Generation of double gene disruptions in Dictyostelium discoideum using a single antibiotic marker selection

Gene targeting is a powerful molecular genetic technique that has been widely used to understand specific gene function in vivo. This technique allows the ablation of an endogenous gene by recombination between an introduced DNA fragment and the homologous target gene. However, when multiple gene disruptions are needed, the availability of only a limited number of marker genes becomes a complication. Here we describe a new approach to perform double gene disruptions in Dictyostelium discoideum by simultaneous transfection of two gene targeting cassettes followed by performing clonal selection against only one marker gene. The subsequent PCR-based screens of blasticidin-resistant clones revealed the integration of both the selected and the nonselected targeting cassettes at their original respective loci creating complete gene disruptions. For the genes we have tested in these studies (myosin heavy chain kinases B and C), the efficiency of the double gene targeting event is found in the range of 2%-5% of all blasticidin-resistant colonies following the transfection step. This approach for the simultaneous disruptions of multiple genes should prove to be a valuable tool for other laboratories interested in creating multiple gene disruptants in Dictyostelium or other organisms where a limited number of selectable markers are available.     

The ble gene of Streptoalloteichus hindustanus as a new selectable marker for Dictyostelium discoideum confers resistance to phleomycin

An expression cassette has been constructed which allows expression of the ble gene isolated from Streptoalloteichus hindustanus in Dictyostelium discoideum. This construct has been shown to confer resistance to the bleomycin related antibiotic phleomycin. since the uptake of phleomycin by the cells is pH dependent, we established conditions that allow selection of phleomycin-resistant transformants. Vectors pfeI and pfeII contain, in addition to the cassette, a 592 bp fragment of the D. discoideum plasmid Ddp2 that enables the plasmids to replicate extrachromosomally in Dictyostelium when transformed into a strain that expresses a Ddp2-specific transacting factor (12). pfeI and pfeII contain various unique restriction enzyme sites for cloning. They differ in the G/C-content of the sequence upstream of the ATG start codon.     

Complementation of a Dictyostelium discoideum thymidylate synthase mutation with the mouse gene provides a new selectable marker for transformation.

A cDNA encoding mouse thymidylate synthase has been inserted 3' to the Dictyostelium discoideum actin 15 promoter in an E. coli-D.discoideum shuttle vector. When this construct was introduced into a D.discoideum thymidylate synthase mutant strain HPS400, stable transformants were obtained at high frequency. These transformants grew in standard axenic medium without requiring exogenous thymidine. This construct provides a second selectable marker for use in transformation of D.discoideum.     

Co-transformation using a negative selectable marker gene for the production of selectable marker gene-free transgenic plants

A negative selectable marker gene, codA, was successfully co-transformed with a GUS reporter gene to develop selectable marker gene-free transgenic plants. The pNC binary vector contained a T-DNA harboring the codA gene next to the nptII gene, while a second binary vector, pHG, contained a GUS reporter gene. Tobacco plants (Nicotiana tabacum cv. Samsun NN) were co-transformed via the mixture method with Agrobacterium tumefaciens LBA4404 strains harboring pNC and pHG, respectively. Seeds harvested from the co-transformants were sown on germination media containing 5-fluorocytosine (5-FC). Analysis of the progeny by GUS staining and PCR amplification revealed that all of the 5-FC-resistant R₁ plants were codA free, and that the codA gene segregated independently of the GUS gene. Because codA-free seedlings developed normally on 5-FC-containing medium, we suggest that co-transformation with negatively selectable markers is a viable method for the production of easily distinguished, selectable marker gene-free transgenic plants.     

Identification of the single gene for calmodulin in Dictyostelium discoideum.

We report the isolation and sequence determination of a cDNA containing most of the coding sequence for Dictyostelium discoideum calmodulin. The cloned cDNA was used as a probe to examine the complexity of D. discoideum genomic DNA. These studies indicated that D. discoideum cells possess a single calmodulin gene.     

A selectable and excisable marker system for the rapid creation of recombinant poxviruses

Background

Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.

Methodology/Principal Findings

Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene.

Conclusion/Significance

The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.     

Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase

Fertile transgenic pearl millet plants expressing a phosphomannose isomerase (PMI) transgene under control of the maize ubiquitin constitutive promoter were obtained using the transformation system described here. Proliferating immature zygotic embryos were used as target tissue for bombardment using a particle inflow gun. Different culture and selection strategies were assessed in order to obtain an optimised mannose selection protocol. Stable integration of the manA gene into the genome of pearl millet was confirmed by PCR and Southern blot analysis. Stable integration of the manA transgene into the genome of pearl millet was demonstrated in T₁ and T₂ progeny of two independent transformation events with no more than four to ten copies of the transgene. Similar to results obtained from previous studies with maize and wheat, the manA gene was shown to be a superior selectable marker gene for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes.Abbreviations 2,4-D: 2,4-Diclorophenoxyacetic acid - IAA: Indole acetic acid - ICRISAT: International Crops Research Institute for the Semi-Arid Tropics - IZEs Immature zygotic embryos Communicated by H. Lörz     

An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes

A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.     

