone formation. A method of screening single gene knockdown candidates for their ability to prevent the formation of urinary stones was successfully developed utilizing Drosophila. METHODS Microdissection facilitates identification and collection of fly stones within the lumen of the Drosophila Malpighian tubule (the functional equivalent of the human renal tubule). In a similar fashion as seen in humans, fly stones frequently obstruct the ureter and result in early death. Utilizing shortened lifespan as a surrogate for stone formation, an unbiased screen of orthologues for human genes known to be associated with nephrolithiasis was undertaken. A double knockdown unbiased screen to identify genes that lengthened lifespan and modulated the stone formation process was developed; microdissection confirmed these results. RESULTS Stone formation was associated with a significantly shortened fly lifespan (mean lifespan 60 days with no stones, 3 days with stones, p<0.05). An unbiased double knockdown screen of more than 80 genes was performed utilizing the GAL4-UAS RNAi system and identified 7 genes that rescued lifespan. Microdissection confirmed that three genes decreased stone formation in the fly, including genes encoding a salt transporter (SLC5A5), a zinc transporter (ZnT35C), and a regulator of oxidative stress (Cyp4d1). These lengthened lifespan to 9, 12, and 11 days (p<0.05) respectively and significantly modulated the stone formation process (see Table). CONCLUSIONS A Drosophila urinary stone model was leveraged to perform large scale genetic screens to identify novel genes that modulate calculi formation. Newly identified genes may represent novel targets that could translate to new therapies to prevent or reduce the formation of urinary stones. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e269-e270 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Thomas Chi More articles by this author Man Su Kim More articles by this author Sven Lang More articles by this author Tiffany Zee More articles by this author Gulinuer Muteliefu More articles by this author Sarah Blaschko More articles by this author David Killilea More articles by this author Katja Bruckner More articles by this author Arnold Kahn More articles by this author Pankaj Kapahi More articles by this author Marshall Stoller More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 244: Cancer stem cells predict engraftment and poor prognosis of primary breast tumor.

Abstract Despite the promise of the cancer stem cell (CSC) model, its clinical relevance remains to be proven in breast cancer (BC) and requires a sensitive xenograft assay to define CSC on key functional properties i.e tumorigenicity, self-renewal and differentiation potential. We report the establishment of primary breast tumor-derived xenografts (PDXs) that retain main primary tumors features (histo-phenotypic, molecular and genomic features, hierarchical organization of the tumoral cell population). We demonstrate that success in engraftment is correlated with the presence of CSC in primary tumor and predicts prognosis in patients (HR= 3.61; 95%CI [1.12-11.65], p=3.20E-02). The xenograft assay demonstrated the hierarchical organization of BC. Analysis of gene expression from functionally validated CSC yield to a breast CSC signature (BCSC-GES) is an independent predictor of patient survival. We then identified a core of genes commonly expressed by BCSC and embryonic, hematopoietic, neural stem cells, the CE-4SC. This shared transcriptional program comprised 19 genes encoding mismatch and nucleotide excision repair proteins, proteins implicated in transport through the endoplasmic reticulum, RNA processing, translation and transcriptional regulators, enzymes of detoxification and mitochondrial/oxidative stress, or enzymes of lipid metabolism. This “universal” stem cell program is also an independent predictor of patient survival. Using a screening of an RNA-interference library specially designed to block the expression of these 19 target genes, we validated the effect of single gene knock-down (KD) on the breast CSC population for 14 genes (KD of 5 genes decreased the CSC population and were called “self-renewers”genes, KD of 9 genes increased the CSC, and were designated as“differentiators”). Hence, this study validated the clinical relevance of CSC model in breast cancer for the first time, and the ability of PDXs to accelerate transfer for personalized CSC therapy into clinics. Citation Format: Emmanuelle Charafe-Jauffret, Christophe Ginestier, Francois Bertucci, Olivier Cabaud, Jose Adelaide, Max Chaffanet, Luc Xerri, Patrice Viens, Daniel Birnbaum. Cancer stem cells predict engraftment and poor prognosis of primary breast tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 244. doi:10.1158/1538-7445.AM2013-244

