Single-copy Integration Research Articles

Agrobacterium-Mediated Transformation of Yeast and Fungi.

Two decades ago, it was discovered that the well-known plant vector Agrobacterium tumefaciens can also transform yeasts and fungi when these microorganisms are co-cultivated on a solid substrate in the presence of a phenolic inducer such as acetosyringone. It is important that the medium has a low pH (5-6) and that the temperature is kept at room temperature (20-25°C) during co-cultivation. Nowadays, Agrobacterium-mediated transformation (AMT) is the method of choice for the transformation of many fungal species; as the method is simple, the transformation efficiencies are much higher than with other methods, and AMT leads to single-copy integration much more frequently than do other methods. Integration of T-DNA in fungi occurs by non-homologous end-joining (NHEJ), but also targeted integration of the T-DNA by homologous recombination (HR) is possible. In contrast to AMT of plants, which relies on the assistance of a number of translocated virulence (effector) proteins, none of these (VirE2, VirE3, VirD5, VirF) are necessary for AMT of yeast or fungi. This is in line with the idea that some of these proteins help to overcome plant defense. Importantly, it also showed that VirE2 is not necessary for the transport of the T-strand into the nucleus. The yeast Saccharomyces cerevisiae is a fast-growing organism with a relatively simple genome with reduced genetic redundancy. This yeast species has therefore been used to unravel basic molecular processes in eukaryotic cells as well as to elucidate the function of virulence factors of pathogenic microorganisms acting in plants or animals. Translocation of Agrobacterium virulence proteins into yeast was recently visualized in real time by confocal microscopy. In addition, the yeast 2-hybrid system, one of many tools that have been developed for use in this yeast, was used to identify plant and yeast proteins interacting with the translocated Agrobacterium virulence proteins. Dedicated mutant libraries, containing for each gene a mutant with a precise deletion, have been used to unravel the mode of action of some of the Agrobacterium virulence proteins. Yeast deletion mutant collections were also helpful in identifying host factors promoting or inhibiting AMT, including factors involved in T-DNA integration. Thus, the homologous recombination (HR) factor Rad52 was found to be essential for targeted integration of T-DNA by HR in yeast. Proteins mediating double-strand break (DSB) repair by end-joining (Ku70, Ku80, Lig4) turned out to be essential for non-homologous integration. Inactivation of any one of the genes encoding these end-joining factors in other yeasts and fungi was employed to reduce or totally eliminate non-homologous integration and promote efficient targeted integration at the homologous locus by HR. In plants, however, their inactivation did not prevent non-homologous integration, indicating that T-DNA is captured by different DNA repair pathways in plants and fungi.

High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing.

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

Disclosing the Parameters Leading to High Productivity of Retroviral Producer Cells Lines: Evaluating Random Versus Targeted Integration.

Gammaretrovirus and lentivirus are the preferred viral vectors to genetically modify T and natural killer cells to be used in immune cell therapies. The transduction efficiency of hematopoietic and T cells is more efficient using gibbon ape leukemia virus (GaLV) pseudotyping. In this context gammaretroviral vector producer cells offer competitive higher titers than transient lentiviral vectors productions. The main aim of this work was to identify the key parameters governing GaLV-pseudotyped gammaretroviral vector productivity in stable producer cells, using a retroviral vector expression cassette enabling positive (facilitating cell enrichment) and negative cell selection (allowing cell elimination). The retroviral vector contains a thymidine kinase suicide gene fused with a ouabain-resistant Na+,K+-ATPase gene, a potential safer and faster marker. The establishment of retroviral vector producer cells is traditionally performed by randomly integrating the retroviral vector expression cassette codifying the transgene. More recently, recombinase-mediated cassette exchange methodologies have been introduced to achieve targeted integration. Herein we compared random and targeted integration of the retroviral vector transgene construct. Two retroviral producer cell lines, 293 OuaS and 293 FlexOuaS, were generated by random and targeted integration, respectively, producing high titers (on the order of 107 infectious particles·ml-1). Results showed that the retroviral vector transgene cassette is the key retroviral vector component determining the viral titers notwithstanding, single-copy integration is sufficient to provide high titers. The expression levels of the three retroviral constructs (gag-pol, GaLV env, and retroviral vector transgene) were analyzed. Although gag-pol and GaLV env gene expression levels should surpass a minimal threshold, we found that relatively modest expression levels of these two expression cassettes are required. Their levels of expression should not be maximized. We concluded, to establish a high producer retroviral vector cell line only the expression level of the genomic retroviral RNA, that is, the retroviral vector transgene cassette, should be maximized, both through (1) the optimization of its design (i.e., genetic elements composition) and (2) the selection of high expressing chromosomal locus for its integration. The use of methodologies identifying and promoting integration into high-expression loci, as targeted integration or high-throughput screening are in this perspective highly valuable.