Characterization of endosome-endosome fusion in a cell-free system using Dictyostelium discoideum.

An in vitro endosome fusion assay using Dictyostelium discoideum is described. The method requires endocytosis of anti-dinitrophenol (DNP) IgG or DNP-derivitized beta-glucuronidase into two sets of cells. After homogenizing the cells, the vesicles were mixed, and fusion was measured by quantitating immune complex formation between DNP-beta-glucuronidase and anti-DNP IgG. Fusion was dependent upon ATP, temperature, pH, ionic strength, and cytosol and sensitive to detergent, dilution, trypsin, N-ethylmaleimide, and guanosine 5'-3-O-(thio)triphosphate. Although weak bases, ionophores, hadacidin, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and caffeine inhibit endocytosis in vivo, these reagents had no affect on in vitro endosome fusion. Comparison of Dictyostelium with mammalian cells showed differences in the temperature, pH, and salt requirements for fusion, possibly reflecting differences in the life-styles of various cell types. Like mammalian cells, Dictyostelium required GTP-binding protein(s) and an N-ethylmaleimide-sensitive factor for endosome fusion. Thus, the mechanism driving endosome fusion may have been conserved throughout evolution. Electron microscopic studies confirmed in vitro endosome fusion and revealed endosomes were being engulfed by other endosomes, resulting in formation of multivesicular elements (i.e. autophagic vesicles). This system may be useful for characterizing mutations, evolution, and developmental regulation along the endocytic pathway.     

Biolistic transformation of soybean using a new selectable marker gene conferring resistance to dinitroanilines

A biolistic transformation of soybean using the construct pAHTUAm, which contains a mutant α-tubulin gene, as a selectable marker gene that confers resistance to dinitroaniline herbicides, as well as the additional construct pAHTUB1, which carries a full-length barley β-tubulin gene that ensures correct co-expression of exogenous tubulin in cells of transgenic soybean lines, is carried out. It is established that 10 μM trifluralin is the most optimal selective concentration to pick up transformed soybean lines. The transgenic nature of the selected regenerants is confirmed by Southern blotting hybridization using a specific probe to the α-tubulin selectable gene     

Excision of selectable marker gene from transgenic tobacco using the GM-gene-deletor system regulated by a heat-inducible promoter

To excise a selectable marker gene from transgenic plants, a new binary expression vector based on the 'genetically modified (GM)-gene-deletor' system was constructed. In this vector, the gene coding for FLP site-specific recombinase under the control of a heat shock-inducible promoter HSP18.2 from Arabidopsis thaliana and isopentenyltransferase gene (ipt), as a selectable marker gene under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter, were flanked by two loxP/FRT fusion sequences as recombination sites in direct orientation. Histochemical staining for GUS activity showed that, upon induction by heat shock, all exogenous DNA, including the selectable marker gene ipt, beta-glucuronidase (gusA) gene and the FLP recombinase gene, between two loxP/FRT sites was eliminated efficiently from primary transgenic tobacco plants. Molecular analysis further confirmed that excision of the marker gene (ipt) was heritable and stable. Our approach provides a reliable strategy for auto-excising a selectable marker gene from calli, shoots or other tissues of transgenic plants after transformation and producing marker-free transgenic plants.     

cAMP induces a rapid and reversible modification of the chemotactic receptor in Dictyostelium discoideum

Stimulation, within 1 min after cAMP stimulation, of aggregation-competent Dictyostelium discoideum amebae was found to cause a rapid (within 1 min) modification of the cell's surface cAMP receptor. The modified receptor migrated on SDS PAGE as a 47,000-mol-wt protein, as opposed to a 45,000-mol-wt protein labeled on unstimulated cells. The length of time this modified receptor could be detected depended upon the strength of the cAMP stimulus: 3-4 min after treatment with 10(-7) M cAMP, cells no longer possessed the 47,000-mol-wt form of the cAMP receptor. Instead, the 45,000-mol-wt form was present. Stimulation of cells with 10(-5) M cAMP, however, resulted in the persistent (over 15 min) expression of the modified receptor. The time course, concentration dependence, and specificity of stimulus for this cAMP-induced shift in the cAMP receptor were found to parallel the cAMP-stimulated phosphorylation of a 47,000-mol-wt protein. In addition, both phenomena were shown to occur in the absence of endogenous cAMP synthesis. The possibility that the cAMP receptor is phosphorylated in response to cAMP stimulation, and the role of this event in cell desensitization, are discussed.     

A protocol for the efficient screening of putatively transformed plants forbar,the selectable marker gene,using the polymerase chain reaction

Amplification of thebar gene usingTaq DNA polymerase in PCR is often not successful, possibly due tobar's high GC content. We describe a PCR protocol in which reliable amplification at a sensitivity of one gene copy per genome (in this study, barley) present in the reaction was achieved using a novel pair of primers and Expand^tm High Fidelity DNA polymerase mix (Boehringer Mannheim). This method should allow for rapid screening of plants putatively transformed withbar.