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PubMed1994

Transformation of Bartonella bacilliformis by electroporation.

Bartonella bacilliformis is a member of the order Rickettsiales, family Bartonellaceae. The bacterium is an intracellular parasite of human erythrocytes. To date, members of the family Bartonellaceae have not been transformed by standard chemical methods. We report the first successful transformation of a member of the Bartonellaceae family, B. bacilliformis, by the method of electroporation. The optimal conditions for electroporation of B. bacilliformis include a field strength of 12.5 kV/cm and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters and the cosmid pEST (RK2 replicon), a transformation efficiency of 7.8 x 10(5) was obtained. Transformants were readily cultured on medium containing kanamycin sulfate at concentrations ranging from 15 to 600 micrograms/mL. Bacterial survival was approximately 31% under optimal electroporation conditions, and the maximal number of transformants was obtained with 80 ng of pEST DNA. Bartonella bacilliformis was verified as the transformed organism by a comparison of transformant protein profiles with those of wild-type B. bacilliformis using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and detection of the exogenous plasmid in DNA from the transformed bacteria by DNA hybridization. Transformations using the plasmids pMK20, pML31, and pUCK18 (containing the replicons ColE1, F, and pMB1, respectively) were not successful.

Bartonella↗
PubMed1992

Double-stranded and single-stranded RNA viruses of Saccharomyces cerevisiae.

Yeast RNA viruses include L-A (and its toxin-encoding satellites M1, M2, ...) and L-BC dsRNA viruses and the single-stranded replicons 20S RNA and 23S RNA. L-A has a single-segment 4.6-kb linear genome encoding a major coat protein (gag) and its RNA-dependent RNA polymerase (pol), the latter expressed as a gag-pol fusion protein formed by a -1 ribosomal frameshift. In vitro replication, transcription, and binding systems for L-A have been used to define cis sites necessary for packaging and replication of viral RNA. Cellular functions that promote viral replication include the MAK3-encoded N-acetyltransferase whose modification of the gag N terminus is necessary for L-A virus assembly. The toxins encoded by the M satellite RNAs are processed by enzymes (KEX1 and KEX2, for killer expression) whose study led to discovery of mammalian hormone-processing enzymes. 20S RNA is an apparently naked circular RNA replicon (with a dsRNA form called W) encoding a RNA polymerase-like molecule. Its copy number is induced 10,000-fold in 1% potassium acetate, and it is subject to the same SKI antiviral system that represses L-A, L-BC, and M dsRNA copy number.

Base Sequence↗
PubMed2006

Increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector.

An improved viral vector for cancer gene therapy should be capable of infecting tumors with high efficiency, inducing specific and high-level expression of transgene in the tumor and selectively destroying tumor cells. In the design of such a vector to treat hepatocellular carcinoma, we took advantage of (a) the high infectivity of adenoviruses for hepatic cells, (b) the high level of protein expression and proapoptotic properties that characterize Semliki Forest virus (SFV) replicon, and (c) tumor selectivity provided by alpha-fetoprotein (AFP) promoter. We constructed a hybrid viral vector composed of a helper-dependent adenovirus containing an SFV replicon under the transcriptional control of AFP promoter and a transgene driven by SFV subgenomic promoter. Hybrid vectors containing murine interleukin-12 (mIL-12) genes or reporter gene LacZ showed very specific and high-level expression of transgenes in AFP-expressing hepatocellular carcinoma cells, both in vitro and in an in vivo hepatocellular carcinoma animal model. Infected hepatocellular carcinoma cells were selectively eliminated due to the induction of apoptosis by SFV replication. In a rat orthotopic liver tumor model, treatment of established tumors with a hybrid vector carrying mIL-12 gene resulted in strong antitumoral activity without accompanying toxicity. This new type of hybrid vectors may provide a potent and safe tool for cancer gene therapy.

Adenoviridae↗
PubMed2005

Activation of interferon-stimulated response element in huh-7 cells replicating hepatitis C virus subgenomic RNA.