Genomic integration of the full-length dystrophin coding sequence in Duchenne muscular dystrophy induced pluripotent stem cells.

The plasmid vectors that express the full-length human dystrophin coding sequence in human cells was developed. Dystrophin, the protein mutated in Duchenne muscular dystrophy, is extraordinarily large, providing challenges for cloning and plasmid production in Escherichia coli. The authors expressed dystrophin from the strong, widely expressed CAG promoter, along with co-transcribed luciferase and mCherry marker genes useful for tracking plasmid expression. Introns were added at the 3' and 5' ends of the dystrophin sequence to prevent translation in E. coli, resulting in improved plasmid yield. Stability and yield were further improved by employing a lower-copy number plasmid origin of replication. The dystrophin plasmids also carried an attB site recognized by phage phiC31 integrase, enabling the plasmids to be integrated into the human genome at preferred locations by phiC31 integrase. The authors demonstrated single-copy integration of plasmid DNA into the genome and production of human dystrophin in the human 293 cell line, as well as in induced pluripotent stem cells derived from a patient with Duchenne muscular dystrophy. Plasmid-mediated dystrophin expression was also demonstrated in mouse muscle. The dystrophin expression plasmids described here will be useful in cell and gene therapy studies aimed at ameliorating Duchenne muscular dystrophy.

BIBAC-GW-based vectors for generating reporter lines for site-specific genome editing in planta

When generating transgenic plants, one of the objectives is to achieve stable expression of the transgene. Transgene silencing can be avoided by single copy integration of the transgene. Binary systems that predominantly result in single copy integrations, such as BIBAC vectors, are also single-copy in E. coli, the organism in which the T-DNA to be delivered to the plant is assembled. Although a low-copy number is important for stable maintenance of large DNA fragments in E. coli, it hampers cloning into the vector due to a low DNA yield. Here we describe BIBAC vectors to which Gateway site-specific recombination sites are added. These sites provide a fast and easy introduction of sequences of interest into any vector. Our Gateway-compatible BIBAC vectors are available with two selectable markers for plants – resistance to Basta (BIBAC-BAR-GW) and DsRed fluorescence in the seed coat (BIBAC-RFP-GW).Using the BIBAC-BAR-GW vector we have generated different fluorescence-based reporter constructs that, when delivered to plant cells, can be used to study and optimize precise, template-dependent site-specific genome editing by CRISPR-Cas9, TALENs or ZFP-nuclease complexes, and oligonucleotide-directed mutagenesis. We have generated 59 reporter lines in A. thaliana with our reporter constructs, and for the lines carrying single T-DNA integrations (32 out of 59) we have determined the integrity of the integrations, their genomic locations and the expression level of the reporters. Similarly to its original counterpart, BIBAC-BAR-GW generates single T-DNA integrations in Arabidopsis with 50% efficiency, and 90% of those are intact. The reporter constructs in the independent transgenic lines exhibit only an up to 3-fold difference in expression level. These features combined with an easy manipulation of the vector due to the added Gateway sites make the BIBAC-GW vectors an attractive tool for generating transgenic plants.

Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Cercospora zeae-maydis

An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis.

Establishment of a simple and efficient Agrobacterium-mediated transformation system for Phytophthora palmivora.

BackgroundAs an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems. Among them is the broad host range pathogen P. palmivora, which infects many economically important plant species. An essential way to dissect their pathogenesis mechanisms is genetic modification of candidate genes, which requires effective transformation systems. Four methods were developed for transformation of Phytophthora spp., including PEG(polyethylene glycol)/CaCl2 mediated protoplast transformation, electroporation of zoospores, microprojectile bombardment and Agrobacterium-mediated transformation (AMT). Among them, AMT has many advantages over the other methods such as easy handling and mainly generating single-copy integration in the genome. An AMT method previously reported for P. infestans and P. palmivora has barely been used in oomycete research due to low success and low reproducibility.ResultsIn this study, we report a simple and efficient AMT system for P. palmivora. Using this system, we were able to reproducibly generate over 40 transformants using zoospores collected from culture grown in a single 100 mm-diameter petri dish. The generated GFP transformants constitutively expressed GFP readily detectable using a fluorescence microscope. All of the transformants tested using Southern blot analysis contained a single-copy T-DNA insertion.ConclusionsThis system is highly effective and reproducible for transformation of P. palmivora and expected to be adaptable for transformation of additional Phytophthora spp. and other oomycetes. Its establishment will greatly accelerate their functional genomic studies.

Molecular Analysis and Effectiveness Assay of AV1 Gene in Transgenic Tobacco for Resistance to Begomovirus

<p>Genetic transformation<br />of tobacco plant using AV1 gene was conducted at<br />the previously experiment and generated transgenic tobacco<br />plants positively carrying the selectable marker nptII gene.<br />The objectives of this experiment were to (1) analyze the<br />presence of Begomovirus AV1 gene in T0 generation putative<br />transgenic tobacco plants using PCR technique with specific<br />primers and its correlation with resistance phenotype, (2)<br />analyze the integration and copy number of the transgene in<br />T0 generation putative transgenic tobacco plants and its<br />correlation with resistance response, (3) screen the T0<br />generation putative transgenic tobacco plants with the target<br />virus infection and to detect the presence of the virus in the<br />transgenic plant tissue using universal primers. PCR<br />detection of AV1 gene in tobacco transgenic was conducted<br />by using specific primer for Begomovirus AV1 gene.<br />Meanwhile, Southern Blot analysis was conducted by using<br />the AV1 gene probe. The effectiveness of AV1 gene in<br />tobacco transgenic was tested by inoculation of target virus<br />using whiteflies vector. Result of the experiments showed<br />that there was a positive correlation between the presence<br />of the AV1 transgene in T0 generation putative transgenic<br />tobacco plants and the resistant phenotype. Transgenic<br />plants with a single copy integration of the transgene<br />exhibited more resistant than the multiple copy one. and<br />non transgenic plant. The resistance as a result of AV1 gene<br />expression was indicated with no symptom in T0 generation<br />transgenic tobacco plants and the accumulation of the virus<br />in the transgenic plants tissue. Northern and Western<br />hybridization analysis need to be perfomed for investigating<br />the presence of mRNA or protein accumulation so that the<br />resistance mechanism of the AV1 gene could be explained<br />more detail.</p>

Detailed Molecular Characterisation of the Transgenic Potato Line, AppA6, Modified with the Apple (Malus domestica) Polygalacturonase Inhibiting Protein 1 (pgip1) Gene

