Conditional Negative Selection Research Articles

Removing auto-activators from yeast-two-hybrid assays by conditional negative selection

Yeast-two-hybrid (Y2H) is widely used as a strategy to detect protein–protein interactions (PPIs). Recent advancements have made it possible to generate and analyse genome-wide PPI networks en masse by coupling Y2H with next-generation sequencing technology. However, one of the major challenges of yeast two-hybrid assay is the large amount of false-positive hits caused by auto-activators (AAs), which are proteins that activate the reporter genes without the presence of an interacting protein partner. Here, we have developed a negative selection to minimize these auto-activators by integrating the pGAL2-URA3 fragment into the yeast genome. Upon activation of the pGAL2 promoter by an AA, yeast cells expressing URA3 cannot grow in media supplemented with 5-Fluoroorotic acid (5-FOA). Hence, we selectively inhibit the growth of yeast cells expressing auto-activators and thus minimizing the amount of false-positive hits. Here, we have demonstrated that auto-activators can be successfully removed from a Marchantia polymorpha cDNA library using pGAL2-URA3 and 5-FOA treatment, in liquid and solid-grown cultures. Furthermore, since URA3 can also serve as a marker for uracil autotrophy, we propose that our approach is a valuable addition to any large-scale Y2H screen.

A bacterial gene codA encoding cytosine deaminase is an effective conditional negative selectable marker in Glycine max.

Research describes the practical application of the codA negative selection marker in Soybean. Conditions are given for codA selection at both the shooting and rooting stages of regeneration. Conditional negative selection is a powerful technique whereby the absence of a gene product allows survival in otherwise lethal conditions. In plants, the Escherichia coli gene codA has been employed as a negative selection marker. Our research demonstrates that codA can be used as a negative selection marker in soybean, Glycine max. Like most plants, soybean does not contain cytosine deaminase activity and we show here that wild-type seedlings are not affected by inclusion of 5-FC in growth media. In contrast, transgenic G. max plants expressing codA and grown in the presence of more than 200μg/mL 5-FC exhibit reductions in hypocotyl and taproot lengths, and severe suppression of lateral root development. We also demonstrate a novel negative selection-rooting assay in which codA-expressing aerial tissues or shoot cuttings are inhibited for root formation in media containing 5-FC. Taken together these techniques allow screening during either the regeneration or rooting phase of tissue culture.

The codA gene as a negative selection marker in Citrus.

The use of positive selectable marker genes is widespread in plant genetic transformation allowing transgenic cells to grow while repressing non-transgenic cells. Negative selectable markers, on the contrary, allow the repression or ablation of transgenic cells. The codA gene of Escherichia coli encodes cytosine deaminase that hydrolyzes 5-fluorocytosine (5-FC) into the cytotoxic compound 5 fluorouracil. We tested the transgenic expression of the bacterial codA gene in citrus as a conditional negative selection marker, with the goal of selecting against plant tissues in which a transgenic cassette has not been successfully removed. We developed transgenic citrus lines containing the selection cassette, codA::nptII, driven by double enhanced CaMV35S promoter, verified by Southern blot analysis, RT-PCR, DsRed expression and subjected these transgenic lines to a 5-FC sensitivity assay. We found that, while non-transgenic citrus were unaffected by the presence of 5-FC, all of the transformed lines displayed symptoms of toxicity, indicating that the codA gene could be used as a negative selectable marker in Citrus, for post-transformation detection of the removal of undesired sequences.

A Mutated Cytosine Deaminase Gene, codA (D314A), as an Efficient Negative Selection Marker for Gene Targeting in Rice

Gene targeting (GT) is a powerful tool manipulating a gene of interest in a given genome specifically and precisely. To achieve efficient GT in higher plants, both positive and negative selection markers are required. In particular, a strong negative selection system is needed for enrichment of cells to eliminate those cells in which random integration of the introduced DNA has occurred in GT experiments. Currently, non-conditional negative selection marker genes are used for GT experiments in rice plants, and no conditional negative selection system is available. In this study, we describe the development of an efficient conditional negative selection system in rice plants using Escherichia coli cytosine deaminase (codA). We found that a mutant codA gene, codA(D314A), acts more efficiently than the wild-type codA for negative selection in rice plants. The codA(D314A) marker was further used as a negative selection marker for GT experiments in rice. Our conditional negative selection system effectively eliminated the cells in which random integration event(s) occurred; the enrichment factor was approximately 100-fold. This enrichment factor was similar to that found when Corynebacterium diphtheriae toxin fragment A was used. Our results suggest the codA(D314A) marker gene as a promising negative selection marker for GT of rice.

