Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker

With increasing application of Hansenula polymorpha in fundamental research and biotechnology, many more genetic manipulations are required. However, these have been restricted for the finiteness of selectable markers. Here, MazF, a toxin protein from Escherichia coli, was investigated as a counter-selectable marker in H. polymorpha. The lethal effect of MazF on yeast cells suggested that it is a candidate for counter-selection in H. polymorpha. Markerless or scarless gene deletion in H. polymorpha was conducted based on selectable markers cassette mazF-zeoR, in which the zeocin resistance cassette and mazF expression cassette were used as positive and counter-selectable markers, respectively. For markerless deletion, the target region can be replaced by CYC1TT via two-step homologous recombination. For scarless deletion, the innate upstream region (5′UP) of target genes rather than CYC1TT mediates homologous recombination to excise both selectable markers and 5′ sequence of target genes. Moreover, scarless deletion can be accomplished by using short homologous arms for the effectiveness of mazF as a counter-selectable marker. The applicability of the strategies in markerless or scarless deletion of PEP4, LEU2, and TRP1 indicates that this study provides easy, time-efficient, and host-independent protocols for single or multiple genetic manipulations in H. polymorpha.     

The selectable antibiotic marker, tetA(C), increases Pseudomonas aeruginosa susceptibility to the herbicide/superoxide generator, paraquat

The presence of the widely used selectable antibiotic marker, tetA(C), unexpectedly increased the sensitivity of Pseudomonas aeruginosa PAO1 to the superoxide-generating herbicide, paraquat. A DNA fragment spanning the first 99 amino acids of TetA(C) was sufficient to confer paraquat sensitivity. The TetA(C)-induced paraquat sensitive phenotype was observed in other Gram-negative bacteria such as Agrobacterium tumefaciens, Salmonella enterica ser. Typhimurium and Xanthomonas campestris suggesting that this is a general property of tetA(C). This finding serves as a cautionary note for those using tetA(C) as a selectable marker for genetic manipulations in studies using paraquat either as a superoxide stress generator or a redox cycling drug.     

The elimination of a selectable marker gene in the doubled haploid progeny of co-transformed barley plants

Following the production of transgenic plants, the selectable marker gene(s) used in the process are redundant, and their retention may be undesirable. They can be removed by exploiting segregation among the progeny of co-transformants carrying both the selectable marker gene and the effector transgene. Here we show that the doubled haploid technology widely used in conventional barley breeding programmes represents a useful means of fixing a transgene, while simultaneously removing the unwanted selectable marker gene. Primary barley co-transformants involving hpt::gfp (the selectable marker) and gus (a model transgene of interest) were produced via Agrobacterium-mediated gene transfer to immature embryos using two respective T-DNAs. These plants were then subjected to embryogenic pollen culture to separate independently integrated transgenes in doubled haploid progeny. A comparison between 14 combinations, involving two Agrobacterium strains carrying various plasmids, revealed that the highest rate of independent co-transformation was achieved when a single Agrobacterium clone carried two binary vectors. Using this principle along with Agrobacterium strain LBA4404, selectable marker-free, gus homozygous lines were eventually obtained from 1.5 per 100 immature embryos inoculated. Compared to the segregation of uncoupled T-DNAs in conventionally produced progeny, the incorporation of haploid technology improves the time and resource efficiency of producing true-breeding, selectable marker-free transgenic barley.     

Development and application of a positive–negative selectable marker system for use in reverse genetics in Plasmodium

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as ‘Hit and Run’ and ‘vector recycling’ which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.     

Identification of Tf1 integration events in S. pombe under nonselective conditions

Integration of retroviral elements into the host genome is a phenomena observed among many classes of retroviruses. Much information concerning the integration of retroviral elements has been documented based on in vitro analysis or expression of selectable markers. To identify possible Tf1 integration events within silent regions of the Schizosaccharomyces pombe genome, we focused on performing an in vivo genome-wide analysis of Tf1 integration events from the nonselective phase of the retrotransposition assay. We analyzed 1000 individual colonies streaked from four independent Tf1 transposed patches under nonselection conditions. Our analysis detected a population of G418^S/neo⁺ Tf1 integration events that would have been overlooked during the selective phase of the assay. Further RNA analysis from the G418^S/neo⁺ clones revealed 50% of clones expressing the neo selectable marker. Our data reveals Tf1's ability to insert within silent regions of S. pombe's genome.     

Co-targeting strategy for precise,scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)—in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.