Administration of PLGA nanoparticles carrying shRNA against focal adhesion kinase and CD44 results in enhanced antitumor effects against ovarian cancer

The two membrane-bound proteins, focal adhesion kinase (FAK) and CD44, are involved in processes critical to cancer progression. FAK has an active role in angiogenesis, cell proliferation and cell apoptosis, whereas the heavily glycosylated CD44 has been implicated in cancer metastasis. Here, using short hairpin RNA (shRNA) against FAK and CD44, we demonstrate that simultaneous knockdown of both these genes inhibits cancer growth more efficiently than knockdown of either gene individually. Plasmids targeting these genes or non-relative control sequences were constructed and delivered to ovarian cancer targets by biodegradable poly D,L-lactide-co-glycolide acid nanoparticles (PLGANPs). Nude mice were utilized in an intraperitoneal model of ovarian carcinomatosis to assess antitumor efficacy in vivo. Single gene knockdown resulted in significantly smaller tumors than those observed in the empty-vector control (P's<0.001). More importantly, knockdown of both genes resulted in tumors smaller than both the empty-vector group (P<0.0001) and the single gene knockdown groups (P's<0.001). Knockdown of both FAK and CD44 resulted in tumors with inhibited angiogenesis, reduced proliferation and increased apoptosis as compared with controls (P's<0.001) and single knockdown groups (P's<0.05). These results indicate that dual knockdown of FAK and CD44 in the tumors of patients with ovarian cancer may have an enhanced therapeutic effect, and point toward a mechanism involving the inhibition of angiogenesis, cellular proliferation and the induction of apoptosis.

Joint estimation of causal effects from observational and intervention gene expression data

BackgroundIn recent years, there has been great interest in using transcriptomic data to infer gene regulatory networks. For the time being, methodological development in this area has primarily made use of graphical Gaussian models for observational wild-type data, resulting in undirected graphs that are not able to accurately highlight causal relationships among genes. In the present work, we seek to improve the estimation of causal effects among genes by jointly modeling observational transcriptomic data with arbitrarily complex intervention data obtained by performing partial, single, or multiple gene knock-outs or knock-downs.ResultsUsing the framework of causal Gaussian Bayesian networks, we propose a Markov chain Monte Carlo algorithm with a Mallows proposal model and analytical likelihood maximization to sample from the posterior distribution of causal node orderings, and in turn, to estimate causal effects. The main advantage of the proposed algorithm over previously proposed methods is its flexibility to accommodate any kind of intervention design, including partial or multiple knock-out experiments. Using simulated data as well as data from the Dialogue for Reverse Engineering Assessments and Methods (DREAM) 2007 challenge, the proposed method was compared to two alternative approaches: one requiring a complete, single knock-out design, and one able to model only observational data.ConclusionsThe proposed algorithm was found to perform as well as, and in most cases better, than the alternative methods in terms of accuracy for the estimation of causal effects. In addition, multiple knock-outs proved to contribute valuable additional information compared to single knock-outs. Finally, the simulation study confirmed that it is not possible to estimate the causal ordering of genes from observational data alone. In all cases, we found that the inclusion of intervention experiments enabled more accurate estimation of causal regulatory relationships than the use of wild-type data alone.

