Restriction Enzyme-mediated Integration Research Articles

Mutation Screening of Dictyostelium Restriction Enzyme-Mediated Integration (REMI) Libraries.

Identifying the mechanisms of action of existing and novel drugs is essential for the development of new compounds for therapeutic and commercial use. Here we provide a technique to identify these mechanisms through isolating mutant cell lines that show resistance to drug-induced phenotypes using Dictyostelium discoideum REMI libraries. This approach provides a robust and rapid chemical-genetic screening technique that enables an unbiased approach to identify proteins and molecular pathways that control drug sensitivity. Mutations that result in drug resistance often occur in target proteins thus identifying the specific protein targets for drugs and bioactive natural products. Following the identification of a list of putative molecular targets user selected compound targets can be analyzed to confirm and validate direct inhibitory effects.

CRISPR/Cas9-based simple transgenesis in Xenopus laevis

Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis.

Mutant resources for functional genomics in Dictyostelium discoideum using REMI-seq technology

BackgroundGenomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology.ResultsWe describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of ‘barcoded’ mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified.ConclusionsWe present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system.

Development of a Positive Selection High Throughput Genetic Screen in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum is a powerful model organism that can be utilized to investigate human health and disease. One particular strength of Dictyostelium is that it can be utilized for high throughput genetic screens. For many phenotypes, one limitation of utilizing Dictyostelium is that screening can be an arduous and time-consuming process, limiting the genomic depth one can cover. Previously, we utilized a restriction enzyme-mediated integration screen to identify suppressors of polyglutamine aggregation in Dictyostelium. However, due to the time required to perform the screen, we only obtained ∼4% genome coverage. Here we have developed an efficient screening pipeline that couples chemical mutagenesis with the 5-fluoroorotic acid counterselection system to enrich for mutations in genes of interest. Here we describe this new screening methodology and highlight how it can be utilized for other biological systems.

Axial Skeletal Malformations in Genetically Modified Xenopus laevis and Xenopus tropicalis.

Skeletal malformations in captive-bred, adult Xenopus spp., have not previously been reported. Here we describe 10 sexually mature, genetically modified laboratory frogs (6 Xenopus laevis and 4 Xenopus tropicalis) with axial skeletal abnormalities. The young adult frogs were described by veterinary staff as presenting with "hunchbacks," but were otherwise considered to be in good health. All affected frogs were genetically engineered using various techniques: transcription activator-like effector nucleases (TALEN) editing using thyroid hormone receptor α TALEN mRNA, restriction enzyme-mediated integration methods involving insertion of the inducible transgene pCAR/TRDN, or via I-SceI meganuclease transgenesis using either pDRTREdpTR-HS4 or pDPCrtTA-TREG-HS4 plasmid sequences. Radiographic findings (6 frogs) and gross necropsy (10 frogs) revealed vertebral column malformations and sacroiliac deformities that resulted in moderate to severe kyphosis and kyphoscoliosis. These findings were confirmed and additional skeletal abnormalities were identified using computed tomography to create a 3D reconstruction of 4 frogs. Additional findings visible on the 3D reconstructions included incomplete vertebral segmentation, malformed transverse processes, and a short and/or curved urostyle. Histopathologic findings included misshapen intervertebral joints with nonconforming articular surfaces, narrowed joint cavities, flattened or irregularly-formed articular cartilage, irregular maturation lines and nonpolarized chondrocytes, excess fibrocartilage, and evidence of irregular bone resorption and growth. While the specific etiology of the vertebral skeletal abnormalities remains unclear, possibilities include: 1) egg/oocyte physical manipulation (dejellying, microinjection, fertilization, etc.), 2) induction and expression of the transgenes, 3) inactivation (knockout) of existing genes by insertional mutagenesis, or 4) a combination of the above. Furthermore, the possibility of undetected changes in the macro or microenvironment, or a feature of the genetic background of the affected frogs cannot be ruled out.

The histidine kinase slnCl1 of Colletotrichum lindemuthianum as a pathogenicity factor against Phaseolus vulgaris L

Colletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes.

