Single Transposon Insertion Research Articles

Mapping of long interspersed element-1 (L1) insertions by TIPseq provides information about sub chromosomal genetic variation in human embryos.

Retrotransposons play important roles during early development when they are transiently de-repressed during epigenetic reprogramming. Long interspersed element-1 (L1), the only autonomous retrotransposon in humans, comprises 17% of the human genome. We applied the Single Cell Transposon Insertion Profiling by Sequencing (scTIPseq) to characterize and map L1 insertions in human embryos. Sixteen cryopreserved, genetically tested, human blastocysts, were accessed from consenting couples undergoing IVF at NYU Langone Fertility Center. Additionally, four trios (father, mother, and embryos) were also evaluated. scTIPseq was applied to map L1 insertions in all samples, using L1 locations reported in the 1000 Genomes as controls. Twenty-nine unknown and unique insertions were observed in the sixteen embryos. Most were intergenic; no insertions were located in exons or immediately upstream of genes. The location or number of unknown insertions did not differ between euploid and aneuploid embryos, suggesting they are not merely markers of aneuploidy. Rather, scTIPseq provides novel information about sub-chromosomal structural variation in human embryos. Trio analyses showed a parental origin of all L1 insertions in embryos. Several studies have measured L1 expression at different stages of development in mice, but this study for the first time reports unknown insertions in human embryos that were inherited from one parent, confirming no de novo L1 insertions occurred in parental germline or during embryogenesis. Since one-third of euploid embryo transfers fail, future studies would be useful for understanding whether these sub-chromosomal genetic variants or de novo L1 insertions affect embryo developmental potential.

Role of Type 4B Secretion System Protein, IcmE, in the Pathogenesis of Coxiella burnetii.

Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes Q fever, a life-threatening zoonotic disease. C. burnetii replicates within an acidified parasitophorous vacuole derived from the host lysosome. The ability of C. burnetii to replicate and achieve successful intracellular life in the cell cytosol is vastly dependent on the Dot/Icm type 4B secretion system (T4SSB). Although several T4SSB effector proteins have been shown to be important for C. burnetii virulence and intracellular replication, the role of the icmE protein in the host-C. burnetii interaction has not been investigated. In this study, we generated a C. burnetii Nine Mile Phase II (NMII) mutant library and identified 146 transposon mutants with a single transposon insertion. Transposon mutagenesis screening revealed that disruption of icmE gene resulted in the attenuation of C. burnetii NMII virulence in SCID mice. ELISA analysis indicated that the levels of pro-inflammatory cytokines, including interleukin-1β, IFN-γ, TNF-α, and IL-12p70, in serum from Tn::icmE mutant-infected SCID mice were significantly lower than those in serum from wild-type (WT) NMII-infected mice. Additionally, Tn::icmE mutant bacteria were unable to replicate in mouse bone marrow-derived macrophages (MBMDM) and human macrophage-like cells (THP-1). Immunoblotting results showed that the Tn::icmE mutant failed to activate inflammasome components such as IL-1β, caspase 1, and gasdermin-D in THP-1 macrophages. Collectively, these results suggest that the icmE protein may play a vital role in C. burnetii virulence, intracellular replication, and activation of inflammasome mediators during NMII infection.

Transposon insertion profiling by sequencing (TIPseq) identifies novel LINE-1 insertions in human sperm.

Long interspersed nuclear element-1 (LINE-1 or L1) comprises 17% of the human genome. Retrotransposons may perturb gene integrity or alter gene expression by altering regulatory regions in the genome. The germline employs a number of mechanisms, including cytosine methylation, to repress retrotransposon transcription throughout most of life. Demethylation during germ cell and early embryo development de-represses retrotransposons. Intriguingly, de novo genetic variation appearing in sperm has been implicated in a number of disorders in offspring, including autism spectrum disorder, schizophrenia, and bipolar disorder. We hypothesize that human sperm exhibit de novo retrotransposition and employ a new sequencing method, single cell transposon insertion profiling by sequencing (scTIPseq) to map them in small amounts of human sperm. Cross-sectional case-control study of sperm samples (n=10 men; ages 32-55 years old) from consenting men undergoing IVF at NYU Langone Fertility Center. scTIPseq identified novel LINE-1 insertions in individual sperm and TIPseqHunter, a custom bioinformatics pipeline, compared the architecture of sperm LINE-1 to known LINE-1 insertions from the European database of Human specific LINE-1 (L1Hs) retrotransposon insertions (euL1db). scTIPseq identified 17 novel insertions in sperm. New insertions were mainly intergenic or intronic. Only one sample did not exhibit new insertions. The location or number of novel insertions did not differ by paternal age. This study for the first time reports novel LINE-1 insertions in human sperm, demonstrating the feasibility of scTIPseq, and identifies new contributors to genetic diversity in the human germ line.