Slit/Robo-mediated axon guidance in Tribolium and Drosophila: Divergent genetic programs build insect nervous systems

As the complexity of animal nervous systems has increased during evolution, developmental control of neuronal connectivity has become increasingly refined. How has functional diversification within related axon guidance molecules contributed to the evolution of nervous systems? To address this question, we explore the evolution of functional diversity within the Roundabout (Robo) family of axon guidance receptors. In Drosophila, Robo and Robo2 promote midline repulsion, while Robo2 and Robo3 specify the position of longitudinal axon pathways. The Robo family has expanded by gene duplication in insects; robo2 and robo3 exist as distinct genes only within dipterans, while other insects, like the flour beetle Tribolium castaneum, retain an ancestral robo2/3 gene. Both Robos from Tribolium can mediate midline repulsion in Drosophila, but unlike the fly Robos cannot be down-regulated by Commissureless. The overall architecture and arrangement of longitudinal pathways are remarkably conserved in Tribolium, despite it having only two Robos. Loss of TcSlit causes midline collapse of axons in the beetle, a phenotype recapitulated by simultaneous knockdown of both Robos. Single gene knockdowns reveal that beetle Robos have specialized axon guidance functions: TcRobo is dedicated to midline repulsion, while TcRobo2/3 also regulates longitudinal pathway formation. TcRobo2/3 knockdown reproduces aspects of both Drosophila robo2 and robo3 mutants, suggesting that TcRobo2/3 has two functions that in Drosophila are divided between Robo2 and Robo3. The ability of Tribolium to organize longitudinal axons into three discrete medial–lateral zones with only two Robo receptors demonstrates that beetle and fly achieve equivalent developmental outcomes using divergent genetic programs.

Therapeutic Potential of Targeted HOXA-TALE Knockdown in AML,

Abstract 3460HOX genes are master regulators of hematopoietic stem and progenitor cell (HSPC) development. HOX interaction with TALE (PBX or MEIS) proteins is pivotal to their function. The prevailing hypothesis is that HOX/TALE expression underlies HSPC self-renewal while dysregulated HOX/TALE expression underpins maintenance of the leukemia initiating cell. Expression profiling studies support this hypothesis and highlight the importance of the HOXA cluster and TALE genes in hematopoiesis and leukemogenesis. The aim of this study was to identify and target clinically relevant HOX and TALE genes in AML cells representative of the particular disease subtype.HOXA cluster and leukemia-associated TALE gene expression was evaluated in 166 favourable and intermediate AML patient samples by Affymetrix microarray and expression of the significantly different genes was confirmed by RQ-PCR assays in an independent patient cohort. HOXA1-A2, A4-A10, PBX3 and MEIS1 displayed significant differential expression (p’0.01) between favourable and intermediate AML subgroups and confirmed an association of HOXA/TALE with nucleophosmin gene mutations (NPM1-mut) previously identified as a favourable prognostic marker in AML. HOXA9 was shown to be differentially expressed and HOXA6 was identified as the most consistently and highly expressed member of the previously reported ‘HOX code’ by microarray analysis. These two HOX genes together with MEIS1 and PBX3 were selected for further study. Immunoprecipitation assays identified HOXA6/9-PBX3-MEIS1 protein interactions in AML cell lines demonstrating potential functional benefit of targeting members of these complexes in the disease setting.The HOXA/TALE axis was targeted in OCI AML3 (NPM1-mut) and U937 (NPM1-WT) AML cells using specific shRNAs (at least two per target) with Nucleofection or viral delivery followed by puromycin selection. Knockdown was validated by RQ-PCR as greater than 70% and western blotting where compatible antibodies were available. Cell viability was assessed using CellTiter-Glo® and direct cell counting whilst cell death was determined using the Caspase-Glo™assay. Cell responses were examined following targeted single gene knockdown of HOXA6, HOXA9, MEIS1 and PBX3, dual knockdown of HOXA6+HOXA9 either alone or in combination with standard-of-care therapeutic agents (Cytarabine or Mylotarg). Altered expression of genes associated with cell cycle and adhesion was identified following targeted gene knockdown using the Roche AmpliChip containing 1500 leukemia-associated probesets.Targeted reduction of HOXA6, HOXA9, MEIS1 PBX3 and combined HOXA6+HOXA9 levels greatly reduced cell growth compared to controls and was additive when combined with IC50 values of the chemotherapies used in the treatment of AML. This effect on cell growth may be due in part to increased apoptosis since increased caspase3/7 activity (up to 3.3 fold for PBX3 in U937 cells) was observed following HOX or TALE knockdown in either cell line. Furthermore, HOX or TALE knockdown resulted in impaired colony formation in both cell lines either due to reduction in number, size or metabolic activity compared to non-silenced controls. In addition, altered cell morphology with reduced blast-like features, was observed by HOXA6, HOXA9, MEIS1 or PBX3 knockdown in both cell lines. Together the data support a key role for HOX-TALE in AML and demonstrate dependency on this axis in leukemia maintenance.In summary, HOXA/TALE knockdown impairs growth and sensitises leukemic cells to chemotherapy independent of NPM1 mutation status. These findings suggest targeting of clinically significant HOXA cluster genes and their critical co-factors or modulation of downstream pathways could offer a novel strategy in the treatment of AML. Disclosures:No relevant conflicts of interest to declare.