Conidiogenesis-related DNA photolyase gene in Beauveria bassiana

Beauveria bassiana is an entomopathogenic fungi used in environmentally mindful pest management. Its main active ingredient, conidia, is commercially available as a fungal biopesticide. Many studies of conidia production have focused on how to optimize culture conditions for maximum productivity and stability against unfavorable abiotic factors. However, understanding of how conidiogenesis-related genes provide improved conidial production remains unclear. In this study, we focus on identifying conidiogenesis-related genes in B. bassiana ERL1170 using a random mutagenesis technique. Transformation of ERL1170 using restriction enzyme-mediated integration generated one morphologically different transformant, ERL1170-pABeG #163. The transformant was confirmed to represent B. bassiana, and the binary vector was successfully integrated into the genome of ERL1170. Compared to the wild type, transformant #163 showed very slow hyphal growth and within 6 days only produced <1 × 106 conidia/0.28 cm2 agar block (wild type: 6.2 × 107 conidia/agar block). Transformant #163 also exhibited different morphology than the wild type, including thicker hyphae with some club-shaped parts. In contrast, the typical morphology of wild type B. bassiana exhibits thread-like hyphae and conidiophore structures and circular conidia. To determine the location of the randomly inserted DNA, we conducted thermal asymmetric interlaced (TAIL) PCR and Escherichia coli cloning to clearly sequence the disrupted region. We identified one colony (colony No. 7) with an insertion site identified as DNA photolyase. This was confirmed through a gene knock-out study. It is possible the gene that encodes for DNA photolyase was disrupted during the insertion process and might be involved in fungal conidiogenesis. This work serves as a platform for exploring the function of a variety of B. bassiana genes involved in pest management and their downstream processing.

Molecular tools for gene manipulation in filamentous fungi.

Functional genomics of filamentous fungi has gradually uncovered gene information for constructing 'cell factories' and controlling pathogens. Available gene manipulation methods of filamentous fungi include random integration methods, gene targeting technology, gene editing with artificial nucleases and RNA technology. This review describes random gene integration constructed by restriction enzyme-mediated integration (REMI); Agrobacterium-mediated transformation (AMT); transposon-arrayed gene knockout (TAGKO); gene targeting technology, mainly about homologous recombination; and modern gene editing strategies containing transcription activator-like effector nucleases (TALENs) and a clustered regularly interspaced short palindromic repeat/associated protein system (CRISPR/Cas) developed in filamentous fungi and RNA technology including RNA interference (RNAi) and ribozymes. This review describes historical and modern gene manipulation methods in filamentous fungi and presents the molecular tools available to researchers investigating filamentous fungi. The biggest difference of this review from the previous ones is the addition of successful application and details of the promising gene editing tool CRISPR/Cas9 system in filamentous fungi.

Random mutagenesis analysis and identification of a novel C2H2-type transcription factor from the nematode-trapping fungus Arthrobotrys oligospora

Arthrobotrys oligospora is a typical nematode-trapping fungus. In this study, 37 transformants of A. oligospora were obtained by REMI (restriction enzyme mediated integration) method and phenotypic properties of nine transformants were analyzed. The nine transformants showed differences in growth, conidiation, trap formation, stress tolerance, and/or pathogenicity among each other and with those of the parental wild-type strain (WT). The insertional sites of the hph cassette were identified in transformants X5 and X13. In X5, the cassette was inserted in the non-coding region between AOL_s00076g273 (76g273) and AOL_s00076g274 (76g274) and the transcription of 76g274, but not 76g273, was enhanced in X5. 76g274p had two conserved domains and was predicted as a nucleoprotein, which we confirmed by its nuclear localization in Saccharomyces cerevisiae using the green fluorescent protein-fused 76g274p. The transcription of 76g274 was stimulated or inhibited by several environmental factors. The sporulation yields of 76g274-deficient mutants were decreased by 70%, and transcription of several sporulation-related genes was severely diminished compared to the WT during the conidiation. In summary, a method for screening mutants was established in A. oligospora and using the method, we identified a novel C2H2-type transcription factor that positively regulates the conidiation of A. oligospora.

