Screenable Marker Research Articles

Stable electrotransformation of symboint candidate diazotrophic bacterium with plasmids carrying selectable and screenable marker genes

Nitrogen-fixing symbioses had been established between the originally asymbiotic soil bacterium Azotobacter vinelandii CCM289 and different lower and higher plant species. Better characterization and further development of such artificial systems require a reliable genetic transformation method for the introduction of marker genes into symbiont candidates. The performance of electroporation was evaluated using pJB3 (4.8 kb), pBl121 (12.8 kb) and pFAJ31.2 (24 kb) plasmid DNAs containing selectable (Ap, Km, Tc) and screenable ( gusA, lacZ) marker genes. The adapted methods for the preparation of transformation-competent azotobacters and their electroporation (18 kV/cm electric field strength, 5 ms time constant, 0°C) provided up to 6.8 × 10 5 transformants per μg plasmid DNA, which is about 10 3 times the transformation efficiency achieved in control experiments. No electrotransformants were obtained with the 24-kb pFAJ31.2. The size of plasmid DNA did not significantly affect the efficiency of transformation. Transformants were able to grow at antibiotic concentrations that were 100–200 times greater than the lowest amounts that completely inhibited the growth of wild-type bacteria. A constitutive expression of gusA gene was observed in transformants with the CaMV 35S promoter- gusA fusion containing pBl121, while lacZ expression was not detected under the control of the lac promoter in pJB3 transformants. Electroporated plasmids were reisolated from transformants in their original form, while non-transformed bacteria did not contain indigenous plasmids. PCR amplification and Southern DNA blot hybridization showed the integration of plasmid DNA into the host genome as well. Transformants retained their nitrogen-fixing ability and had normal morphological and growth characteristics. Experimental findings proved the stable maintenance of plasmid DNA in azotobacters, making possible the routine transformation and detection of these symbiont candidates.

The green fluorescent protein (GFP) as a vital screenable marker in rice transformation

An engineered green fluorescent protein (GFP) from the jellyfish Aequora victoria was used to develop a facile and rapid rice transformation system using particle bombardment of immature embryos. The mgfp4 gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright-green fluores- cence easily detectable and screenable in rice tissue 12—22 days after bombardment. Visual screening of transformed rice tissue, associated with a low level of antibiotic selection, drastically reduced the quantity of tissue to be handled and the time required for the recovery of transformed plants. GFP expression was observed in primary transformed rice plants (T 0 ) and their progeny (T 1 ). We describe various techniques to observe GFP in vitro and in vivo. The advantages of this new screenable marker in rice genetic engineering programmes are discussed.

Agrobacterium-mediated transformation of sugarcane using GFP as a screenable marker

The use of the green fluorescent protein (GFP) and in vivo screening for transgenic cells has enabled the first visual selection of transgenic sugarcane (Saccharum L. hybrid) transformed by Agrobacterium-mediated techniques. Selection of GFP-positive embryogenic callus transformed using Agrobacterium tumefaciensstrain AGL0 was performed by fluorescence microscopy for 6 weeks after transformation. The use of GFP as a screenable marker aided the rapid segregation of individual transformation events and facilitated the initial visual selection of transgenic sugarcane callus without antibiotics, herbicides or an assay. The binary vector used in transformation was pTO134, which contains a synthetic gfp gene (sgfpS65T) and the bialaphos resistance gene (bar) both controlled by CaMV 35S promoters. The appearance of GFP-expressing cells observed on embryogenic callus was 5.3%. Subsequently, GFP-positive calli grew in the presence of a level of the herbicide bialaphos, that was toxic to untransformed calli. GFP-positive shoots were regenerated and one sugarcane plant expressed GFP in the initial stages. Molecular analysis confirmed the presence of the sgfpS65T coding region in the genome of regenerated plants. Visual screening for Agrobacterium-mediated transformation events using GFP was an efficient technique, which may be applied for the transformation of other plants.

Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants

A set of plasmids has been constructed utilizing the promoter, 5' untranslated exon, and first intron of the maize ubiquitin (Ubi-1) gene to drive expression of protein coding sequences of choice. Plasmids containing chimaeric genes for ubiquitin-luciferase (Ubi-Luc), ubiquitin-beta-glucuronidase (Ubi-GUS), and ubiquitin-phosphinothricin acetyl transferase (Ubi-bar) have been generated, as well as a construct containing chimaeric genes for both Ubi-GUS and Ubi-bar in a single plasmid. Another construct was generated to allow cloning of protein coding sequences of choice on Bam HI and Bam HI-compatible restriction fragments downstream of the Ubi-1 gene fragment. Because the Ubi-1 promoter has been shown to be highly active in monocots, these constructs may be useful for generating high-level gene expression of selectable markers to facilitate efficient transformation of monocots, to drive expression of reference reporter genes in studies of gene expression, and to provide expression of biotechnologically important protein products in transgenic plants.

