Efficiency Of Gene Deletion Research Articles

Gene Editing of Candida glycerinogenes by Designed Toxin-Antitoxin Cassette.

Candida glycerinogenes is an industrial yeast with excellent multistress resistance. However, due to the diploid genome and the lack of meiosis and screening markers, its molecular genetic operation is limited. Here, a gene editing system using the toxin-antitoxin pair relBE from the type II toxin-antitoxin system in Escherichia coli as a screening marker was constructed. The RelBE complex can specifically and effectively regulate cell growth and arrest through a conditionally controlled toxin RelE switch, thereby achieving the selection of positive recombinants. The constructed editing system achieved precise gene deletion, replacement, insertion, and gene episomal expression in C.glycerinogenes. Compared with the traditional amino acid deficiency complementation editing system, this editing system produced higher biomass and the gene deletion efficiency was increased by 3.5 times. Using this system, the production of 2-phenylethanol by C.glycerinogenes was increased by 11.5-13.5% through metabolic engineering and tolerance engineering strategies. These results suggest that the stable gene editing system based on toxin-antitoxin pairs can be used for gene editing of C.glycerinogenes to modify metabolic pathways and promote industrial applications. Therefore, the constructed gene editing system is expected to provide a promising strategy for polyploid industrial microorganisms lacking gene manipulation methods.

Improving the Editing Efficiency of CRISPR-Cas9 by Reducing the Generation of Escapers Based on the Surviving Mechanism.

Due to the high specificity in targeting DNA and highly convenient programmability, CRISPR-Cas-based antimicrobials applied for eliminating specific strains such as antibiotic-resistant bacteria in the microbiome were gradually developed. However, the generation of escapers makes the elimination efficiency far lower than the acceptable rate (10-8) recommended by the National Institutes of Health. Here, a systematic study was carried out providing insight into the escaping mechanisms in Escherichia coli, and strategies for reducing the escapers were devised accordingly. We first showed an escape rate of 10-5-10-3 in E. coli MG1655 under the editing of pEcCas/pEcgRNA established previously. Detailed analysis of the escapers obtained at ligA site in E. coli MG1655 uncovered that the disruption of cas9 was the main cause of the generation of survivors, notably the frequent insertion of IS5. Hence, the sgRNA was next designed to target the "perpetrator" IS5, and subsequently the killing efficiency was improved 4-fold. Additionally, the escape rate in IS-free E. coli MDS42 was also tested at the ligA site, ∼10-fold decrease compared with MG1655, but the disruption of cas9 was still observed in all survivors manifested in the form of frameshifts or point mutations. Thus, we optimized the tool itself by increasing the copy number of cas9 to retain some cas9 that still has the correct DNA sequence. Fortunately, the escape rates dropped below 10-8 at 9 of the 16 tested genes. Furthermore, the λ-Red recombination system was added to generate the pEcCas-2.0, and a 100% gene deletion efficiency was achieved at genes cadA, maeB, and gntT in MG1655, whereas those genes were edited with low efficiency previously. Last, the application of pEcCas-2.0 was then expanded to the E. coli B strain BL21(DE3) and W strain ATCC9637. This study reveals the mechanism of E. coli surviving Cas9-mediated death, and a highly efficient editing tool is established based on the mechanism, which will accelerate the further application of CRISPR-Cas.

Establishment of a visual gene knockout system based on CRISPR/Cas9 for the rare actinomycete Nonomuraea gerenzanensis.

To develop a modified CRISPR/Cas9 system with the β-glucuronidase (GusA) reporter and a dual sgRNA cassette for Nonomuraea gerenzanensis (N. gerenzanensis). With the aid of a visual GusA reporter, the complicated and tedious process of cloning and gene identification could be abandoned entirely in the genetic editing of N. gerenzanensis. Moreover, introducing a dual sgRNA cassette into the CRISPR/Cas9 system significantly improved gene deletion efficiency compared to the single sgRNA element. Furthermore, the length of the homologous flanking sequences set to the lowest value of 500bp in this system could still reach the relatively higher conjugation transfer frequency. The enhanced CRISPR/Cas9 system could efficiently perform genetic manipulation on the rare actinomycete N. gerenzanensis.