Conditional Negative Selection of Gene-Modified Hematopoietic Stem Cells

In this issue of Molecular Therapy, Barese and colleagues from the National Institutes of Health present a landmark study conducted in a clinically relevant large-animal model to evaluate the utility of the herpes simplex virus thymidine kinase (HSVtk) gene for conditional elimination of transplanted long-term repopulating hematopoietic cells, potentially representing true hematopoietic stem cells (HSCs).1 This paper provides proof of concept for this approach, is rich in highly relevant experimental findings, and presents a stimulating discussion that will certainly motivate the field to pay even more attention to the important principle of conditional negative selection of gene-modified cells.

A bacterial haloalkane dehalogenase (dhlA) gene as conditional negative selection marker for rice callus cells

The dhlA gene of Xanthobacter autotrophicus encodes dehalogenase that hydrolyzes dihaloalkanes such as 1,2-dichloroethane (DCE) into cytotoxic halogenated alcohol and an inorganic halide. As plants do not contain dehalogenase activity, they grow normally in the presence of DCE. We tested the transgenic expression of the bacterial dhlA gene in rice as a conditional negative selection marker. We developed 24 transgenic callus lines containing dhlA gene driven by rice actin-1 promoter, verified the expression of dhlA by Northern blot analysis, and subjected these transgenic lines to DCE treatment. We found that, while untransformed callus (Nipponbare) was unaffected by the DCE treatment, most of the transformed lines displayed symptoms of toxicity, indicating that dhlA is an effective conditional negative selection marker gene for rice in vitro cultures.

A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum

Rapid progress in fungal genome sequencing presents many new opportunities for functional genomic analysis of fungal biology through the systematic mutagenesis of the genes identified through sequencing. However, the lack of efficient tools for targeted gene replacement is a limiting factor for fungal functional genomics, as it often necessitates the screening of a large number of transformants to identify the desired mutant. We developed an efficient method of gene replacement and evaluated factors affecting the efficiency of this method using two plant pathogenic fungi, Magnaporthe grisea and Fusarium oxysporum. This method is based on Agrobacterium tumefaciens-mediated transformation with a mutant allele of the target gene flanked by the herpes simplex virus thymidine kinase ( HSVtk) gene as a conditional negative selection marker against ectopic transformants. The HSVtk gene product converts 5-fluoro-2′-deoxyuridine to a compound toxic to diverse fungi. Because ectopic transformants express HSVtk, while gene replacement mutants lack HSVtk, growing transformants on a medium amended with 5-fluoro-2′-deoxyuridine facilitates the identification of targeted mutants by counter-selecting against ectopic transformants. In addition to M. grisea and F. oxysporum, the method and associated vectors are likely to be applicable to manipulating genes in a broad spectrum of fungi, thus potentially serving as an efficient, universal functional genomic tool for harnessing the growing body of fungal genome sequence data to study fungal biology.

The bacterial cytosine deaminase gene used as a conditional negative selection marker in transgenic rice plants

The bacterial gene cod A encodes cytosine deaminase (CD), which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a compound that is toxic to cell growth. Consequently, CD can potentially be used as a conditional negative selection marker when 5-FC is applied. Rice seedlings and calli are sensitive to low concentrations of 5-FU, while the substrate of CD, 5-FC, is not toxic. To test whether the cod A gene can be used as a conditional negative selection marker in rice, we developed transgenic rice plants to contain the cod A coding sequence fused with the phloem-specific promoter of rice tungro bacilliform virus. The transgenic rice plants containing the cod A gene can be killed by the addition of 5-FC to the medium. The first generation of rice transgenic lines with the cod A gene showed a 3:1 segregation pattern of 5-FC sensitivity. We therefore conclude that the cod A gene from E. coli can be used as a conditional negative selection gene in rice.