Use of the green fluorescent protein as a marker in transfected Leishmania

We have tested the suitability of the green fluorescent protein (GFP) of Aequorea victoria as a marker for studies of gene expression and protein targeting in the trypanosomatid parasite Leishmania. Leishmania promastigotes expressing GFP from episomal pXG vectors showed a bright green fluorescence distributed throughout the cell, readily distinguishable from control parasites. Transfection of a modified GFP gene containing GC-rich synonymous codons and the S65T mutation (GFP+) yielded a much higher fluorescence. FACS analysis revealed a clear quantitative separation between GFP-transfected and control parasites, with pXG-GFP + transfectants showing fluorescence signals more than 100-fold background. Episomal DNAs could be recovered from small numbers of fixed cells, showing that GFP could be used as a convenient screenable marker for FACS separations. GFP was fused to the C-terminus of the LPG1 protein, which retained its ability to restore LPG expression when expressed in the lpg − R2D2 mutant of L. donovani. The LPG1(GFP) fusion was localized to a region situated between the nucleus and kinetoplast; its pattern was similar to that of LPG2, which is known to be located in the Golgi apparatus. This is notable as LPG1 participates in the biosynthesis of the glycan core of the LPG GPI anchor, whereas protein GPI anchor biosynthesis occurs in the endoplasmic reticulum. These studies suggest that the GFP will be a broadly useful marker in Leishmania.

Escherichia coli β-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii

We have developed several protocols for the use of β-galactosidase (βGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent βGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chlorophenol red-β-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing βGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of βGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites

Stable transformation via electroporation into maize Type II callus and regeneration of fertile transgenic plants

Maize Type II callus tissue was used as the plant material for genetic transformation via electroporation. Plasmid DNA containing a selectable marker gene (either neomycin phosphotransferase (npt-II) or phosphinothricin acetyl transferase (bar)), and a screenable marker gene (gus A) was incubated with the tissue prior to electroporation. Electroporated callus tissue was placed on selection medium containing kanamycin sulfate or Bast(™). No kanamycin resistant colonies were recovered whereas four independent Basta(™) resistant callus isolates were recovered from a total of 544 cuvettes electroporated. After 8 to 16 weeks on the Basta(™) containing medium, selected calli were isolated and maintained in individual selection plates for 4 to 6 weeks until sufficient tissue accumulated. Enzyme assays and DNA analyses were performed to verify the transformation events. Several plants were regenerated from individual callus isolates. The plants derived from one callus isolate were male sterile while those derived from the other isolates were both male and female fertile. Most plants showed Basta(™) resistance. DNA analyses confirmed the presence of the introduced bar gene(s) in the primary regenerants and their progeny. The integration patterns of the inserted DNA appeared to be complex.

Non‐destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow‐scan CCD camera and an optimized assay

A flexible, comparatively inexpensive system based on a liquid nitrogen‐cooled slow‐scan CCD (charge coupled device) camera is presented, which can be employed for quantitative low‐light (bioluminescence, chemiluminescence or fluorescence) imaging. Using this camera system and the firefly luciferase (LUC) as a screenable marker, transgenic tobacco lines have been produced by direct gene transfer. Bioluminescence emitted from single tobacco cells transiently expressing the firefly luciferase gene (Luc) as well as from stably transformed calli, regenerated shoots, plantlets and T1 seedlings could be monitored in vivo with no effect on the viability of the material analysed. The patterns of light emission from sections through Luc‐expressing leaves and bioluminescent single protoplasts isolated from such leaves were also imaged microscopically. The assay used to detect in vivo LUC activity was optimized by quantifying bioluminescence emitted from Luc‐expressing tobacco protoplasts and leaves incubated in different substrate solutions and determining the kinetics of light emission during incubation in the substrate solution.

Efficient selection of recombinant adenoviruses by vectors that express beta-galactosidase.