Interferon-alpha (IFN(alpha)) binds to receptors on the cell surface, which initiate a cascade of signal transduction pathways that leads to transcription of selected genes. This transduction pathway involves binding of transcription factors to a common cis-acting DNA sequence called IFN-stimulated response element (ISRE). To test whether these signaling pathways are functional in hepatitis C virus (HCV)-replicating cells, we studied the regulation of ISRE-mediated transcription of firefly luciferase gene in stable replicon cell lines. A plasmid construct was prepared (pISRELuc) which contains four tandem repeats of 9-27 ISRE sequences positioned directly upstream of the herpes virus 1 thymidine kinase promoter TATA box that drives the expression of firefly luciferase. Regulation of ISRE-mediated expression of firefly luciferase by IFN(alpha) was studied by transfecting this clone into Huh-7 cells replicating HCV subgenomic HCV RNA. The significance of ISRE-mediated transcriptional activation was studied in a replicon cell line by pretreatment of cells with actinomycin D, which inhibits cellular DNA-dependent RNA transcription. IFN treatment activates ISRE-mediated expression of luciferase, indicating that this pathway is functional in Huh-7 cells. Activation of ISRE-mediated transcription of luciferase is relatively high in two Huh-7 stable cell lines replicating HCV subgenomic RNA. Inhibition of ISRE-mediated transcription of luciferase by actinomycin D also makes HCV replication totally resistant to IFN(alpha). These in vitro studies suggest that activation of IFN-inducible genes is important in mounting a successful antiviral response against HCV.

Cell Line, Tumor↗
PubMed2004

Synthesis of beta-enantiomers of N4-hydroxy-3'-deoxypyrimidine nucleosides and their evaluation against bovine viral diarrhoea virus and hepatitis C virus in cell culture.

N4-Hydroxycytidine (NHC) was recently reported to have anti-pestivirus and anti-hepacivirus activity. It is thought that this nucleoside acts as a weak alternative substrate for the hepatitis C virus (HCV) polymerase. In addition to NHC, 3'-deoxyuridine (3'-dU) was found to inhibit bovine diarrhoea virus (BVDV) production by 1 log10 at 37.2 microM. These initial findings prompted the synthesis of beta-D and beta-L analogues of (i) base-modified 3'-deoxy-NHC; (ii) 3'-deoxyuridine; and 3'-deoxycytidine. The antiviral activity of these 42 nucleosides was evaluated against BVDV and HCV bicistronic replicon in cell culture. Among the NHC analogues, the antiviral activity observed for the beta-L-3'-deoxy-5-fluoro-derivative 1-(3-deoxy-beta-L-erythro-pentofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one and the beta-D-3'-deoxy-5-iodo-derivative 1-(3-deoxy-beta-D-erythro-pentofuranosyl)-5-iodocytosine in the replicon system (1 log10 reduction at 100 microM) was due to the concomitant toxicity towards intracellular ribosomal RNA levels (CC90 equal or lower than the EC90). In conclusion, none of the newly synthesized derivatives exhibited enhanced antiviral activity compared to the parent nucleoside NHC.

Animals↗
PubMed2026

Spatiotemporal genomic analysis and risk assessment of the plasmids carrying blaOXA-48-like genes based on a large-scale international dataset.

BACKGROUND: The spread of OXA-48-like carbapenemases represents a major public health challenge. Although previous studies have investigated OXA-48-like carbapenemases risk factors, nosocomial dissemination, and plasmid dynamics, an integrated plasmid-centered framework combining complete plasmid mining, transmission-unit analysis, phylogenetic reconstruction, and machine learning-based risk assessment remains limited. METHODS: We systematically collected 747 complete plasmid sequences carrying blaOXA-48-like genes from the NCBI database, establishing the largest collections of complete plasmid sequences to date. Using an integrative framework of population genomics, phylogenetic dating, and machine learning, this study aimed to characterize the dissemination patterns, plasmid replicon diversity, transmission units, mobile genetic elements, co-resistance profiles, and risk classification of these plasmid. RESULTS: Plasmids carrying blaOXA-48-like genes were detected across 50 countries on six continents, with blaOXA-48 predominating in Europe, blaOXA-181 in South Asia, and blaOXA-232 largely in Asia. IncL and ColKP3/IncX3 replicons, together with Tn1999.2 and other MGEs, were central drivers of plasmid maintenance and spread. Sixteen transmission units were defined, with AA068_Cluster3 estimated to have originated in the Netherlands around 2005 before expanding to Europe, the Middle East, Asia, and North America. Co-resistance analyses revealed frequent modules involving aminoglycoside and quinolone resistance, with qnrS1 and aph(3'')-Ib most prevalent. Notably, high-risk transposon structures were often identified in non-clinical environments, underscoring their cross-ecological transmission potential. Machine learning-based classification models showed good internal performance for predefined composite-risk categories, with plasmid mobility, clinical/non-clinical source composition, and host background contributing to the classification results. CONCLUSIONS: This study provides a large-scale plasmid-centered genomic analysis of publicly available complete plasmid sequences carrying blaOXA-48-like genes, integrating transmission-unit inference, phylogeographic reconstruction, mobile genetic element and co-resistance profiling, and composite genomic risk stratification. This gene-centered framework may support future One Health-oriented antimicrobial resistance surveillance and prioritization of plasmids with higher dissemination and resistance potential.