Current safety assessment of genetically modified crops requires detailed information about the insertion of the transgene and the effect of its expression on the biochemistry and physiology of the host plant. Whilst the intended effect of the transformation can be verified through phenotypic screening, molecular approaches are required to observe unintended effects. We investigated the molecular details of the integration of a polygalacturonase inhibiting protein 1 gene from Malus domestica (Mdpgip1), overexpressed in Solanum tuberosum (cv BP1) for enhanced resistance against Verticillium wilt. Genome walking studies of the selected AppA6 transformant revealed that the T-DNA containing the Mdpgip1 transgene under control of the CaMV 35S promoter was inserted into the genome without any non-T-DNA sequences from the pCAMBIA2300 vector. Sequence data indicate that the insertion of the Mdpgip1 transgene was in a gene-rich region of chromosome 1, adjacent to the photosystem Q B gene but without disruption of structural genes. Transcriptome-based cDNA-representational difference analysis revealed the distinctive expression of Mdpgip1 in the transgenic AppA6 line, verifying the intended effect. Protein extracts from the transgenic plants inhibited the activities of Verticillium dahliae polygalacturonases in in vitro studies, showing that the transgene is expressed to produce an active PGIP defense protein. cDNA-AFLP fingerprinting revealed genes that were differentially expressed, including genes encoding tryptophan/tyrosine permease, Ef-Tu domain and SKP1-like 1A proteins. qRT-PCR indicated that the Mdpgip1 transgene insertion resulted in increased expression in the AppA6 transgenic of the xyloglucan endotransglycosylase (xth) gene and an endogenous Stpgip1 gene. These unintended changes were either caused by the constitutive expression of the Mdpgip1 transgene or transformation-related somaclonal variation. The results indicate that the stable, single copy integration of the Mdpgip1 gene in the AppA6 transgenic line did not disrupt any structural genes but caused unintended effects that affected gene expression compared to the parental counterpart under the non-stressed experimental conditions investigated.

Over-expression of fHbp in Arabdopsis for development of meningococcal serogroup B subunit vaccine.

Due to lack of commercial vaccine against the serogroup B (MenB) of Neisseria meningitides, the incidence of meningococcal disease remains high. To solve the issue, transgenic plants are used as bioreactors to produce a plant-derived fHbp subunit vaccine. In this study, the fHbp gene was optimized according to the codon usage bias of Arabidopsis thaliana, synthesized artificially, cloned into an expression vector, driven by a seed-specific promoter, and introduced into A. thaliana by Agrobacterium-mediated floral-dip transformation. Transgenic plants were identified by glufosinate selection, quickstix strips for PAT/bar tests and PCR analysis. The five plants showing higher expression of recombinant fHbp were screened through indirect ELISA. Southern blot analysis showed that the transgenic line rHF-22 had a single-copy integration and the highest expression of fHbp. Recombinant fHbp was purified from seeds of rHF-22 by nitrilotriacetic acid-mediated affinity chromatography, and the purity was 82.5%. BALB/c mice were tested for fHbp vaccine protection from lethal MenB infection, and the relative percent survival was found to be 80%. This study indicates that the recombinant fHbp produced from seeds of rHF-22 is a potential candidate for commercial MenB vaccine. It also provides a reference for safe, cheap and large-scale production of other plant-made vaccines.

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae.

Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering. In order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evolution but also on-demand gene amplification in yeast, which can be broadly applicable for metabolic engineering.

HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

BackgroundHIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability.ResultsA fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8.ConclusionsThis is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.

Agrobacterium-Mediated Stable Genetic Transformation of Populus angustifolia and Populus balsamifera.

The present study demonstrates Agrobacterium tumefaciens-mediated stable genetic transformation of two species of poplar – Populus angustifolia and Populus balsamifera. The binary vector pCAMBIA-Npro-long-Luc containing the luciferase reporter gene was used to transform stem internode and axillary bud explants. Putative transformants were regenerated on selection-free medium using our previously established in vitro regeneration method. Explant type, genotype, effect of pre-culture, Agrobacterium concentration, a time period of infection and varying periods of co-culture with bacteria were tested for the transformation frequency. The highest frequency of transformation was obtained with stem internode explants pre-cultured for 2 days, infected with Agrobacterium culture at the concentration of OD600 = 0.5 for 10 min and co-cultivated with Agrobacterium for 48 h. Out of the two genotypes tested, P. balsamifera exhibited a higher transformation rate in comparison to P. angustifolia. The primary transformants that exhibited luciferase activity in a bioluminescence assay under the CCD camera when subjected to polymerase chain reaction and Southern blot analysis revealed a stable single-copy integration of luc in their genomes. The reported protocol is highly reproducible and can be applied to other species of poplar; it will also be useful for future genetic engineering of one of the most important families of woody plants for sustainable development.