Adenovirus serotype 5 vectors which contain the Excherichia coli beta-galactosidase gene driven by the cytomegalovirus immediate-early promoter as a screenable marker have been made and successfully used in the construction of recombinant adenoviruses. The beta-galactosidase gene has been introduced into viruses in which the E3 region is maintained or deleted and in which the cis-acting packaging sequence has been reiterated at the right end of the chromosome. A unique BstBI site has been introduced 3' of the beta-galactosidase gene. Cotransfection of BstBI-digested vector DNA and a plasmid containing the left end of the viral chromosome followed by staining with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) results in clear plaques when overlap recombination has occurred and blue plaques when ligation of the viral arms has occurred within the host cell. The beta-galactosidase-expressing viruses grow to lower titers than do the parental viruses, leading to a relative growth advantage for viruses resulting from overlap recombination. Combined with color selection based on the beta-galactosidase gene, this system permits efficient production and selection of recombinant viruses after cotransfection of BstBI-digested viral DNA with a plasmid including left-end viral sequences and the gene of interest. The beta-galactosidase-expressing viral DNAs were used to construct viruses containing BstBI sites on either side of the cis-acting packaging element as a means of testing their utility.

Targeted disruption of the TGA3 locus in Arabidopsis thaliana

A major drawback to study gene functions in plant systems is the lack of an effective gene knockout strategy. With a large number of plant genes isolated and the accelerating pace by which this collection is growing, the need for their functional analyses at the whole plant level has become increasingly urgent. Here evidence is reported for the first successful disruption of a non-selectable gene in Arabidopsis thaliana by creating a mutant of the TGA3 locus via targeted insertion of the bacterial neo gene conferring kanamycin (Km) resistance. A beta-glucuronidase (GUS) expression unit outside the region of homology was used as a screenable marker to distinguish homologous recombination events from those of ectopic insertions. PCR amplification coupled with Southern blot screening identified two putative homologous recombination events among 2580 Kmr calli. One callus line was subsequently isolated and the structure of the targeted TGA3 allele confirmed by Southern blot analyses. This study demonstrates the feasibility of targeting a non-selectable locus in Arabidopsis. Combined with future improvements in negative selection strategies and efficient transformation methodologies, gene replacement studies in plants could become a routine technique.

Molecular and genetic characterization of elite transgenic rice plants produced by electric-discharge particle acceleration

The recovery of transgenic rice plants expressing a number of exogenous genes was reported previously. Using immature embryo explants as the target tissue, plasmids containing both selectable and screenable marker genes were introduced into elite rice varieties via electric-discharge particle acceleration. Co-integration, copy number, expression, and inheritance of these genes were analyzed. A 100% co-integration frequency was confirmed by Southern-blot analyses of R0 plants. The majority of transgenic plants contained between one and ten copies of exogenous DNA and molecular and genetic analyses of progeny indicated that all copies in almost all R0 plants were inherited as a single dominant hemizygous locus. Co-expression of unselected genes ranged from 30-66% for gus/hmr constructs, depending on the promotor used, and up to 90% for bar/hmr constructs. The integrative structures of two unlinked transgenic loci of a rare R0 plant were analyzed in detail by Southern-blot analysis of its progeny.

A plant transformation vector with a minimal T-DNA.

Plant transformation, via Agrobacterium tumefaciens, is usually performed with binary vectors. Most of the available binary vectors contain within the T-DNA (which is transferred to the plant genome) components not required for the intended modification. These additional sequences may cause potential risks during field testing of the transgenic plants or even more in the case of commercialization. The aim of this study was to produce a plant transformation vector which only contains a selectable and screenable marker gene and a multiple cloning site for insertion of promoter::foreign gene::terminator cassettes from other plasmids.

Agrobacterium-mediated transformation of Stylosanthes guianensis and production of transgenic plants

Transgenic plants of the tropical forage legume Stylosanthes guianensis (Aubl.) Sw. CIAT 184 were regenerated from kanamycin- and phosphinotricin-resistant calli derived from Agrobacterium-inoculated leaf sections. Inoculations were carried out using the disarmed Agrobacterium tumefaciens strain EHA 101 harboring the binary vector pGV1040. pGV1040 T-DNA contains the bar and nptII genes as selectable markers, and the gusA gene as a screenable marker. Kanamycin was deleterious to plant regeneration, consequently phosphinotricin resistance was used for selection. Presence of the bar, npt II and gusA genes was detected during the regeneration process as well as in the transformed regenerated plants. Mendelian inheritance of the bar gene in the selfed progeny of the transformed plants was demonstrated.

Electroporation of rapeseed protoplasts - transient and stable transformation.

Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β-glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead-type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10-5 and a relative transformation frequency of 0.3% could be achieved. X-ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.