Plasmids↗
PubMed1987

Recovery of DNA synthesis from inhibition by ultraviolet light in mammalian cells.

In general mammalian cells recover from DNA synthesis inhibition by ultraviolet light (u.v.) before most of the pyrimidine dimers have been removed from the genome. This is a complex phenomenon whose biological significance has not been fully assessed. In Chinese hamster V79 cells this recovery seems to be directly coupled to an enhanced rate of double-stranded DNA elongation. The presence of the DNA polymerase alpha inhibitor, aphidicolin, after u.v. irradiation produces two different responses. At low concentration, sufficient to inhibit 95% of DNA replication but having no effect on excision repair, the drug has no effect on the recovery. This shows that ongoing replicative DNA synthesis is not required for recovery. At higher concentrations of aphidicolin, sufficient to block excision repair, the recovery phenomenon was prevented. The recovery was also prevented by actinomycin D at a concentration that inhibits 60% of RNA synthesis. In quantitative autoradiography experiments in which previously irradiated cells were fused with unirradiated cells the nuclei of the latter exhibited a higher resistance to inhibition by u.v. than nuclei from non-fused cells. These results indicate that: (1) even the low repair rate exhibited by V79 cells (relative to human cells) is important for recovery; although most of the dimers remain in the V79 genome after recovery of DNA synthesis, either the removal of lesions from some important region of chromatin or the activity of the repair process itself is important for the recovery; (2) the recovery mechanism is induced and depends on RNA synthesis and the production of specific factors. Finally, we have observed that cells previously treated with fluorodeoxyuridine become more resistant to inhibition by u.v. After irradiation these cells replicate DNA faster than untreated cells. Since it has been shown that this drug activates unused origins of replication in Chinese hamster cells, reducing the average replicon size, we assume that the acquired resistance has to do with the operation of a larger number of smaller replicons. This may also be the mechanism whereby recovery from inhibition occurs after u.v. irradiation.

Animals↗
PubMed1981

DNA replications in Chironomus polytene chromosomes during treatment with ethanol.

DNA replication of Chironomus polytene chromosomes was investigated The results show that during treatment with ethanol there is an accumulation of newly synthesized DNA with the size expected of replicons. These and previous results indicate that it is possible that in polytene chromosomes the termination of replication and merging of adjacent replicons is more sensitive to damage than in initiation and/or elongation steps.

Animals↗
PubMed1989

[Genetic and biochemical studies on the mechanism of mammalian chromosome replication].

It has been clarified that basic mechanism of deoxyribonucleic acid (DNA) replication is conserved from bacteria to higher cells. What distinguishes prokaryotic and eukaryotic modes of DNA replication most clearly is that bacterial chromosomes form a single replicon copied from a single initiation point and eukaryotic chromosomes consist of multiple replicons that initiate at multiple points. Thus, eukaryotes have to coordinate orderly replication of the genome. In order to understand this complex problem as a whole, three approaches were chosen. First approach is a genetic one. Certain number of temperature-sensitive (ts) mutants were isolated from mouse FM3A cells. One of the ts mutants, designated as tsFT20, was shown to contain heat-labile DNA polymerase alpha (pol. alpha). By the use of this mutant strain, it was proved that pol. alpha is essential for mammalian DNA replication. In addition, the human gene for pol. alpha on the X chromosome was assigned. Second approach is an enzymological one. FM3A cells were used for the identification and characterization of enzymes and proteins supposed to be involved in DNA replication. Four DNA-dependent ATPases, three pol. alpha stimulation factors, DNA topoisomerases I and II have been identified, as well as a stimulation factor for the assembly of nucleosome. DNA helicase activity was detected in two of the DNA-dependent ATPase (B and C1). Third approach is the reconstitution of DNA replication in cell-free system. By use of polyoma virus DNA as a template, cell-free extract from FM3A cells supported DNA replication in the presence of polyoma virus large T-antigen. This cell-free system will be useful for the analysis of the function of replication enzymes and proteins as well as the characterization of ts mutants.