Efficient Generation of Mice with Consistent Transgene Expression by FEEST.

Transgenic mouse models are widely used in biomedical research; however, current techniques for producing transgenic mice are limited due to the unpredictable nature of transgene expression. Here, we report a novel, highly efficient technique for the generation of transgenic mice with single-copy integration of the transgene and guaranteed expression of the gene-of-interest (GOI). We refer to this technique as functionally enriched ES cell transgenics, or FEEST. ES cells harboring an inducible Cre gene enabled the efficient selection of transgenic ES cell clones using hygromycin before Cre-mediated recombination. Expression of the GOI was confirmed by assaying for the GFP after Cre recombination. As a proof-of-principle, we produced a transgenic mouse line containing Cre-activatable tTA (cl-tTA6). This tTA mouse model was able to induce tumor formation when crossed with a transgenic mouse line containing a doxycycline-inducible oncogene. We also showed that the cl-tTA6 mouse is a valuable tool for faithfully recapitulating the clinical course of tumor development. We showed that FEEST can be easily adapted for other genes by preparing a transgenic mouse model of conditionally activatable EGFR L858R. Thus, FEEST is a technique with the potential to generate transgenic mouse models at a genome-wide scale.

Expression and Control of Codon-Optimized Granulocyte Colony-Stimulating Factor in Pichia pastoris.

Granulocyte colony-stimulating factor (GCSF) has therapeutic applications due to its proven efficacy in different forms of neutropenia and chemotherapy-induced leucopenia. The original 564-bp nucleotide sequence from NCBI was codon optimized and assembled by overlapping PCR method comprising of 16 oligos of 50-nt length with 15 base overhang. The synthetic gene (CO-GCSF) was cloned under glucose utilizing glyceraldehyde 3-phosphate dehydrogenase (GAP) and methanol-utilizing alcohol oxidase (AOX1) promoters and expressed in Pichia pastoris SMD1168 strain. Constitutive expression under GAP resulted in cellular toxicity while AOX1 promoter controlled expression was stable. Variation in the levels of expression was observed among the transformant colonies with transformant #2 secreting up to ∼4 mg/L of GCSF. The molecular mass of the expressed GCSF in P. pastoris was ∼19.0 kDa. Quatitation of the expressed protein was carried out by a highly reproducible gel densitometric method. Effect of several operational and nutritional conditions was studied on GCSF production and the results suggest a general approach for increasing the yield of GCSF several folds (2- to 5-fold) over the standard conditions employed currently. Cultivation of the single-copy integrant in the chemically defined medium in a 5-L fermenter resulted in a volumetric productivity of ∼0.7 mg/L/h at the end of the induction phase, which was about 4-fold higher than attained in the shake flask.

Transgene integration complexity and expression stability following biolistic or Agrobacterium-mediated transformation of sugarcane

Sugarcane (Saccharum spp. hybrids) accounts for 80% of the table sugar produced worldwide and is also a prime feedstock for biofuel production. However, very few studies are available for directly comparing Agrobacterium tumefaciens-mediated transfer of T-DNA (AMT) and biolistic transfer of minimal expression cassettes (BLT MC) regarding transgene complexity and expression stability. In this study, the transformation efficiency, transgene integration pattern, expression level, and expression stability were compared in the commercially important sugarcane cultivar CP88-1762. A total of 312 transgenic lines derived from AMT and 250 lines derived from BLT MC were identified by PCR from genomic DNA using nptII-specific primers. Lines were analyzed with both qPCR (TaqMan®) and NPTII ELISA to determine the nptII transgene copy number and expression level. The results of Southern blot analysis on selected lines were highly correlated to the qPCR results. There were no significant differences between the two transformation systems for transformation efficiency, frequency of single copy integration, or level and stability of transgene expression when carried out with the same expression cassette, tissue culture, and selection procedure in 12 independent experiments. These findings suggested that both BLT MC and AMT provide suitable platforms for generation of elite sugarcane events.