A transgene-free strategy allows precise editing of genes lacking a selectable phenotype by electroporation of CRISPR/Cas9 ribonucleoproteins and single-stranded oligodeoxynucleotide templates.     

Development of a plant transformation selection system based on expression of genes encoding gentamicin acetyltransferases

The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plants. A new selectable marker system has been developed based on bacterial gentamicin-3-N-acetyltransferases [AAC(3)]. These enzymes inactivate aminoglycoside antibiotics by acetylation. Two examples of AAC(3) enzymes have been manipulated to be expressed in plants. Chimeric AAC(3)-III and AAC(3)-IV genes were assembled using the constitutively expressed cauliflower mosaic virus 35S promoter and the nopaline synthase 3′ nontranslated region. These chimeric genes were engineered into vectors for Agrobacterium-mediated plant transformation. Petunia hybrida and Arabidopsis thaliana tissue transformed with these vectors grew in the presence of normally lethal levels of gentamicin. The transformed nature of regenerated Arabidopsis plants was confirmed by DNA hybridization analysis and inheritance of the selectable phenotype in progeny. The chimeric AAC(3)-IV gene has also been used to select transformants in several additional plant species. These results show that the bacterial AAC(3) genes will serve as useful selectable markers in plant tissue culture.     

Cloning of Phanerochaete chrysosporium leu2 by Complementation of Bacterial Auxotrophs and Transformation of Fungal Auxotrophs

A Phanerochaete chrysosporium cDNA library was constructed in an expression vector that allows expression in both Escherichia coli and Saccharomyces cerevisiae. This expression vector, λYES, contains the lacZ promoter for expression in E. coli and the GAL1 promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the β-isopropylmalate dehydrogenase from P. chrysosporium was characterized further. The genomic clone (gleu2) was subsequently isolated and was used successfully as a selectable marker to transform P. chrysosporium auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous P. chrysosporium transformation procedures. The method described here allows other genes to be isolated from P. chrysosporium for use as selectable markers.     

An optimized method for suicide vector-based allelic exchange in Klebsiella pneumoniae

Klebsiella pneumoniae is an important and versatile bacterium that can be found in diverse environments and is also a frequent cause of human infections. Limited data exists on the mechanisms of interaction between K. pneumoniae and the human host and of adaptations to other environments. Coupled with the high genetic diversity of this species, these factors highlight the necessity for substantial further K. pneumoniae-focused molecular genetics studies.In this report we describe a simple and efficient experimental protocol for suicide vector-based allelic exchange in K. pneumoniae. The protocol has been validated by mutating multiple loci in four distinct K. pneumoniae strains, including highly capsulated and/or multi-antibiotic resistant clinical isolates. Three key enhancements are reported:(1) Use of pDS132-derived conjugative plasmids carrying improved cloning sites, (2) Performance of sacB counterselection at 25 °C as opposed to higher temperatures, and (3) Exploitation of Flp-recombinase-mediated deletion of FRT (Flp recombinase target) flanked resistance cassettes to allow for reiterative manipulations with a single selectable marker. This study also highlights a problem that may be encountered when the aacC1 gentamicin resistance marker is used in K. pneumoniae and suggests alternative markers.The protocol developed in this study will help investigate the plethora of uncharacterized genes present in the K. pneumoniae pan-genome and shed further light upon clinically and industrially important phenotypes observed in this ubiquitous species.     

The Cre/loxP system in Giardia lamblia: genetic manipulations in a binucleate tetraploid protozoan

The bacteriophage-derived Cre/loxP system is a valuable tool that has revolutionised genetic and cell biological research in many organisms. We implemented this system in the intestinal parasite Giardia lamblia, an evolutionarily diverged protozoan whose binucleate and tetraploid genome organisation severely limits the application of reverse genetic approaches. We show that Cre-recombinase is functionally expressed in G. lamblia and demonstrate “recycling” of selectable markers. Providing the means for more complex and versatile genetic modifications, this technique massively increases the scope of functional investigations in G. lamblia and other protozoa with similar limitations with respect to genetic manipulation.     

Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1

Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd) or human dihydrofolate reductase (hdhfr). In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP) and human dihydrofolate reductase (hDHFR), was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP) and blasticidin-S deaminase (BSD). Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which thioredoxin peroxidase-1 (Bbtpx-1) gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO) parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.     

Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequene of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nulceotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These results promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.     

Agrobacterium-mediated transformation of Lolium rigidum Gaud.

