Inducible Plasmid Research Articles

Optimization of protein trans-splicing in an inducible plasmid display system for high-throughput screening and selection of soluble proteins

Directed evolution is widely used to optimize protein folding and solubility in cells. Although the screening and selection of desired mutants is an essential step in directed evolution, it generally requires laborious optimization and/or specialized equipment. With a view toward designing a more practical procedure, we previously developed an inducible plasmid display system, in which the intein (auto-processing) and Oct-1 DNA-binding (DBD) domains were used as the protein trans-splicing domain and DNA-binding module, respectively. Specifically, the N-terminal (CfaN) and C-terminal (CfaC) domains of intein were fused to the C-terminal end of the His-tag and the N-terminal end of Oct-1 DBD to generate His6-CfaN and CfaC-Oct-1, respectively. For such a system to be viable, the efficiency of protein trans-splicing without the protein of interest (POI) should be maximized, such that the probability of occurrence is solely dependent on the solubility of the POI. To this end, we initially prevented the degradation of l-arabinose (the inducer of the PBAD promoter) by employing an Escherichia coli host strain deficient in the metabolism of l-arabinose. Given that a low expression of His6-CfaN, compared with that of CfaC-Oct-1, was found to be conducive to the generation to a soluble product of the protein trans-splicing event, we designed the expression of His6-CfaN and CfaC-Oct-1 to be inducible from the PBAD and PT7 promoters, respectively. The optimized system thus obtained enabled in vitro selection of the plasmid–protein complex with high yield. We believe that the inducible plasmid display system developed in this study would be applicable to high-throughput screening and/or selection of protein variants with enhanced solubility.

Microbots Gene Delivery System Based on Bifidobacteria in a Tumor Model

In cancer, the use of microbots based on anaerobic bacteria as specific transporters targeting tumor tissues has been explored since most solid tumors exhibit hypoxic regions. The aim of this study was to develop and characterize magnetic microbots based on Bifidobacteria and iron oxide fluorescent magnetic nanoparticles complexed with chitosan and a hypoxia inducible plasmid. In addition, the efficiency of the microbots for gene delivery to solid tumors was evaluated in an in vivo model by florescence and luminescence. To elaborate microbots, iron oxide fluorescent magnetic nanoparticles complexed with chitosan and a hypoxia-inducible plasmid called nanocomplex (NCs) with a size of 302 nm and a ζ potential of +16 mV were obtained and loaded onto Bifidobacteria membranes. Microbots with a diameter between 1–2 µm were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Microbots were injected intravenously through the tail vein to tumor-bearing mice, and then a magnet was placed to focus them to the tumor area. Forty-eight hours after injection, the biodistribution was determined by florescence and luminescence. The greatest luminescence and fluorescence emitted were found in tumor tissue compared with the normal organs. We created a vector that can be directed by a magnet and deliver genes whose expression is regulated by hypoxic microenvironment of tumor.

Minimized backbone and novel microaerobic promoters boost plasmid DNA production

A novel microaerobically inducible pUC18-derived plasmid was produced in Escherichia coli chromosomally expressing Vitreoscilla hemoglobin. The plasmid contains a copy of the positive replication control molecule rnaII under the control of the Pvgb promoter. The plasmid was unstable in the engineered strain. Sequencing showed that the plasmid was fragmented during preculture development, probably due to interactions between the host and vector genetic elements. Therefore, the rnaII copy under the control of the microaerobic promoters Pvgb, Pyfid, or Pyfidm was inserted in the minimal plasmid pUC57mini. Such plasmids were successfully produced in the engineered strain. The maximum plasmid DNA yield from biomass was 16.6 ± 0.1 mg g−1 in microaerobic cultures of the engineered strain using the Pyifd promoter. This was 14-fold greater than that using the original pUC57mini and the wild-type strain. Our results show the importance of considering host-vector interactions during the biodesign process and the potential of microaerobic induction for intensifying pDNA manufacturing.