Detecting Photoactivatable Cre-mediated Gene Deletion Efficiency in Escherichia coli.

Gene deletion is one of the standard approaches in genetics to investigate the roles and functions of target genes. However, the influence of gene deletion on cellular phenotypes is usually analyzed sometime after the gene deletion was introduced. Such lags from gene deletion to phenotype evaluation could select only the fittest fraction of gene-deleted cells and hinder the detection of potentially diverse phenotypic consequences. Therefore, dynamic aspects of gene deletion, such as real-time propagation and compensation of deletion effects on cellular phenotypes, still need to be explored. To resolve this issue, we have recently introduced a new method that combines a photoactivatable Cre recombination system and microfluidic single-cell observation. This method enables us to induce gene deletion at desired timings in single bacterial cells and to monitor their dynamics for prolonged periods. Here, we detail the protocol for estimating the fractions of gene-deleted cells based on a batch-culture assay. The duration of blue light exposure significantly affects the fractions of gene-deleted cells. Therefore, gene-deleted and non-deleted cells can coexist in a cellular population by adjusting the duration of blue light exposure. Single-cell observations under such illumination conditions allow the comparison of temporal dynamics between gene-deleted and non-deleted cells and unravel phenotypic dynamics provoked by gene deletion.

Increasing the λ-Red mediated gene deletion efficiency in Escherichia coli using methyl phosphotriester-modified DNA

BackgroundPhage λ-Red recombineering is a powerful genetic tool to edit the bacterial genome. With the demand of the multiplex genome editing, the efficiency of recombineering is worthy to be further improved. MethodsThe methyl phosphotriester (MPTE)-modified DNA (MPTEDNA) was used as a supplemental molecule during the electroporation of linear DNA, where the MPTEDNA was single-stranded, 69-nt long, had 5 methylation sites on its 5′-end region, and had a sequence complementary to the dsDNA for recombineering. Significant findingsThis study is the first to demonstrate that MPTEDNAs enhance the transformation efficiency (TE) for ldhA deletion and for frdABCD deletion by 6- and 12-fold, respectively, with a low total dsDNA loading of 40–70 ng. It is suggested that MPTEDNA acts a protective agent and forms the stable ssDNA-MPTEDNA duplex, where the 3′end of ssDNA is critical in both ssDNA-annealing model and recA-dependent double-strand invasion recombination model. When the duplex reaches the target gene site, the ssDNA intermediate is interchanged and annealed due to a higher melting temperature (Tm) between the ssDNA and the target gene site than that between ssDNA and MPTEDNA. The DNA duplex formation with the DNA protective agent may apply to other genomic or biomedical studies.

Development of versatile and efficient genetic tools for the marine-derived fungus Aspergillus terreus RA2905.

Marine-derived Aspergillus terreus produces a variety of structurally novel secondary metabolites, most of which show unique biological activities. However, the lack of efficient genetic tools limits the discovery of new compounds, the elucidation of involved biosynthesis mechanism, as well as the strain engineering efforts. Therefore, in this study, we first established both an effective PEG-mediated chemical transformation system of protoplasts and an electroporation system of conidia in a marine-derived fungus A. terreus RA2905. To overcome the insensitivity of RA2905 to fungicides, the uracil auxotrophy strain (pyrG gene deletion mutant, ΔpyrG) was constructed using PEG-mediated transformation system, and using ΔpyrG as the genetic background, the methyltransferase gene laeA-overexpression transformants were further constructed through both PEG- and electroporation-mediated transformations, which showed enhanced terrein production. Besides, in this study, an efficient CRISPR/Cas9 genome-editing system was established for the first time in A. terreus, and a higher gene deletion efficiency of 71% for APSES transcription factor gene stuA could be achieved when using short homologous arms compared with conventional long homologous ones. In addition, using a non-integrative Cas9 plasmid, another efficient and marker-free genome-editing system was established, which allowing repeatable and unlimited genetic manipulation in A. terreus. Using the marker-free genome-editing system, we successfully developed the ΔpyrGΔku70 double-deletion mutant in RA2905, which could further improve gene deletion efficiency. In conclusion, efficient genetic manipulation systems along with a variety of functional mutants were developed in this study, which would significantly expedite both theoretical and applied researches in not only A. terreus but also other marine-derived filamentous fungi.