Frequencies and Variations of Magnaporthe oryzae Avirulence Genes in Hunan Province, China.

Rice blast caused by Magnaporthe oryzae poses significant threaten to rice production. For breeding and deploying resistant rice varieties, it is essential to understand the frequencies and genetic variations of avirulence (AVR) genes in the pathogen populations. In this study, 444 isolates were collected from Hunan Province, China in 2012, 2015, and 2016, and their pathogenicity was evaluated by testing them on monogenic rice lines carrying resistance genes Pita, Pizt, Pikm, Pib, or Pi9. The frequencies of corresponding AVR genes AVRPizt, AVRPikm, AVRPib, AVRPi9, and AVRPita were characterized by amplification and sequencing these genes in the isolates. Both Pi9 and Pikm conferred resistance to >75% of the tested isolates, while Pizt, Pita, and Pib were effective against 55.63, 15.31, and 3.15% of the isolates, respectively. AVRPikm and AVRPi9 were detected in 90% of the isolates and AVRPita, AVRPizt, and AVRPib were present in 26.12, 66.22, and 79% of the isolates, respectively. Sequencing of AVR genes showed that most mutations were single nucleotide polymorphisms, transposon insertions, and insertion mutations. The variable sites of AVRPikm and AVRPita were mainly located in the coding sequence regions (CDS), and most were synonymous mutations. A 494-bp Pot2 transposon sequence insertion was found at the 87 bp position upstream of the start codon in AVRPib. Noteworthy, although no mutations were found in CDS of AVRPi9, a GC-rich inserted sequence of ∼200 bp was found at the 1,272 bp position upstream of the start codon in three virulent isolates. As AVRPikm and AVRPi9 were widely distributed with low genetic variation in the pathogen population, Pikm and Pi9 should be promising genes for breeding rice cultivars with blast resistance in Hunan.

P–003 Transposon insertion profiling by sequencing (TIPseq) identifies novel LINE–1 insertions in human sperm

Abstract Study question Do human sperm contain novel LINE–1 insertions and are they affected by paternal age? Summary answer Human sperm contain novel LINE–1 insertions. Their location or number are not affected by paternal age. What is known already LINE–1 comprises 17% of the human genome and some LINE–1s are the only autonomous retrotransposons in humans. Retrotransposons influence genomic instability and/or regulation if new retrotransposition events disrupt coding or regulatory regions in the host genome. Demethylation during germ cell development de-represses retrotransposons. Advanced paternal age is associated with genomic instability. Previously we showed that sperm LINE–1 copy number decreases with paternal age. We hypothesize that human sperm exhibit de novo retrotransposition and that sperm from older men contain increased novel LINE–1 insertions. Study design, size, duration Cross-sectional case–control study with semen samples collected between February to July 2020. Participants/materials, setting, methods Normospermic sperm samples (n = 10; 5 <35 years old and 5 ≥45 years old) obtained from consenting men undergoing IVF at NYU Fertility Center were submitted to a novel method, single cell Transposon Insertion Profiling by Sequencing (scTIPseq) to identify and map LINE–1 insertions in human sperm. TIPseqHunter, a custom bioinformatics pipeline, compared the architecture of sperm LINE–1 to known LINE–1 insertions from the European database of human specific LINE–1 (L1Hs) retrotransposon insertions in humans (euL1db). Main results and the role of chance TIPseq identified 17 novel insertions in sperm, 8 from older (≥ 45 years) and 9 in younger men (<35 years). New insertions were mainly intergenic or intronic, including AC007402 (2/10), TMEM163 (2/7), CTTNBP2NL (3/5), AC107023 (3/3), TMC2 (2/19), MacroD2 (2/6), RAB3C (3/4), LINC02664 (1/1), AC079052 (2/3) and AC017091 (4/4). One novel insertion (<35 years old) hits a known regulatory element. Only one sample (≥ 45 years old) did not exhibit any new insertion. The location or number of novel insertions did not differ by paternal age. Limitations, reasons for caution The small sample-size and use of normospermic specimens limit interpretation of paternal age effect on LINE–1. Besides, the novel insertions could be polymorphic sites that have low allele frequency and thus have not yet been described. Wider implications of the findings: This study for the first time reports novel LINE–1 insertions in human sperm, demonstrating that scTIPseq method is a feasible technique, and identifying new contributions to genetic diversity in the human germ line. Further studies are needed to evaluate the impact of these insertions on sperm function. Trial registration number Not applicable

Identification of genes required for the fitness of Rhodococcus equi during the infection of mice via signature-tagged transposon mutagenesis.