Immunization of chicken with flagellin adjuvanted Salmonella enteritidis bacterial ghosts confers complete protection against chicken salmonellosis

The present study describes the generation of Salmonella enteritidis (SE) ghosts with a surface decorated Salmonella Typhimurium (ST) flagellin (FliC) antigen for immune enhancement and strain-specific protection. The ghosts were generated by biological means using pJHL184::fliC temperature inducible plasmid where the lysis occurs by phage PhiX174 lysis gene E expression. Being an inactivated strain, no environmental contamination was observed by fecal shedding upon inoculation into the chicken. To test the protective immune responses, ghost vaccination was conducted via the intramuscular route using chicken as the model organism. The development of antigen-specific humoral, cell-mediated, and protective immune responses was assessed. Compared to vector alone and phosphate-buffered saline (PBS) control groups, pJHL184::fliC ghost could generate significantly high antigen-specific IgY and cell-mediated immune (CMI) responses measured by a peripheral blood mononuclear cell proliferation, flow cytometer, and cytokine responses elicited by stimulated splenic T-cells (P < 0.05). The adjuvant effect induced by FliC was demonstrated by elicitation of Toll-like receptor 5 (TLR5). To test the protection efficacy, chickens were challenged with both SE and ST wild type (WT) strains, and the protection efficacy was assessed by determining the presence of challenging strains in the spleen and liver, and by assessing the histopathological alterations. Complete clearance of the challenged strain and least inflammatory signs were evident in the SE ghosts vaccinated group compared to the vector and PBS control. The elimination of both SE and ST in chicken organs ensures the intramuscular immunization of the present SE ghost vaccine can reduce SE and ST contamination levels in chicken that can be beneficial to prevent enteric infections in humans.

CRISPR Interference To Inducibly Repress Gene Expression in Chlamydia trachomatis.

The ability to inducibly repress gene expression is critical to the study of organisms, like Chlamydia, with reduced genomes in which the majority of genes are likely to be essential. We recently described the feasibility of a CRISPR interference (CRISPRi) system to inducibly repress gene expression in Chlamydia trachomatis. However, the initial system suffered from some drawbacks, primarily leaky expression of the anhydrotetracycline (aTc)-inducible dCas9 ortholog and plasmid instability, which prevented population-wide studies (e.g., transcript analyses) of the effects of knockdown. Here, we describe various modifications to the original system that have allowed us to measure gene expression changes within a transformed population of C. trachomatis serovar L2. These modifications include (i) a change in the vector backbone, (ii) the introduction of a weaker ribosome binding site driving dCas9 translation, and (iii) the addition of a degradation tag to dCas9 itself. With these changes, we demonstrate the ability to inducibly repress a target gene sequence, as measured by the absence of protein by immunofluorescence analysis and by decreased transcript levels. Importantly, the expression of dCas9 alone (i.e., without a guide RNA [gRNA]) had minimal impact on chlamydial growth or development. We also describe complementation of the knockdown effect by introducing a transcriptional fusion of the target gene 3' to dCas9. Finally, we demonstrate the functionality of a second CRISPRi system based on a dCas12 system that expands the number of potential chromosomal targets. These tools should provide the ability to study essential gene function in Chlamydia.

Inclusion Membrane Growth and Composition Are Altered by Overexpression of Specific Inclusion Membrane Proteins in Chlamydia trachomatis L2.

ABSTRACTChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections. This obligate intracellular bacterium develops within a membrane-bound vacuole called an inclusion, which sequesters the chlamydiae from the host cytoplasm. Host-pathogen interactions at this interface are mediated by chlamydial inclusion membrane proteins (Incs). However, the specific functions of most Incs are poorly characterized. Previous work from our laboratories indicated that expressing an IncF fusion protein at high levels in C. trachomatis L2 negatively impacted inclusion expansion and progeny production. We hypothesize that some Incs function in the structure and organization of the inclusion membrane and that overexpression of those Incs will alter the composition of endogenous Incs within the inclusion membrane. Consequently, inclusion biogenesis and chlamydial development are negatively impacted. To investigate this, C. trachomatis L2 was transformed with inducible expression plasmids encoding IncF-, CT813-, or CT226-FLAG. Overexpression of IncF-FLAG or CT813-FLAG, but not CT226-FLAG, altered chlamydial development, as demonstrated by smaller inclusions, fewer progeny, and increased plasmid loss. The overexpression of CT813-FLAG reduced the detectable levels of endogenous IncE and IncG in the inclusion membrane. Notably, recruitment of sorting nexin-6, a eukaryotic protein binding partner of IncE, was also reduced after CT813 overexpression. Gene expression studies and ultrastructural analysis of chlamydial organisms demonstrated that chlamydial development was altered when CT813-FLAG was overexpressed. Overall, these data indicate that disrupting the expression of specific Incs changed the composition of Incs within the inclusion membrane and the recruitment of associated host cell proteins, which negatively impacted C. trachomatis development.