Single-plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis

Lactococcus lactis is a food-grade lactic acid bacterial species that is widely used in food and medical industries. Due to its relatively small genome and simple metabolism, L. lactis is commonly engineered to produce large quantities of recombinant proteins. The most common single-gene knockout strategy in L. lactis involves RecA-dependent homologous double-crossover recombination, which is relatively time-consuming and laborious. In this study, a precise and efficient genome-editing plasmid for L. lactis NZ9000 genome engineering, pLL, was established based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. By studying the effects of different single guide RNA (sgRNA) promoters, the efficiency of gene deletion was optimized. For LLNZ_02045 (ldh), gene deletion efficiency of up to 50% was achieved. Effective sequential gene deletion of LLNZ_11240 (upp) and LLNZ_04580 (upp1) was also demonstrated using this tool. Additionally, the gene that encodes for uracil phosphoribosyltransferase was identified using this system. Similar robust gene deletion efficiencies of sgRNA that targeted different regions of a single gene suggested that gene deletion was not affected by the location of sgRNA binding. Thus, our study established a new gene-editing tool that may allow further investigation and understanding of the L. lactis NZ9000 genome.

Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic Arabidopsis.

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.

A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH

Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive abilities and diverse metabolic activities, and are therefore widely utilized in bioconversion, biosynthesis and bioremediation. However, due to the high GC-content of the genome (~70%), together with low transformation and recombination efficiency, the efficient genome editing of Rhodococcus remains challenging. In this study, we report for the first time the successful establishment of a CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, making it feasible to obtain enough colonies for screening of mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61, the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated triple-plasmid recombineering system was developed with high efficiency of gene deletion, insertion and mutation. Finally, this new genome editing method was successfully applied to engineer R. ruber for the bio-production of acrylamide. By deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained with reduced byproduct formation and enhanced catalytic stability. Compared with the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells could be recycled from 1 batch to 4 batches.

High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins

Efficient gene deletion methods are essential for the high-throughput study of gene function. Compared to most ascomycete model systems, gene deletion is more laborious in mushroom-forming basidiomycetes due to the relatively low incidence of homologous recombination (HR) and relatively high incidence of non-homologous end-joining (NHEJ). Here, we describe the use of pre-assembled Cas9-sgRNA ribonucleoproteins (RNPs) to efficiently delete the homeodomain transcription factor gene hom2 in the mushroom-forming basidiomycete Schizophyllum commune by replacing it with a selectable marker. All components (Cas9 protein, sgRNA, and repair template with selectable marker) were supplied to wild type protoplasts by PEG-mediated transformation, abolishing the need to optimize the expression of cas9 and sgRNAs. A Δku80 background further increased the efficiency of gene deletion. A repair template with homology arms of 250 bp was sufficient to efficiently induce homologous recombination. This is the first report of the use of pre-assembled Cas9 RNPs in a mushroom-forming basidiomycete and this approach may also improve the genetic accessibility of non-model species.

Efficient gene disruption by posttransformational directed internal homologous recombination in Pichia pastoris

In this study, we designed an improved method to construct gene-deficient strain in non-conventional yeast Pichia pastoris. This method achieved high efficiency of gene deletion without disruption of NHEJ-related genes. The disruption efficiency of Och1 reached about 80%. This simple method will be a new useful molecular tool for P. pastoris.