Adenosine Triphosphatases↗
PubMed2003

Inheritance of Oryza sativa endornavirus in F1 and F2 hybrids between japonica and indica rice.

We have found a 14 kbp double-stranded RNA (dsRNA) in many cultivars of japonica rice (Oryza sativa L.) but not in any cultivars of indica rice. This dsRNA is an RNA replicon with plasmid-like properties and is proposed to be a novel dsRNA virus, Oryza sativa endornavirus (OSV). Reciprocal crosses between the OSV-carrier japonica variety (Nipponbare) and the OSV-free indica variety (IR 26 or Kasalath) were performed to investigate whether OSV can be transmitted to F1 hybrids. When IR 26 and Nipponbare were used, efficient transmission of OSV from ova (93%) and pollen (89%) was observed. When Kasalath and Nipponbare were used, the OSV transmission efficiency to F1 progeny was 68% from ova and 20% from pollen. The transmission of OSV to F2 progeny plants was also complicated, showing non-Mendelian inheritance. These results suggest that the dsRNA replicon (OSV) is unstable in indica rice plants.

Chimera↗
PubMed2025

Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6.

The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating across the entire viral genome and their functional impact is not well understood. To address the contribution of these mutations, we reconstructed genomes of recent Omicron variants with disabled Spike expression (replicons) to systematically compare their RNA replication capabilities independently from Spike. We also used a single reference replicon and complemented it with various Omicron variant Spike proteins to quantify viral entry capabilities in single-round infection assays. Viral entry and RNA replication were negatively correlated, suggesting that as variants evolve reduced entry functions under growing immune pressure on Spike, RNA replication increases as a compensatory mechanism. We identified multiple mutations across the viral genome that enhanced viral RNA replication. NSP6 emerged as a hotspot with a distinct L260F mutation independently arising in the BQ.1.1 and XBB.1.16 variants. Using mutant and revertant NSP6 viral clones, the L260F mutation was validated to enhance viral replication in cells and increase pathogenesis in mice. Notably, this mutation reduced host lipid droplet content by NSP6. Collectively, a systematic analysis of RNA replication of recent Omicron variants defined NSP6's key role in viral RNA replication that provides insight into evolutionary trajectories of recent variants with possible therapeutic implications.

SARS-CoV-2↗
PubMed2004

Delivery and expression of heterologous genes in mammalian cells using self-replicating alphavirus vectors.

The RNA genomes of alphaviruses have been exploited to create highly efficient vectors for transient expression of foreign genes in mammalian cells and for use as vehicles for genetic vaccines. For Semliki Forest virus (SFV), a representative alphavirus, three strategies for in vitro and in vivo gene-delivery have been developed. One method relies on the packaging of recombinant vectors into suicidal viral particles and infection of target cells, whereas the other two methods are based on direct transfection of target cells, either by using naked DNA encoding the SFV replicon placed downstream of an RNA polymerase II dependent promoter, or by using in vitro transcribed RNA encoding the SFV replicon. All three approaches result in the delivery of a selfreplicating SFV vector into target cells, with expression of foreign genes being driven from a highly efficient viral subgenomic promoter.

Alphavirus↗
PubMed2006

Nucleoside analog inhibitors of hepatitis C virus replication.