Thetms2 gene as a negative selection marker in rice

A conditional negative selection marker is essential for high throughput insertional mutagenesis with any two-element transposon tagging system. Thetms2 gene encodes indoleacetic acid hydrolase (IAAH) which converts naphthaleneacetamide (NAM) to the potent auxin naphthaleneacetic acid, a phytotoxic derivative. This gene, under the control of the manopine synthase gene 2 promoter fromAgrobacterium tumefaciens and exogenously applied NAM, have been used effectively as a negative selector inAc/Ds insertional mutagenesis ofArabidopsis thaliana (Sundaresan et al., 1995). In this study we show thattms2 can also be used as a negative selector in rice. T1 transgenic seedlings expressing thistms2 gene under the control of themas2’ promoter showed significant reduction in shoot and root growth in the presence of 5–10 μM NAM under specified growth conditions compared to plants not containing this gene.

Negative selection systems for transgenic barley (Hordeum vulgare L.): comparison of bacterial codA- and cytochrome P450 gene-mediated selection.

Efficient negative selection systems are increasingly needed for numerous applications in plant biology. In recent years various counter-selectable genes have been tested in six dicotyledonous species, whereas there are no data available for the use of negative selection markers in monocotyledonous species. In this study, we compared the applicability and reliability of two different conditional negative selection systems in transgenic barley. The bacterial codA gene encoding cytosine deaminase, which converts the non-toxic 5-fluorocytosine (5-FC) into the toxic 5-fluorouracil (5-FU), was used for in vitro selection of germinating seedlings. Development of codA-expressing seedlings was strongly inhibited by germinating the seeds in the presence of 5-FC. For selecting plants in the greenhouse, a bacterial cytochrome P450 mono-oxygenase gene, the product of which catalyses the dealkylation of a sulfonylurea compound, R7402, into its cytotoxic metabolite, was used. T1 plants expressing the selectable marker gene showed striking morphological differences from the non-transgenic plants. In experiments with both negative selectable markers, the presence or absence of the transgene, as predicted from the physiological appearance of the plants under selection, was confirmed by PCR analysis. We demonstrate that both marker genes provide tight negative selection; however, the use of the P450 gene is more amenable to large-scale screening under greenhouse or field conditions.

A conditional negative selection for Arabidopsis expressing a bacterial cytosine deaminase gene

A conditional negative selection for Arabidopsis expressing a bacterial cytosine deaminase gene

A conditional negative selection for Arabidopsis expressing a bacterial cytosine deaminase gene.

The enzyme activity for cytosine deaminase, which converts cytosine to uracil in bacterial, is usually undetected in higher plants and animals. The enzyme also catalyzes conversion of non-toxic 5-fluorocytosine (5-FC) to 5- fluorouracil (5-FU), a toxic compound for plant growth. The gene encoding cytosine deaminase (codA) from Escherichia coli was fused to cauliflower mosaic virus (CaMV) 35S promoter (P35S), and cloned into a binary vector pLABR101. The resulting plasmid pLABR102 contained two marker genes for plants: a positive marker gene, bialaphos resistance (bar) gene driven by the promoter from nopaline synthase gene (Pnos) and a negative one, P35S-codA. The binary vector pLABR102 was transformed into Arabidopsis thaliana via Agrobacterium-mediated transformation. In transgenic progenies (T3) of the second (T2) generation heterozygous for a single T-DNA insertion, a 3:1 segregation ratio was observed on both bialaphos (resistance to sensitive) and 5-FC (sensitive to unaffected). From T2 plants homozygous for the T-DNA insert, on the other hand, no segregation was detected: all the T3 seedlings were resistant to bialaphos and sensitive to 5-FC. PCR and Northern analyses showed that the 5-FC sensitivity in transgenic descendants was caused by the integration and expression of the chimeric codA gene in the Arabidopsis genome. The results indicated that cytosine deaminase from E. coli is functional and useful for negative selection in Arabidopsis, and that sensitivity to 5-FC as well as the positive bialaphos resistance are dominant traits in Arabidopsis.

Use of suicide genes in gene therapy

Gene therapy encompasses a broad range of treatment modalities that may eventually be applied to a variety of genetic as well as acquired diseases. In addition to compensating for a defective gene--and enhancing a cellular function--gene transfer can allow for a conditional negative selection of target cells. Indeed, the transfer of a gene encoding a susceptibility factor can make a cell specifically sensitive to a drug. After genomic integration, such as potentially destructive gene is endogenously expressed and has therefore been coined a suicide gene. This review describes current experimental approaches and prospects for using suicide genes for the treatment of human diseases.