Abstract 66: Reduction of the Number of Endothelial Cells Harboring Mutations of AVM Causative Genes Lessens Brain AVM Severity

Background and purpose: Mutation of arteriovenous malformation (AVM) causal genes in the endothelial cells (ECs) is essential for AVM formation. However, the relationship of the mutant EC-dose and AVM severity is not clear. We tested whether reduction of mutant ECs reduces bAVM severity using three mouse models that mimic human hereditary hemorrhagic telangiectasia (HHT), a familiar form of bAVM. Majority of HHT patients have Alk1 or endoglin ( Eng ) gene mutation. Methods: Model 1 is Pdgfb iCreER; Alk1 2f/2f mouse that expresses an EC-specific estrogen inducible cre recombinase and has its Alk1 gene exon 4-6 floxed. Alk1 gene deletion was induced by treating the mice with different doses of tamoxifen (TM, 1.25, 0.75, or 0.01 mg/25g of body weight). Model 2 is RosaCreER; Eng 2f/2f mouse that expresses a estrogen inducible cre globally and has exon 5 and 6 of its Eng gene floxed. Model 3 is Pdgfb iCreER; Eng 2f/2f mice. The EC Eng deletion in Model 2 & 3 was induced by three doses of TM (TM 2.5M/25g) treatment. The icre in Pdgfb iCreER transgene has a codon mutant, which is more efficient in deletion of floxed alleles than wild-type cre. BAVMs were induced in TM treated mice through intra-brain injection of AAV-VEGF. Gene deletion efficiency was quantified by qPCR and immunostaining. BAVM phenotypes were analyzed histologically. Results: In model 1, reduction of TM dose reduces the number of Alk1 null ECs and the severity of bAVM. The number of abnormal vessels in bAVM is positively correlated with the TM doses (R=0.8) and negatively correlated with Alk1 positive ECs (R=-0.9). Reduction of TM has also reduced mouse mortality. Compared to Rosa-CreER-mediated global Eng deletion, pdgfb- icreER-induced EC Eng deletion did not increase the number of abnormal vessels, but reduced vascular mural cell-coverage and increased hemorrhage in the bAVM lesion. Conclusion: Reduction of mutant ECs reduces bAVM severity.

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

September 2016-This Month in JoVE: Introducing JoVE Genetics, JoVE Biochemistry, and JoVE Cancer Research

This month we are pleased to introduce three new sections of the JoVE family: Genetics, Biochemistry, and Cancer Research. JoVE Genetics contains methodologies for exploring all aspects of genes and heredity-from human genetics to model organisms, epigenetics to evolutionary genetics, and gene editing to gene therapy. This section features a method for genotyping pufferfish species by liquid chromatography/mass spectrometry, described by Miyaguchi. This technique can aid in the appropriate identification and differentiation of toxic species, which is not only important for public health but also for forensics and investigations of food fraud. Also in JoVE Genetics, Yu et al. report methods for genetically engineering the unconventional yeast Yarrowia lipolytica with improved gene deletion efficiency. The engineered Y. lipolytica strains have potential applications in biofuel and biochemical production. JoVE Biochemistry comprises methods that advance our understanding of biomolecule structure and function, as well as their interactions and transformations during biological processes. This month, Gunning et al. present a method of meat authentication using multiple reaction monitoring (MRM) mass spectrometry, which identifies peptides and gives relative quantitation for detecting adulterant species in meat mixtures. This method is sensitive enough to detect 1% horsemeat in beef products. Also in JoVE Biochemistry, Head and Liu describe a method for identifying small molecule-binding proteins using photoaffinity labeling. The target proteins are bound and covalently labeled within the live cellular environment, which helps preserve native protein structure and binding conditions. JoVE Cancer Research encompasses a broad range of techniques used to advance the understanding and treatment of cancer. This includes methodologies for studying carcinogenesis, developing innovative diagnostics and therapeutics, and uncovering the mechanisms of drug resistance. This month in JoVE Cancer Research, Ansari et al. report a method of targeted cell isolation via glass surface functionalization. This method can identify biomarkers of resistance or susceptibility to anti-angiogenic therapies. Also in this section, Domogauer et al. present a mixed cell culture model that mimics the tumor microenvironment. With this model, the intercellular communication within the tumor microenvironment can be studied under various conditions. You've just had a sneak peek of the articles in the newest sections of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae

CRISPR-Cas has become a powerful technique for genetic engineering of yeast. Here, we present an improved version by using only one single plasmid expressing Cas9 and one or two guide-RNAs. A high gene deletion efficiency was achieved even with simultaneous recombination cloning of the plasmid and deletion in industrial strains.