Insecticide detoxification indicator strains as tools for enhancing chemical discovery screens

Insecticide discovery screens carried out on whole organisms screen for potency resulting from chemical activity at the target site. However, many potentially insecticidal compounds are naturally detoxified in vivo and do not make it to the target site. It is hypothesised that insect strains with their xenobiotic detoxification machinery compromised could be used to identify such compounds that normally fail to show up in screens; these compounds could then be more rationally designed to increase their bioavailability. This strategy was tested with transgenic Drosophila lines with altered expression of Cyp6g1 and Dhr96. It was observed that Cyp6g1 knockdown transgenic lines have increased susceptibility to the test compound imidacloprid, while Dhr96 knockdown transgenic lines are resistant. Evidence was found for a systemic response to xenobiotic exposure, uncovered by piperonyl butoxide treatment and by gene expression profiling. Sex-specific gene expression regulated by DHR96 was also observed. The results confirm that this approach to chemical discovery could identify compounds that escape traditional screens. The complexity of the system means that a panel of single and multiple gene knockdown transgenic lines may be required.

Abstract 229: A cross-species approach to identifying genetic modifiers of BRCA1-associated breast cancer risk

Abstract Background: Although carriers of germline mutations of BRCA1 have a high risk of developing breast and ovarian cancers, penetrance is not complete, and it is likely that unidentified genes modify the risk for individual mutation carriers. We hypothesize that genes that modify the penetrance of cancer in BRCA1 carriers interact with BRCA1 mutations to confer increased sensitivity to DNA damage at the cellular level, and that cellular sensitivity to DNA damage can be used as an intermediate phenotype to predict breast cancer susceptibility in humans. Objective: Our goal is to test a cross-species systems biology approach to predict gene-gene interactions underlying breast cancer susceptibility, using sensitivity to DNA damage as an intermediate phenotype. Experiment Procedure: The yeast RAD9 gene is one putative homolog of the human breast tumor suppressor gene BRCA1. We conducted a genome-wide screen to identify genes that interact with a rad9 mutation to confer enhanced sensitivity to DNA damage. The yeast screen identified 25 interactions, from which we selected 10 candidate gene-gene interactions to be tested in human breast epithelial cells. To determine if the gene-gene interactions discovered in yeast are conserved in human mammary epithelial cells, we built stable human mammary epithelial cells (MCF10A) capable of single- or double-gene knockdown via lentivirus-mediated RNA interference. Cells confirmed for intended gene knockdown were subject to a clonogenic assay to determine if increased sensitivity to DNA damage can be observed in double gene vs. single gene knockdowns. Results: Among the 10 pairs of interactions tested in MCF10A cells, two genes, HLTF and MGMT, were found to interact with BRCA1 to confer enhanced DNA damage sensitivity, based on a clonogenic assay. Cells with combined knockdown of BRCA1 and HLTF show increased sensitivity to a chronic low dose of MMS treatment compared with those with only single knockdown of BRCA1 or HLTF. In addition, distinct morphological features were observed in double knockdown cells with MMS treatment. Cells became enlarged and showed a flat, senescence-like morphology. Even a low concentration of 0.001% MMS induced a strong and irreversible growth arrest in cells with combined knockdown of BRCA1 and HLTF, but not in the single knockdown cells. Conclusion: Our results suggest that it is feasible to use a cross-species (yeast-to-human) comparative systems genetics strategy to identify gene-gene and pathway-pathway interactions. Ongoing studies are aimed at determining whether any of the observed gene-gene interactions contribute to breast cancer penetrance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 229. doi:10.1158/1538-7445.AM2011-229