High-frequency germinal transposition of DsALS in Arabidopsis.

We have established an efficient transposontagging system in Arabidopsis thaliana using the Activator/Dissociation (Ac/Ds) elements from maize. This system consists of two components, a stable trans-activator, Acst, that supplies transposase, and a cis-responsive Ds element. Ds and Acst were constructed with different selectable and screenable markers to facilitate monitoring of Ds excisions and insertions as well as segregation of Ds and Acst. Fusions of the 35S, rbcS, or CHS promoters to Ac transposase were used to trans-activate DsALS, a Ds element carrying an herbicide-resistance gene. The ALS gene encoding acetolactase synthase, which confers resistance to chlorsulfuron, functioned as a versatile marker for selection of plants grown in tissue culture as well as in soil. Thirty-five Acst lines were crossed to two DsALS lines, and the resulting progeny were assayed for germinal transposition of DsALS. Trans-activation of DsALS by Acst resulted in germinal excision frequencies of up to 64% when using 35S promoter-Ac transposase fusions, up to 67% when using rbcS-transposase fusions, and up to 1% when using CHS-transposase fusions. Amongst progeny bearing terminal excisions, Southern analysis revealed that 45% from 35S-Acst crosses and 29% from rbcS-Acst crosses carried reintegrated DsALS elements. The Ac/Ds system we have developed should prove to be an effective tool for stable gene tagging in Arabidopsis.

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2-4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was verified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T0 plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T1 and T2 progenies. Enzyme analyses on T1 progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T0 plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.

Factors affecting transient gene expression in electroporated Glycine max protoplasts

Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 μF and a protoplast concentration of 1-3 × 10(6)/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.

FOREIGN GENE TRANSFER TO SWEET POTATO (IPOMOEA BATATAS)

Development of a gene transfer system will enable rapid introduction of agronomically useful genes into elite cultivars of sweet potato. We compared microprojectile bombardment and Agrobacterium cocultivation approaches to introduce foreign genes into the genome of two sweet potato cultivars. Chimeric marker genes (gusA and kan) were successfully introduced into cvs. Jewel and TIS-70357 using both approaches. However, transgenic plants were generated in vitro using only the Agrobacterium approach. Callus and root isolates with stable expression of gusA gene were obtained using the microprojectile method. Expression of the screenable marker gusA gene was detected by histochemical assays. Integration of the introduced gene into the genome of sweet potato was confirmed by polymerase chain reaction (PCR) amplification of the kan gene and Southern blot analyses. Transgenic sweet potato plants from two cultivars are being raised and studied for quantitative expression and localization of the introduced genes. These results show that foreign genes can be successfully introduced and expressed in sweet potato. Current efforts are directed at optimizing several variables to increase the transformation efficiencies and to generate transgenic cultivars with foreign genes of agricultural importance.

Use of a novel cassette to label phenotypically a cryptic plasmid of Bacillus subtilis and map loci involved in its stable maintenance

In order to facilitate studies on the maintenance of cryptic plasmids from Gram-positive bacteria we have constructed a novel cassette cAPG1000 (5.0 kb) which carries both a selectable marker (chloramphenicol resistance from Staphylococcus aureus plasmid pC194) and a screenable marker (the xylE gene from the TOL plasmid of Pseudomonas putida expressed from a cloned promoter of Bacillus phage SPO2) and which is flanked by terminators to prevent transcription from the cassette activating or inhibiting loci adjacent to the site of insertion. To demonstrate the usefulness of this cassette we have mapped loci required for stable maintenance of an 8.6 kb cryptic plasmid endogenous to Bacillus subtilis (pPOD2000) from the properties of cAPG1000 insertion and insertion/deletion derivatives. We have identified the replication region as well as separate regions required for segregational and structural stability. The segregational mechanism is very efficient since it allows no detectable loss despite the fact that bacteria carrying the plasmid have a greatly increased mean generation time.

Bialaphos selection of stable transformants from maize cell culture

Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for β-glucuronidase (GUS). Bombarded cells were selected on medium containing the herbicide bialaphos, which is cleaved in plant cells to yield phosphinothricin (PPT), an inhibitor of glutamine synthetase. The bialaphos-resistant callus contained the bar gene and expressed PAT as assayed by PPT inactivation. Transformants that expressed high levels of PAT grew more rapidly on increasing concentrations of bialaphos than transformants expressing low levels of PAT. Fifty percent of the bialaphos-resistant transformants tested (8 of 16) expressed the nonselected gene encoding GUS.