Mycobacteriophage-repressor-mediated immunity as a selectable genetic marker: Adephagia and BPs repressor selection.

Mycobacteriophages provide an abundance of systems for use in mycobacterial genetics, including manipulation of Mycobacterium tuberculosis. Because of the dearth of antibiotic resistance cassettes and biosafety concerns in constructing recombinant virulent M. tuberculosis strains, we developed the use of mycobacteriophage-encoded repressor genes that can be selected in the presence of lytic versions of their cognate phages. The phage Adephagia repressor gene (43) was identified through its ability to confer immunity to Adephagia superinfection, together with the mapping of mutations in gene 43 that confer a clear-phage phenotype. Plasmid transformants containing either Adephagia 43 or the previously identified BPs repressor 33 can be readily selected following electroporation using engineered lytic derivatives of Adephagia and BPs, respectively. Selection is as efficient as antibiotic selection, can be used with either single-copy integration vectors or with extrachromosomal vectors, and works similarly in both Mycobacterium smegmatis and M. tuberculosis.

Red fluorescent proteins for imaging Zymoseptoria tritici during invasion of wheat

The use of fluorescent proteins (FPs) in plant pathogenic fungi provides valuable insight into their intracellular dynamics, cell organization and invasion mechanisms. Compared with green-fluorescent proteins, their red-fluorescent “cousins” show generally lower fluorescent signal intensity and increased photo-bleaching. However, the combined usage of red and green fluorescent proteins allows powerful insight in co-localization studies. Efficient signal detection requires a bright red-fluorescent protein (RFP), combined with a suitable corresponding filter set. We provide a set of four vectors, suitable for yeast recombination-based cloning that carries mRFP, TagRFP, mCherry and tdTomato. These vectors confer carboxin resistance after targeted single-copy integration into the sdi1 locus of Zymoseptoria tritici. Expression of the RFPs does not affect virulence of this wheat pathogen. We tested all four RFPs in combination with four epi-fluorescence filter sets and in confocal laser scanning microscopy, both in and ex planta. Our data reveal that mCherry is the RFP of choice for investigation in Z. tritici, showing highest signal intensity in epi-fluorescence, when used with a Cy3 filter set, and laser scanning confocal microscopy. However, mCherry bleached significantly faster than mRFP, which favors this red tag in long-term observation experiments. Finally, we used dual-color imaging of eGFP and mCherry expressing wild-type strains in planta and show that pycnidia are formed by single strains. This demonstrates the strength of this method in tracking the course of Z. tritici infection in wheat.

The Novel PIEZO1 Mutation p.L2023V Is Causal for Hereditary Xerocytosis Resulting in Delayed Channel Inactivation and a Dehydrated Red Blood Cell Phenotype