Overexpression of NrCN improved TMV resistance in selection marker-free tobacco generated by Gene-Deletor system

Two expression cassettes, the Nicotiana tabacum Ubi.U 4 promoter-driven tobacco mosaic virus (TMV)-resistant gene NrCN element and Arabidopsis thaliana senescence-expressive gene SAG 12 promoter-driven LoxP/FRT site-specific recombinase gene FLP expression fragment, were both introduced into the TMV-sensitive tobacco variety K326 via Agrobacterium tumefaciens-mediated genetic transformation. In T0 transgenic plants inoculated TMV, the hypersensitive response (HR) and systemic HR (SHR) occurred at the inoculated leaf and the upper non-inoculated leaf, respectively. The chlorophyll, H2O2 and salicylic acid (SA) content but malonyldialdehyde (MDA) content in transgenic inoculation plants was significantly higher than non-transgenic plants. Meanwhile, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were exactly significantly higher than non-transgenic ones at the early stage after TMV inoculation, but the CAT activity in the former declined and was significantly lower than the latter at the later stage. In addition, the gene relative expression analysis of TMV-resistance-related protein components showed that the expressions of NrCN, NTF6, and PR-1a in genetically modified tobacco (GMT) plants were significantly upregulated after inoculation compared with wild-type tobaccos. Analysis of foreign gene deletion efficiency in transgenic plants indicated the foreign genes (e.g., recombinase gene FLP and screening reporter gene Bar::GUS) deletion events occurred in determinated GMT plant leaf with the development and maturity of the leaves. As a result, the transgenic plants became sensitive to herbicides. Therefore, the introduction of a plant expression vector (Gene-Deleter system with site-specific sequences LoxP/FRT and NrCN-contianing) into TMV-sensitive tobacco variety K326 enhanced the TMV resistance of sensitive tobacco plants. This study provides a basis for screening and acquisition of new varieties of marker-free and safe transgenic antiviral tobacco.

Xenotransplantation literature update, November–December 2014

Xenotransplantation literature update, <scp>N</scp>ovember–<scp>D</scp>ecember 2014

Immunologic applications of conditional gene modification technology in the mouse.

Since the success of homologous recombination in altering mouse genome and the discovery of Cre-loxP system, the combination of these two breakthroughs has created important applications for studying the immune system in the mouse. Here, we briefly summarize the general principles of this technology and its applications in studying immune cell development and responses; such implications include conditional gene knockout and inducible and/or tissue-specific gene over-expression, as well as lineage fate mapping. We then discuss the pros and cons of a few commonly used Cre-expressing mouse lines for studying lymphocyte development and functions. We also raise several general issues, such as efficiency of gene deletion, leaky activity of Cre, and Cre toxicity, all of which may have profound impacts on data interpretation. Finally, we selectively list some useful links to the Web sites as valuable mouse resources.

Importance of codB for new codA-based markerless gene deletion in Gluconobacter strains

For the detailed molecular analysis, genomic modification, and application of acetic acid bacteria such as Gluconobacter in biotechnological processes, a simple markerless deletion system is essential. The available methods have either low efficiencies or their applicability is restricted to strains containing an upp mutation. We now developed a method based on counterselection by cytosine deaminase, encoded by the codA gene from Escherichia coli, in the presence of the fluorinated pyrimidine analogue 5-fluorocytosine (FC). The codA-encoded enzyme converts nontoxic FC to toxic 5-fluorouracil, which is channeled into the metabolism by the uracil phosphoribosyltransferase, encoded by the chromosomal upp gene of Gluconobacter. We found that the presence of E. coli codB, encoding a cytosine permease, was needed for a high efficiency of gene deletion. The system is applicable in wild-type strains because no preceding deletions are required. Based on the fact that a codA gene is absent and an upp gene is present in almost all acetic acid bacteria sequenced so far, the method should also be applicable for other genera of the Acetobacteraceae.