Of the 30 compounds currently marketed in the United States for treatment of viral infections, 15 are nucleoside analogs, demonstrating the utility of this class of compound as a source of antiviral drugs. The success of nucleoside analogs in treating other viral infections provides a compelling rationale for the significant effort that is currently being devoted to the discovery and development of nucleoside analogs to treat infection by hepatitis C virus (HCV) that may lead to improvements in response rates compared to currently available therapies. Several different approaches have been adopted to identify promising analogs, including the use of surrogate viruses in cell culture assays, screening in the cell-based bicistronic HCV replicon assay, and screening nucleoside triphosphates for the ability to inhibit the activity of the HCV RNA-dependent RNA polymerase in vitro. Several classes of ribonucleoside analogs with modifications of the ribose inhibit HCV replication. Nucleoside analogs incorporating a 2'-C-methyl modification are potent inhibitors in the replicon assay in the absence of cytotoxicity, and appear to exert their inhibition by acting as functional chain terminators of RNA synthesis. NM283, a prodrug of 2'-C-methylcytidine, has entered clinical trials and demonstrated viral load reductions in subjects infected with genotype 1 HCV, a genotype known to be difficult to treat effectively with currently approved therapies. Overall, results to date offer encouragement that improved therapies to treat HCV infection including newly developed nucleoside analogs may become available within the next few years.

Animals↗
PubMed1997

Plasmids in Lactobacillus.

This review describes Lactobacillus plasmids on distribution, structure, function, vector construction, vector stability, application, and prospective. About 38% of species of the genus Lactobacillus were found to contain plasmids with different sizes (from 1.2 to 150 kb) and varied numbers (1 or more). Some Lactobacillus plasmids with small sizes were highly similar to those of single strand plasmids from other Gram-positive bacteria. The extensive sequence homologies of plus origins, replication initiation proteins, minus origins, cointegration sites, and the presence of single strand intermediates supported the fact that these small Lactobacillus plasmids replicate with a rolling-circle replication mechanism. Some Lactobacillus plasmid replicons were of broad host range that could function in other Gram-positive bacteria, and even in Escherichia coli, while replicons of other Gram-positive bacteria also function in Lactobacillus. Although most Lactobacillus plasmids are cryptic, some plasmid-encoded functions have been discovered and applied to vector construction and Lactobacillus identification, detection, and modification.

DNA, Bacterial↗
PubMed2026

Genomic surveillance reveals escalating antimicrobial resistance and plasmid diversity in clinical Salmonella 1,4,[5],12:i:- ST34 isolates from Guizhou Province, China.

INTRODUCTION: Salmonella 1,4,[5],12:i:- ST34 has emerged as a significant public health issue due to its association with various antimicrobial resistance genes (ARGs) and transferable plasmids. However, its genomic characteristics and potential influence on public health in Guizhou have not been comprehensively assessed. METHODS: From 2019 to 2023, a 5-year surveillance was conducted in nine cities (prefectures) of Guizhou Province. We integrated phenotypic and genomic analyses of 281 clinical Salmonella 1,4,[5],12:i:- ST34 isolates to investigate the prevalence of ARGs and plasmids and to analyze the molecular epidemiology and evolution. RESULTS: The isolates exhibited resistance to first-line antibiotics, with 22.4% for ciprofloxacin, 11.4% for azithromycin, 18.5% for ceftazidime, and 39.1% for cefotaxime. ARGs showed substantial agreement with phenotypes for tetracycline, macrolides, third-generation cephalosporins (3GCs), carbapenems, and colistin (80.8-100.0% consistency; Kappa: 0.50-1.00). Plasmid analysis identified IncQ1 (84.3%) and IncHI2/IncHI2A (26.3%) as the main replicons, with the variety of plasmid replicons increasing from 7 to 21 over the 5 years. ARGs associated with resistance to critically important antibiotics (CIAs) were frequently predicted to be located on plasmid-associated contigs, with significant associations observed between IncHI2/IncHI2A plasmids and ARGs conferring resistance to fluoroquinolones, macrolides, and cephalosporins (P < 0.05). Molecular typing divided 281 isolates into 37 cgSTs, with cgST52428 being the most common. Molecular epidemiological analysis revealed that Guizhou isolates primarily clustered together, sharing close genetic ties with those from Sichuan and Guangdong, and exhibited the highest genetic similarity to pork-derived isolates. Phylogenetic analysis revealed clustering of CIA-resistant ARGs and plasmids in Clades 4 and 5, with a significant association between IncHI2/IncHI2A plasmids and CIA-resistant ARGs (&#x3c7;2 = 112.12, P < 0.001). Additionally, class 1 integron was associated with higher ARG burdens, while virulence-associated genes were conserved and predominantly chromosome-associated. Gene-content analysis revealed that isolates in Clades 4 and 5 harbored the largest mean gene complements, and cgST52428 isolates also harbored the largest among dominant cgSTs. DISCUSSION: This study presents a comprehensive genomic profile of Salmonella 1,4,[5],12:i:- ST34 in Guizhou, providing essential data for exploring the resistance characteristics and investigating the molecular epidemiology of Salmonella 1,4,[5],12:i:-.