Enhanced Production of Itaconic Acid through Development of Transformed Fungal Strains of Aspergillus terreus.

Metabolic engineering with a high-yielding mutant, A. terreus AN37, was performed to enhance the production of itaconic acid (IA). Reportedly, the gene cluster for IA biosynthesis is composed of four genes: reg (regulator), mtt (mitochondrial transporter), cad (cis-aconitate decarboxylase), and mfs (membrane transporter). By overexpressing each gene of the IA gene cluster in A. terreus AN37 transformed by the restriction enzyme-mediated integration method, several transformants showing high productivity of IA were successfully obtained. One of the AN37/cad transformants could produce a very high amount of IA (75 g/l) in shake-flask cultivations, showing an average of 5% higher IA titer compared with the high-yielding control strain. Notably, in the case of the mfs transformants, a maximal increase of 18.3% in IA production was observed relative to the control strain under the identical fermentation conditions. Meanwhile, the overexpression of reg and mtt genes showed no significant improvements in IA production. In summary, the overexpressed cis-aconitate decarboxylase (CAD) and putative membrane transporter (MFS) appeared to have positive influences on the enhanced IA productivity of the respective transformant. The maximal increases of 13.6~18.3% in IA productivity of the transformed strains should be noted, since the parallel mother strain used in this study is indeed a very high-performance mutant that has been obtained through intensive rational screening programs in our laboratory.

Diacylglycerol acyl transferase: A pathogenicity related gene in Colletotrichum gloeosporioides.

To gain more insight into the molecular mechanisms of Colletotrichum gloeosporioides pathogenesis, restriction enzyme-mediated integration (REMI) mutagenesis identified the mutants of C. gloeosporioides impaired in pathogenicity. Transformants screened for defects in pathogenicity using detached leaves and fruits. Of the 20 REMI transformants tested, two mutants (H4 and H7) showed reduced pathogenicity on leaves of apple, kiwi, mango, peach, and fruits of guava, apple, and capsicum. One tagged gene from the genome sequence of mutant H4 was recovered by inverse PCR. Sequence analysis of the tagged site in mutant H4 revealed insertion in diacylglycerol acyltransferase gene which encodes diacylglycerol acyltransferase enzyme, catalyzing the steps involved in the biosynthesis of triacylglycerol, an important component of biological membranes and source of energy. Therefore, tagging of diacylglycerol acyltransferase gene in mutant H4 resulted in reduced pathogenicity, indicating possible role of this gene in pathogenicity of C. gloeosporioides.

REMI-seq generation of a genome-wide mutant resource for dictyostelium functional genomics

We are combining Restriction Enzyme Mediated Integration (REMI) mutagenesis with NGS technology to create a genome-wide set of gene knockout mutants. Using this process, we aim to create a near total collection of Dictyostelium mutants as a new resource for the Dictyostelium community. The resource will comprise individual, sets and large pools of mutants, with the loci of REMI mutations searchable via dictyBase. To date, we have generated and stored more than 11,000 insertion mutants by REMI. Insertion sites are identified by sequencing the insert/genomic DNA junction. The junctions are captured using MmeI, a type IIC restriction enzyme; indexed with sequencing adapters and amplified by PCR. Following size selection, the target DNA is sequenced using an Illumina MiSeq. A Bioinformatic pipeline has been designed to interpret the data and catalogue the mutants. So far, the REMI insertion sites for ~1,000 mutants have been identified, including 280 mutants with ‘in-gene’ insertions that are not currently catalogued in the dictyBase stock centre. 84% of the stocks contain a single mutant with a single insertion. 63% have in-gene insertions and 4% contain insertions attributed to hotspots. This new resource will produce a step change in Dictyostelium genetics. The principle benefits will be the on-line availability of independent and multi-allelic mutants for nearly all Dictyostelium genes, the ease of whole genome phenotypic screens and lastly the capacity to conduct complex phenotyping of protein families.