Sloppy models, parameter uncertainty, and the role of experimental design.

Computational models are increasingly used to understand and predict complex biological phenomena. These models contain many unknown parameters, at least some of which are difficult to measure directly, and instead are estimated by fitting to time-course data. Previous work has suggested that even with precise data sets, many parameters are unknowable by trajectory measurements. We examined this question in the context of a pathway model of epidermal growth factor (EGF) and neuronal growth factor (NGF) signaling. Computationally, we examined a palette of experimental perturbations that included different doses of EGF and NGF as well as single and multiple gene knockdowns and overexpressions. While no single experiment could accurately estimate all of the parameters, experimental design methodology identified a set of five complementary experiments that could. These results suggest optimism for the prospects for calibrating even large models, that the success of parameter estimation is intimately linked to the experimental perturbations used, and that experimental design methodology is important for parameter fitting of biological models and likely for the accuracy that can be expected from them.

Gene silencing in a human organotypic skin model

Here we present a simple and highly reproducible method which allows the study of the effects of a single gene knockdown in an organotypic skin model. Human keratinocytes (KC) were transfected with backbone-modified short interfering RNAs (siRNAs) specific for vascular endothelial growth factor (VEGF) and matriptase-1. Twenty-four hours later the transfected cells were seeded onto fibroblast collagen suspensions and allowed to build up a multilayered epidermis by culture at the air/medium interface for 7 days. Protein expression of both targeted genes remained down-regulated by more than 80% up to 8 days after transfection. As expected, VEGF knockdown by siRNA did not alter epidermis formation in our organotypic skin model. By contrast ablation of matriptase-1 led to aberrant KC differentiation and impaired filaggrin processing and resulted in an epidermal phenotype closely resembling that of matriptase-1 deficient mouse skin. Our results suggest that siRNA-mediated gene silencing is highly efficient in an organotypic skin model and readily allows the assessment of the roles of individual genes during terminal KC differentiation.

Knocking‐down the NMDAR1 subunit in a limited amount of neurons in the rat hippocampus impairs learning

Amplicon vectors derived from herpes simplex virus type 1 were built to modify NMDA receptors by expressing antisense RNA for the essential NR1 subunit. Their ability to modify endogenous levels of NR1 was tested in cultures of rat embryo neocortical neurons. We studied behaviour and tested for expression in adult rats injected with those vectors into the dorsal hippocampus to find out which cells and how many appear involved in memory formation. Rats injected with vectors expressing NR1 antisense performed significantly worse than control rats in an inhibitory avoidance task. Immunohistochemistry was performed in brain slices from the same animals. The transduced cells represented 6-7% of pyramidal neurons in CA1, showing that a single gene knockdown of NR1 in a small number of neurons significantly impaired memory formation. Perhaps neurons undergoing synaptic plasticity are more susceptible to NR1 knockdown, and hence NMDAR are particularly required in those neurons undergoing synaptic plasticity during learning, or perhaps, and more likely, there is not a high level of redundancy in the hippocampal circuits involved, leading to the idea that a certain level of NR1 expression/availability appears necessary for memory formation in most of CA1 pyramidal neurons.