Lolium rigidum Gaud. is an annual grass grown for forage but also an economically damaging crop weed. A single genotype somatic embryogenic callus line, VLR1-60, was identified from a herbicide susceptible L. rigidum population, VLR1, and proved to be amenable to Agrobacterium tumefaciens-mediated transformation. Somatic embryogenic calli were continuously induced from the meristematic region of VLR1-60 plants multiplied in vitro and the basic tolerance level of VLR1-60 to hygromycin B was determined. A hygromycin phosphotransferase gene was used as a selectable marker for hygromycin B selection. Somatic embryogenic calli derived from in vitro grown vegetative tillers were co-cultivated with the A. tumefaciens strain EHA105 harbouring binary vector carrying reporter genes and selectable marker in the presence of acetosyringone for 3 days. Inoculated calli were recovered on callus proliferation medium containing Timentin? but lacking hygromycin and were then subcultured onto media with hygromycin concentrations increased progressively through time for selection of transformed plant cells. Putative transgenic plants were recovered and integration of transgenes was confirmed by Southern hybridization analysis and by detection of DsRed or GUS activity in transgenic plants. The frequency of plant transformation was 1.3 %. The ability to transform L. rigidum will provide opportunities for functional characterization of genes to improve forage quality and increase our understanding of the evolution of herbicide resistance and of the basic genetics underlying traits that make L. rigidum a damaging crop weed.     

Broad-specificity amino acid racemase,a novel non-antibiotic selectable marker for transgenic plants

The broad-specificity amino acid racemase (Bsar) from Pseudomonas putida catalyzes the racemization of various amino acids, offering a flexible and feasible platform to develop a new non-antibiotic selectable marker system for plant transformation. In the present study, we demonstrated that a Bsar variant, Bsar-R174K, that is useful as a selectable marker gene in Arabidopsis and rice that were susceptible to l-lysine and D-alanine. The introduction of wild-type Bsar, Bsar-R174K or Bsar-R174A into E. coli lysine or asparagine auxotrophs was able to rescue the growth of these microorganisms in minimal media supplemented with selectable amino acid enantiomers. The transformation of Arabidopsis with Bsar or Bsar variants based on d-alanine selection revealed that Bsar-R174K had the greatest efficiency (2.40%), superior to kanamycin selection-based transformation (1.10%). Whereas, l-lysine-based selection exhibited lower efficiency for Bsar-R174K (0.17%). The progenies of selected Bsar-R174K transgenic Arabidopsis revealed normal growth properties. In addition, Bsar-R174K transgenic rice was obtained on l-lysine medium with an efficiency of 0.9%, and the progenies of the transgenic rice revealed morphologically normal phenotypes comparable with their wild-type counterparts. This study presents the first report of broad range amino acid racemase Bsar-R174K as a non-antibiotic selectable marker system applied in transgenic plants.     

Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.)

Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l⁻¹ 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l⁻¹ hygromycin and 200 mg l⁻¹ cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato.     

Agrobacterium-mediated co-transformation of rice using two selectable marker genes derived from rice genome components

A method for Agrobacterium-mediated co-transformation of rice (Oryza sativa L.) was developed using rice-derived selection markers. Two T-DNAs were efficiently introduced into separate loci using selectable marker gene cassettes consisting of the mutated acetolactate synthase gene (mALS) under the control of the callus-specific promoter (CSP) (CSP:mALS) and the ferredoxin nitrite reductase gene (NiR) under the control of its own promoter (NiR P:NiR). The CSP:mALS gene cassette confers sulfonylurea herbicide resistance to transgenic rice callus. The NiR P:NiR construct complements NiR-deficient mutant cultivars such as ‘Koshihikari’, which are defective in the regulation of nitrogen metabolism. In the present study, the CaMV35S:GUS and CaMV35S:GFP gene cassettes were co-introduced into the ‘Koshihikari’ genome using our system. Approximately 5–10 independent transgenic lines expressing both the GUS and GFP reporters were obtained from 100 Agrobacterium co-inoculated calli. Furthermore, transgenic ‘Koshihikari’ rice lines with reduced content of two major seed allergen proteins, the 33 and 14–16?kDa allergens, were generated by this co-transformation system. The present results indicate that the generation of selectable antibiotic resistance marker gene-free transgenic rice is possible using our rice-derived selection marker co-transformation system. Key message An improved rice transformation method was developed based on Agrobacterium-mediated co-transformation using two rice genome-derived selectable marker gene cassettes.