ST34↗
PubMed2006

Checkpoint regulation of replication dynamics in UV-irradiated human cells.

At any moment during S phase, regions of genomic DNA are in various stages of replication (i.e., initiation, chain elongation, and termination). These stages may be differentially inhibited after treatment with various carcinogens that damage DNA such as UV. We used visualization of active replication units in combed DNA fibers, in combination with quantitative analyses of the size distributions of nascent DNA, to evaluate the role of S-checkpoint proteins in UV-induced inhibition of DNA replication. When HeLa cells were exposed to a low fluence (1 J/m(2)) of 254 nm UV light (UVC), new initiation events were severely inhibited (5-6-fold reduction). A larger fluence of UVC (10 J/m(2)) resulted in stronger inhibition of the overall rate of DNA synthesis without decreasing further the frequency of replicon initiation events. Incubation of HeLa cells with caffeine and knockdown of ATR or Chk1 kinases reversed the UVC-induced inhibition of initiation of new replicons. These findings illustrate the concordance of data derived from different experimental approaches, thus strengthening the evidence that the activation of the intra-S checkpoint by UVC is dependent on the ATR and Chk1 kinases.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2003

Two ways to induce innate immune responses in human PBMCs: paracrine stimulation of IFN-alpha responses by viral protein or dsRNA.

In order to study mechanisms of induction of IFN-alpha by Newcastle disease virus (NDV), we used two replicon systems which are based respectively on DNA and RNA of the Semliki forest virus (SFV) and transfected these into baby hamster kidney cells (BHK) which do not produce interferon-alpha. Co-incubation of BHK cells which were transfected with the two vector systems, with human PBMCs, showed that production of IFN-alpha takes place by two different ways. When using the DNA-based SFV vector, only transfectants expressing cell surface HN molecules of NDV (and not the mock-transfected cells) elicited such a response via interaction of these HN molecules with viral receptors on PBMCs. In contrast, BHK cells transfected with RNA which had been in vitro transcribed from the RNA-based SFV vector without foreign gene as insert (mock-transfected) elicited a comparable IFN-alpha response. Transfer by transfection of poly(I:C), an analogue of double stranded RNA (dsRNA), into the BHK cells induced also by itself the production of IFN-alpha. Therefore induction of "danger signals" (as double-strand RNA replicative intermediates) might be responsible for this discrepancy observed in IFN-alpha induction in PBMCs between the two studied SFV vector systems based on transfection of DNA and on RNA. These observations highlight two ways of IFN-alpha induction which additively may explain the high interferonogenic capacity of NDV as virus: i) via cell-surface expressed HN after transfection of the DNA-based SFV replicon and ii) via transfection of self-amplifying RNA.

Animals↗
PubMed2003

Replication and episomal maintenance of Epstein-Barr virus-based vectors in mouse embryonal fibroblasts enable synthetic lethality screens.

Recently, we demonstrated the establishment of chemical and genetic synthetic lethality screens in cultured human cells. Here, we report the establishment of this method in mouse embryonal fibroblasts (MEF). The method employs an immortalized mammalian cell line, deficient in a gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest and the use of a novel green fluorescent protein (GFP)-based double-label fluorescence system. The crucial part in this endeavor has been the identification of a DNA replicon that could stably replicate in MEFs while under selection for survival and gets spontaneously lost relatively fast in the absence of such a pressure. Here, we show for the first time that EBV-based replicons but not polyoma virus-based ones can replicate and be stably maintained in MEFs. In the chemical screen, selective pressure imposed by synthetic lethal drugs prevented the spontaneous loss of the GFP-marked episome, enabling drug identification. Retention or spontaneous loss over time of the episomal survival plasmid could be sensitively detected in a large-scale blind test in the presence or absence of synthetic lethal chemicals, respectively. Establishing the synthetic lethality screen should thus permit high throughput screening for chemicals, which are synthetically lethal with any mouse mutant/knockout gene of interest. Moreover, it forms the basis for a genetic synthetic lethality screen in MEFs, an important new tool for mouse functional genomics.

Animals↗