Metabolic engineering of Rhodococcus jostii RHA1 for production of pyridine-dicarboxylic acids from lignin

Genetic modification of Rhodococcus jostii RHA1 was carried out in order to optimise the production of pyridine-2,4-dicarboxylic acid and pyridine-2,5-dicarboxylic acid bioproducts from lignin or lignocellulose breakdown, via insertion of either the Sphingobium SYK-6 ligAB genes or Paenibacillus praA gene respectively. Insertion of inducible plasmid pTipQC2 expression vector containing either ligAB or praA genes into a ΔpcaHG R. jostii RHA1 gene deletion strain gave 2–threefold higher titres of PDCA production from lignocellulose (200–287 mg/L), compared to plasmid expression in wild-type R. jostii RHA1. The ligAB genes were inserted in place of the chromosomal pcaHG genes encoding protocatechuate 3,4-dioxygenase, under the control of inducible Picl or PnitA promoters, or a constitutive Ptpc5 promoter, producing 2,4-PDCA products using either wheat straw lignocellulose or commercial soda lignin as carbon source. Insertion of Amycolatopsis sp. 75iv2 dyp2 gene on a pTipQC2 expression plasmid led to enhanced titres of 2,4-PDCA products, due to enhanced rate of lignin degradation. Growth in minimal media containing wheat straw lignocellulose led to the production of 2,4-PDCA in 330 mg/L titre in 40 h, with > tenfold enhanced productivity, compared with plasmid-based expression of ligAB genes in wild-type R. jostii RHA1. Production of 2,4-PDCA was also observed using several different polymeric lignins as carbon sources, and a titre of 240 mg/L was observed using a commercially available soda lignin as feedstock.

Inducible plasmid display system for high-throughput selection of proteins with improved solubility

Soluble expression of enzymes inside the cell is a prerequisite for the successful biotransformation of valuable products. Some key enzymes involved in biotransformation processes, however, are hardly expressed in their soluble forms. Here, we propose an inducible plasmid display, which is a molecular evolution strategy coupled with a high-throughput screening and/or selection method, as a simple and powerful tool for improving the solubility of target enzymes. Specifically, the Oct-1 DNA-binding domain and intein (i.e., auto-processing domain) were employed as anchoring and protein trans-splicing motifs to develop the system, in which the probability of protein trans-splicing is dependent on the soluble property of target proteins. The applicability of inducible plasmid display was investigated using an α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori, a highly insoluble and unstable enzyme in the cytoplasmic space of Escherichia coli, as a model protein. One round of the overall inducible plasmid display process, which consists of in vivo production of FucT2 mutants and in vitro screening, enabled soluble expression of FucT2 and selection of plasmids containing the corresponding genetic information. The inducible plasmid display developed in this study will contribute to the rapid and efficient screening and/or selection of soluble proteins.

Dual-function oleaginous biocatalysts for non-sterile cultivation and solvent-free biolipid bioextraction to reduce biolipid-based biofuel production costs