Multiple roles of a putative vacuolar protein sorting associated protein 74, FgVPS74, in the cereal pathogen Fusarium graminearum.

Fusarium graminearum, a member of the F. graminearum species complex, is a filamentous ascomycetous group that causes serious diseases in cereal crops. A screen of insertional mutants of F. graminearum, generated using a restriction enzyme-mediated integration method, identified a mutant designated R7048 showing pleiotropic phenotypes in several mycological traits. The vector insertion site in the R7048 genome was identified as the KpnI site within an ORF annotated as FGSG_06346 (designated FgVPS74), which showed similarity to vacuolar protein sorting-associated protein 74 in the baker yeast. Both targeted gene deletion and complementation analyses confirmed that FgVPS74 was involved in hyphal growth, conidiation, sexual development, mycotoxin production, and virulence towards host plants in F. graminearum. Electron microscopy analysis revealed no significant changes in morphology of the vacuole or other organelles, but a greater number of mitochondria were produced in the ΔFgVPS74 strain compared to the wild-type progenitor. Expression of a GFP-tagged FgVPS74 construct under its native promoter in the ΔFgVPS74 strain exhibited localization of GFP signal to putative vesicle structures, but not to the vacuolar membrane. Taken together, these findings demonstrated that a functional vacuolar protein-sorting pathway mediated by FgVPS74 is crucial for fungal growth and development in F. graminearum.

Current Technologies and Related Issues for Mushroom Transformation

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

Biological control of Asian tiger mosquito, Aedes albopictus (Diptera: Culicidae) using Metarhizium anisopliae JEF-003 millet grain

Mosquitoes have been becoming serious vectors worldwide thus effective and safe control strategies should be established. Entomopathogenic fungi can be alternative controlling agents by substituting chemical insecticides. Herein we assayed 12 soil-borne entomopathogenic fungi against Asian tiger mosquito (Aedes albopictus) larvae in laboratory conditions and tried to establish an effective application method using millet granular formulation (GR). Twelve fungal isolates which belong to 6 genera (Beauveria, Cordyceps, Metarhizium, Paecilomyces, Purpureocillium and Verticillium) were assayed; M. anisopliae JEF-003 showed the fastest mosquitocidal activity, approximately 73% mortality rate at 2days post-inoculation (dpi.) and >90% mortality rate at 5dpi. Conidia of M. anisopliae JEF-003 also showed a dosage dependent activity at 1×105, 1×106 and 1×107conidiaml−1. Hyphal growth of M. anisopliae JEF-003 in the Ae. albopictus larvae was observed by infection of an M. anisopliae JEF-003 EGFP-transformant, which was generated by restriction enzyme-mediated integration (REMI) method. GR of M. anisopliae JEF-003 showed high virulence to Ae. albopictus larvae (>90% mortality) after 5days of application. These results suggest that M. anisopliae JEF-003 has a potential to control A. albopictus larvae and GR can be practically used for management of the serious vector in water environment.

Biological control of Ascosphaera apis in honey bees using restricted enzyme mediated integration (REMI) transformed Trichoderma atroviride mutants

Ascosphaera apis (Onygenales: Ascosphaeraceae) is a chalkbrood causing pathogen in honeybees. Recently, chemicals have been used against this disease. Consequently, the abuse of chemicals has caused serious impacts on the environment and honey quality and on honeybee colonies themselves. Therefore, the search for biological control options should be the priority for controlling chalkbrood disease. In this experiment, we employed restriction enzyme mediated integration (REMI) to construct mutants (transformants) with growth inhibition on A. apis in honeybees using the Trichoderma atroviride T23 wild strain. The integration of the plasmid pBluescript KS-EH1 into the REMI mutants was confirmed by PCR and Southern hybridization. As a result, ten single-copied T. atroviride T23 transformants were successfully identified. Furthermore, culture filtrates of the transformants, their wild strain and Shabaijing (a commercial fungicide, ethyl ethylsulfonothiolate) were tested to compare their in vitro growth inhibition capability on A. apis. The results demonstrated that transformants Tat5 and Tat6 had the highest growth inhibition activity with the minimal fungicidal concentration (v/v) of 3.5–4.5%. Thus, the Trichoderma transformants constructed by REMI could be used as potential biological agents against the growth of fungal diseases in bee hives. Further refinement and strategy development for the integrative use of this result are vital future plans.