The regulation of cell volume is important for the maintenance of integrity in all cells and is especially critical for the highly specialized red blood cell (RBC) which must withstand pressure changes within the vasculature and remain deformable to traverse small vessels. Disorders that interfere with volume homeostasis result in the premature destruction of RBCs. One protein that appears to play a prominent role in RBC hydration is the recently described nonselective cation channel PIEZO1 which is involved in mechanotransduction. Mutations of PIEZO1 have been associated with an autosomal dominant form of hereditary hemolytic anemia (HHA) characterized by RBC dehydration known as hereditary xerocytosis (HX) (Zarychanski et al., Blood 2012;120:1908). There is evidence that PIEZO1 may also be responsible for a channel activity that participates in the dehydration of sickle cells which exacerbates sickling and vaso-occlusive events in patients with Sickle Cell Disease (reviewed in Gallagher, Curr Opin Hematol 2013, 20:201).Using a Next-Generation sequencing panel containing 27 hemolytic anemia associated genes, we identified and characterized a novel PIEZO1 mutation p.L2023V which results in delayed channel inactivation and a dehydrated RBC phenotype. This single amino acid substitution at a highly conserved site was detected in the heterozygous state in a 15 year old Caucasian young man (CQ15) with chronic hemolysis well compensated with reticulocytosis, along with heterozygosity for the SLC4A1 p.E40K mutation known as band 3 Montefiore. Hematologic characteristics included macrocytosis, elevated MCHC, and a smear showing occasional stomatocytes. Several family members also had hemolysis and jaundice and were given a diagnosis of hereditary pyropoikilocytosis prior to our evaluation. The L2023V mutation was considered possibly damaging by the PolyPhen-2 algorithm with a score of 0.777. The mother was heterozygous for the PIEZO1 L2023V mutation while the father carried the band 3 Montefiore mutation. Ektacytometry was used to evaluate RBC deformability; the mother’s RBCs had a profile of classic xerocytosis with decreased Omin and Ohyp, the father had a very mild spherocytosis profile, while the patient’s RBCs had mixed ektacytometry characteristics with decreased Ohyp and an intermediate Omin (Figure 1A). Intracellular cation values determined by flame emission spectroscopy for CQ15 and his mother demonstrated K+ loss. The mutation p.L2023V is located at a site predicted to be at the border between membrane and cytoplasm in the carboxy terminal part of the protein, similar to the previously described p.R2456H mutation in a kindred with HX (Zarychanski et al., 2012).To enable detailed physiologic study of the L2023V mutation, we prepared an HEK293 cell line with a stable, single copy integrant of the variant with an inducible promoter. Whole cell patch clamp studies were performed on HEK293 cells expressing wild type PIEZO1 and PIEZO1 L2023V. Traces of mechanically activated currents were recorded from the cells and were normalized to peak current (Figure 1B). The inactivation time constant tau (in ms) was determined from mono-exponential fits for wild type and mutant PIEZO1 channels. The difference in average inactivation time between wild type PIEZO1 and PIEZO1 L2023V was highly significant (Student’s t-test; p<0.0001) and is predicted to lead to cellular dehydration.Patients often present with a complex clinical picture and laboratory results and may have combinations of potentially damaging genetic variants identified by Next-Generation sequencing. The examination of clinical, laboratory, and genetic data from family members and, in some cases, in vitro studies are required to clarify the relative contribution of these variants and to arrive at an accurate diagnosis. The data presented here further our understanding of the role of PIEZO1 in RBCs and its potential pathological contribution in HHAs with associated cellular dehydration and may facilitate the future development of therapeutic targets for treatment of these conditions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A conditional knockout toolkit for Caenorhabditis elegans based on the Cre/loxP recombination.

Conditional knockout (cKO) based on site-specific recombination (SSR) technology is a powerful approach for estimating gene functions in a spatially and temporally specific manner in many model animals. In Caenorhabditis elegans (C. elegans), spatial- and temporal-specific gene functions have been largely determined by mosaic analyses, rescue experiments and feeding RNAi methods. To develop a systematic and stable cKO system in C. elegans, we generated Cre recombinase expression vectors that are driven by various tissue-specific or heat-shock promoters. Validation using Cre-mediated fluorescence protein inactivation or activation systems demonstrated successful Cre-dependent loxP excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/loxP-based cKO system, we generated sid-1 deletion mutants harboring floxed sid-1 single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. sid-1 mutants that were rescued by the floxed sid-1 SCI were then crossed with the Pdpy-7::Cre strain for cKO in the hypodermis. The sid-1 cKO animals were resistant to bli-3 RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to unc-22 RNAi, which leads to twitching of the body wall muscle. Our system, which is based on the combination of a transgenic Cre collection, pre-existing deletion mutants, and UV/TMP SCI methods, provided a systematic approach for cKO in C. elegans.