Triacylglycerols (TAGs) are starting materials for the production of biolipid-based fuels such as biodiesel and biojet fuel. While various microorganisms can produce TAGs from renewable resources, the cultivation of TAG-producing microorganisms under sterilization conditions to avoid microbial contamination and application of solvent to extract TAGs from the TAG-filled microorganisms are costly. To overcome these challenges, this study reports the feasibility of a non-sterile cultivation of an oleaginous bacterium Rhodococcus opacus PD631SpAHB under saline conditions, followed by the use of a solvent-free, phage-lysis-protein-based bioextraction approach for TAGs release. The engineered strain PD631SpAHB was developed by introducing a recombinant plasmid carrying a phage lytic gene cassette (pAHB) into Rhodococcus opacus PD631 via transformation, followed by adaptive evolution under saline conditions. This newly developed strain is a salt-tolerant strain with the inducible plasmid pAHB to enable TAGs release into the supernatant upon induction. Cell lysis of PD631SpAHB was confirmed by the decrease of the optical density of cell suspension, by the loss of cell membrane integrity, and by the detection of TAGs in the culture medium. Up to 38% of the total TAGs accumulated in PD631SpAHB was released into supernatant after the expression of the lytic genes. PD631SpAHB strain is a promising candidate to produce TAGs from non-sterile growth medium and release of its TAGs without solvent extraction - a new approach to reduce the overall cost of biolipid-based biofuel production.

The hns Gene of Escherichia coli Is Transcriptionally Down-Regulated by (p)ppGpp.

Second messenger nucleotides, such as guanosine penta- or tetra-phosphate, commonly referred to as (p)ppGpp, are powerful signaling molecules, used by all bacteria to fine-tune cellular metabolism in response to nutrient availability. Indeed, under nutritional starvation, accumulation of (p)ppGpp reduces cell growth, inhibits stable RNAs synthesis, and selectively up- or down- regulates the expression of a large number of genes. Here, we show that the E. coli hns promoter responds to intracellular level of (p)ppGpp. hns encodes the DNA binding protein H-NS, one of the major components of bacterial nucleoid. Currently, H-NS is viewed as a global regulator of transcription in an environment-dependent mode. Combining results from relA (ppGpp synthetase) and spoT (ppGpp synthetase/hydrolase) null mutants with those from an inducible plasmid encoded RelA system, we have found that hns expression is inversely correlated with the intracellular concentration of (p)ppGpp, particularly in exponential phase of growth. Furthermore, we have reproduced in an in vitro system the observed in vivo (p)ppGpp-mediated transcriptional repression of hns promoter. Electrophoretic mobility shift assays clearly demonstrated that this unusual nucleotide negatively affects the stability of RNA polymerase-hns promoter complex. Hence, these findings demonstrate that the hns promoter is subjected to an RNA polymerase-mediated down-regulation by increased intracellular levels of (p)ppGpp.

Using inducible lentiviral vectors to generate induced pluripotent stem cell line ZOCi001-A from peripheral blood cells of a patient with CRB1-/- retinitis pigmentosa.

We isolated peripheral blood mononuclear cells from a patient diagnosed with early-onset non-syndromic retinitis pigmentosa caused by compound heterozygous mutations in CRB1 (NM_001257965): c.1369C>T (p.R457X) and c.2027C>T (p.T676M). These cells were transfected with four inducible plasmids encoding human OCT4, SOX2, KLF4 or C-MYC together. Transfected cells were induced to form pluripotent stem cells (iPSCs) expressing the pluripotent stem cell markers SOX2, OCT4, KLF4, SSEA4 and NANOG and presenting a normal karyotype. These cells could be differentiated into three germ layers as well as retinal organoids, and thus provide a valuable cellular model for the study of development of major retina diseases.

Microbes in Oncology: Controllable Strategies for Bacteria Therapy

Abstract Bacterial therapy is an emerging method of tumor treatment. By utilizing wild-type bacteria or engineered bacteria to treat solid tumors, bacterial therapy has recently attracted attention due to its high therapeutic specificity. Although many bacterial strains have been tested in animal models or have even advanced to clinical trials, the efficacy of bacterial therapy remains undesirable. The lack of efficient control methods could cause side effects as well as insufficient therapeutic efficiency, both of which are urgent problems for bacterial therapy. Therefore, some studies have constructed bacteria with inducible plasmid or adsorption with responsive nanoparticles, which improved controllability and specificity during bacterial therapy. Herein, we introduce the unique advantages of bacteria in cancer treatment and highlight the issues associated with the application of bacterial therapy, focusing on the incorporation of various methodologies in the advancement of some controllable strategies in bacterial therapy.

Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli.

To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli. Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined. Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX)=16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX=32-64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline. Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.

Design of a microaerobically inducible replicon for high-yield plasmid DNA production.

A pUC-derived replicon inducible by oxygen limitation was designed and tested in fed-batch cultures of Escherichia coli. It included the addition of a second inducible copy of rnaII, the positive replication control element. The rnaII gene was expressed from Ptrc and cloned into pUC18 to test the hypothesis that the ratio of the positive control molecule RNAII to the negative control element, RNAI, was the determinant of plasmid copy number per chromosome (PCN). The construct was evaluated in several E. coli strains. Evaluations of the RNAII/RNAI ratio, PCN and plasmid yield normalized to biomass (YpDNA/X ) were performed and the initial hypothesis was probed. Furthermore, in high cell-density cultures in shake flasks, an outstanding amount of 126 mg/L of plasmid was produced. The microaerobically inducible plasmid was obtained by cloning the rnaII gene under the control of the oxygen-responsive Vitreoscilla stercoraria hemoglobin promoter. For this plasmid, but not for pUC18, the RNAII/RNAI ratio, PCN and YpDNA/X efficiently increased after the shift to the microaerobic regime in fed-batch cultures in a 1 L bioreactor. The YpDNA/X of the inducible plasmid reached 12 mg/g at the end of the fed-batch but the original pUC18 only reached ca. 6 mg/g. The proposed plasmid is a valuable alternative for the operation and scale-up of plasmid DNA production processes in which mass transfer limitations will not represent an issue.

Inducible Plasmid Self-Destruction (IPSD) Assisted Genome Engineering in Lactobacilli and Bifidobacteria.

Genome engineering is essential for application of synthetic biology in probiotics including lactobacilli and bifidobacteria. Several homologous recombination system-based mutagenesis tools have been developed for these bacteria, but still have many limitations in different species or strains. Here we developed a genome engineering method based on an inducible self-destruction plasmid delivering homologous DNA into bacteria. Excision of the replicon by induced recombinase facilitates selection of homologous recombination events. This new genome editing tool called inducible plasmid self-destruction (IPSD) was successfully used to perform gene knockout and knock-in in lactobacilli and bifidobacteria. Due to its simplicity and universality, the IPSD strategy may provide a general approach for genetic engineering of various bacterial species.

SelProm: A Queryable and Predictive Expression Vector Selection Tool for Escherichia coli.

The rapid prototyping and optimization of plasmid-based recombinant gene expression is one of the key steps in the development of bioengineered bacterial systems. Often, multiple genes or gene modules need to be coexpressed, and for this purpose compatible, inducible plasmid systems have been developed. However, inducible expression systems are not favored in industrial processes, due to their prohibitive cost, and consequently the conversion to constitutive expression systems is often desired. Here we present a set of constitutive-expression plasmids for this purpose, which were benchmarked using fluorescent reporter genes. To further facilitate the conversion between inducible and constitutive expression systems, we developed SelProm, a design tool that serves as a parts repository of plasmid expression strength and predicts portability rules between constitutive and inducible plasmids through model comparison and machine learning. The SelProm tool is freely available at http://selprom.synbiochem.co.uk .

Identification of the two-component guaiacol demethylase system from Rhodococcus rhodochrous and expression in Pseudomonas putida EM42 for guaiacol assimilation

A diversity of softwood lignin depolymerization processes yield guaiacol as the main low molecular weight product. This key aromatic compound can be utilized as a carbon source by several microbial species, most of which are Gram positive bacteria. Microbial degradation of guaiacol is known to proceed initially via demethylation to catechol, and this reaction is catalyzed by cytochrome P450 monooxygenases. These enzymes typically require a set of redox partner proteins, whose number and identities were not described until very recently in the case of guaiacol. In this work we identified two proteins involved in guaiacol demethylation by the actinomycete Rhodococcus rhodochrous. Additionally, we constructed four different polycistronic operons carrying combinations of putative redox partners of this guaiacol demethylation system in an inducible expression plasmid that was introduced into the Gram negative host Pseudomonas putida EM42, and the guaiacol consumption dynamics of each resulting strain were analyzed. All the polycistronic operons, expressing a cytochrome P450 together with a putative ferredoxin reductase from R. rhodochrous and putative ferredoxins from R. rhodochrous or Amycolatopsis ATCC 39116 enabled P. putida EM42 to metabolize and grow on guaiacol as the sole carbon source.