Transgenic animal technology: Recent advances and applications: A Review

Transgenic animal technologies are one of the fastest growing techniques in the biotechnology areas. It is used to incorporate exogenous genes into the animal genome by genetic engineering technology so that these genes can be inherited and expressed by offspring. Two fundamental determinants of the success of any transgenic exploitation of a livestock species are the transport of DNA across the plasma membrane of the recipient cell, and the transport of that DNA across the nuclear membrane to gain access to the chromosomes. A variety of transgenic technologies are available like microinjection method, chemical method mediated transgenesis, somatic cell nuclear transfer, restriction enzyme-mediated integration, retrovirus-mediated gene transfer, sperm mediated gene transfer and embryonic stem cell mediated method etc. Use of transgenic animals will provide solutions for drug research, xenotransplantation, disease resistance and tissue repair etc. This review focuses on various methods of transgenesis and its application.

Construction and evaluation of a transformant library of Lasiodiplodia theobromae generated through Restriction Enzyme-Mediated Integration

Grapevine dieback, caused by Lasiodiplodia theobromae , is an important trunk disease worldwide. Transformants of L. theobromae were generated in an attempt to identify potential pathogenicity-related genes. Lasiodiplodia theobromae strain JZB 0300251, a highly virulent isolate, was selected for the genetic transformation. Based on optimised conditions, the Restriction Enzyme-Mediated Integration (REMI) methodology was established in L. theobromae using pUCATPH (a plasmid carrying a hygromycin B marker). A total of 6,036 transformants were generated with four restriction enzymes, respectively and the transformant library was evaluated based on 200 randomly selected transformants. Mutants that exhibited various degrees of virulence and different growth rates were obtained. The study provides basic results that will lead to increased understanding of the role of the pathogenicity-related genes involved in the infection process of L. theobromae .

Production of antibacterial Bombyx mori cecropin A in mealworm-pathogenic Beauveria bassiana ERL1170.

Efforts are underway to produce antimicrobial peptides in yellow mealworms (Tenebrio molitor), which can be developed as more effective and safer animal feed additives. In this work, we expressed Bombyx mori (Bm) cecropin-A in mealworms by the infection of transformed entomopathogenic Beauveria bassiana ERL1170. The active domain of Bm cecropin A gene was tagged with a signal sequence of B. bassiana for extracellular secretion, and the fragment was inserted into ERL1170 by the restriction enzyme-mediated integration method. Transformant D-6 showed antibacterial activity against Bacillus subtilis and Listeria monocytogenes. Against T. molitor larvae, D-6 had similar mortality to wild-type, and D6-infected mealworm suspension showed strong antibacterial activity against the two bacteria, but not in the wild-type-infected mealworms, thereby increasing the value of mealworms as animal feed additives.

Biodegradation of neonicotinoid insecticide, imidacloprid by restriction enzyme mediated integration (REMI) generated Trichoderma mutants

REMI (restriction enzyme-mediated integration) technique was employed to construct Trichoderma atroviride strain T23 mutants with degrading capability of neonicotinoid insecticide, imidacloprid. The plasmid pBluescript II KS-hph used for integration in REMI mutants was confirmed by PCR and Southern hybridization. Among 153 transformants, 57% of them have showed higher neonicotinoid insecticide, imidacloprid, degradation ability than the wild strain T23 (p<0.01). More specifically, seven single-copied T. atroviride T23 transformants have confirmed a 30% higher degradation rate than their parent isolate. Among all transformed mutants, a 95% imidacloprid degradation rate was identified as the highest. This study, thus, provided an effective approach for improving neonicotinoid insecticide-degrading capability using REMI transformed T. atroviride mutants.