Analyzing Diguanylate Cyclase Activity In Vivo using a Heterologous Escherichia coli Host.

Bacterial biofilms are notorious for their deleterious effects on human health and industrial biofouling. Key processes in biofilm formation are regulated by the second messenger signal cyclic dimeric guanosine monophosphate (c-di-GMP); accumulation of c-di-GMP promotes biofilm formation, while lowering c-di-GMP promotes motility. Complex networks of modular enzymes are involved in regulating c-di-GMP homeostasis. Understanding how these enzymes function in bacterial cells can help enlighten how bacteria use environmental cues to modulate c-di-GMP and cell physiology. In this article, we describe a workflow that utilizes Escherichia coli as a heterologous host to allow the researcher to identify genes encoding potential c-di-GMP-metabolizing proteins, to express the gene of interest from an inducible plasmid, and to directly detect changes in intracellular c-di-GMP using ultra-performance liquid chromatography-tandem mass spectrometry. © 2018 by John Wiley & Sons, Inc.

Over expression of the constitutive expression refractory Mycobacterium tuberculosis Rv3488 protein with an acid-inducible expression system

As inducible protein expression plasmids available for mycobacterial species are limited, here we demonstrate the utility of an acid-inducible promoter driving gene and subsequent protein expression of a difficult to express protein. We wanted to assess the use of an acid inducible promoter expression system to produce proteins in a mycobacterial system, specifically proteins that when overexpressed interfere with mycobacterial cell growth. Overexpression of those types of proteins would require a tightly regulated promoter system. We employed the Mycobacterium tuberculosis lipF minimal acid-inducible promoter (mpr) which had previously been shown to be upregulated by acidic stress >100 X and to have a low basal level of expression in the absence of acidic stress. It is active from pH 4.3 up to pH 6.4 making this an acid range that is compatible with mycobacterial growth or survival and active at acidic ranges encountered in vivo within acidified phagosomes of macrophages. We therefore cloned the M. tuberculosis gene Rv3488, whose product had been unable to be expressed constitutively, into a plasmid downstream of the lipF mpr promoter and overexpressed this gene in the presence of acidic pH in Mycobacterium smegmatis. Sustained overexpression of the gene leads to inhibition of replication of mycobacterial cells as well as inhibition of carotenoid pigment synthesis, while short-term overexpression leads to appropriate protein expression.

Antisense techniques provide robust decrease in GnRH receptor expression with minimal cytotoxicity in GT1-7 cells

ABSTRACTThe episodic pattern of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus is driven by an integrated network of cells termed the GnRH pulse generator. Cultured and immortalized GnRH neurons also produce a pulsatile pattern of GnRH secretions when grown in the absence of other cell types, suggesting the presence of an intrinsic oscillator mediating GnRH secretion. The mechanisms underlying such pulsatility comprise one of the most tantalizing problems in contemporary neuroendocrinology. In order to study the mechanism by which GnRH is produced in a pulsatile fashion, the autocrine effect of GnRH on GnRH-producing neurons must be eliminated. This may be performed by downregulating the expression of the GnRH receptor. Treatment with three 21-mer exogenous phosphorothioates and transient transfections with an inducible plasmid containing an antisense construct to the GnRH receptor gene decreased GnRH receptor expression further. This resulted in less cytotoxicity compared to inhibition of RNA or protein synthesis with actinomycin D, α-amanitin, puromycin, and cycloheximide. This study shows methods and optimized conditions established for the generation of a stable GT1-7 cell line containing an inducible construct allowing the downregulation of GnRH receptor expression.Abbreviations: ANOVA: analysis of the variance; DMEM: Dulbecco’s modified Eagle’s medium; GnRH: gonadotropin-releasing hormone; RXR: retinoid X receptor