Rhodococcus equi is a Gram-positive facultative intracellular bacterium that causes pyogranulomatous pneumonia in foals and immunocompromised people. In the present study, signature-tagged transposon mutagenesis was applied for the negative selection of R. equi mutants that cannot survive in vivo. Twenty-five distinguishable plasmid-transposon (plasposon) vectors by polymerase chain reaction (PCR), each containing a unique oligonucleotide tag, were constructed and used to select the transposon mutants that have in vivo fitness defects using a mouse systemic infection model. Of the 4,560 transposon mutants, 102 mutants were isolated via a real-time PCR-based screening as the mutants were unable to survive in the mouse model. Finally, 50 single transposon insertion sites were determined via the self-cloning strategy. The insertion of the transposon was seen on the virulence plasmid in 15 of the 50 mutants, whereas the remaining 35 mutants had the insertion of transposon on the chromosome. The chromosomal mutants contained transposon insertions in genes involved in cellular metabolism, DNA repair and recombination, gene regulation, non-ribosomal peptide synthesis, and unknown functions. Additionally, seven of the chromosomal mutants showed a reduced ability to multiply in the macrophages in vitro. In this study, we have identified several biosynthetic pathways as fitness factors associated with the growth within macrophages and survival in mice.

SINGLE CELL TIPSEQ, A NEW METHOD TO MAP LINE-1 INSERTIONS, PROVIDES INFORMATION ABOUT SUB CHROMOSOMAL GENETIC VARIATION IN HUMAN EMBRYOS

One third of euploid embryo transfers fail, implicating sub-chromosomal genetic variation. Retrotransposons play important roles during early development, when they are transiently de repressed during epigenetic re programming. Long interspersed element-1 (L-1), the only autonomous retrotransposon in humans, comprises 17% of the human genome. L-1’s repetitive nature limits the utility of conventional assays-positional information is necessary to establish the novelty of L-1 insertions. Recently we developed Single Cell Transposon Insertion Profiling by Sequencing (scTIPseq) to characterize L-1 insertions in individual cells (McKerrow et al, Phil Trans R Soc Lond Bio, 2020).

Development of Transposon Mutagenesis for Chlamydia muridarum.

Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements for Chlamydia infection in vivoIMPORTANCE Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted Chlamydia species that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.

Construction of a Sequence-Defined Transposon Mutant Library in Staphylococcus aureus.

Transposon mutagenesis is one of the most widely used strategies to generate a large number of random mutations within a bacterial genome and then to precisely identify the mutated sites. The generation of sequence-defined transposon mutant libraries that are composed of a collection of different mutants, each containing a single transposon insertion mutation within nearly all of the nonessential genes within the genome, is a rapid and reliable way to enhance the study of gene function. In this chapter, we describe the process to generate a sequence-defined transposon mutant library in Staphylococcus aureus utilizing the mariner-based bursa aurealis transposon.

RNA Helicase Mediates Competitive Fitness of Listeria monocytogenes on the Surface of Cantaloupe

Listeria monocytogenes is a foodborne pathogen that is implicated in numerous outbreaks of disease (listeriosis) via fresh produce. The genetic features of L. monocytogenes that allow adherence and growth on produce remain largely uncharacterized. In this study, two non-motile transposon mutants were characterized for attachment, growth, and survival on the surface of cantaloupe rind. One of the mutants, L1E4, harbored a single transposon insertion in a DEAD-box RNA helicase gene (lmo0866 homolog), while the other, M1A5, harbored an insertion in a gene from a flagellum biosynthesis and chemotaxis gene cluster (lmo0694 homolog). When inoculated alone, neither mutant was significantly impaired in growth or survival on the surface of cantaloupe at either 25 or 37 °C. However, when co-inoculated with the wildtype parental strain, the RNA helicase mutant L1E4 had a clear competitive disadvantage, while the relative fitness of M1A5 was not noticeably impacted. Genetic complementation of L1E4 with the intact RNA helicase gene restored relative fitness on cantaloupe. The findings suggest that the DEAD-box RNA helicase encoded by the lmo0866 homolog is critical for relative fitness of L. monocytogenes on cantaloupe. Mutant L1E4 was pleiotropic, being not only non-motile but also cold-sensitive and with reduced hemolytic activity, warranting further studies to elucidate the role of this helicase in the competitive fitness of L. monocytogenes on produce.

The Listeria monocytogenes Key Virulence Determinants hly and prfA are involved in Biofilm Formation and Aggregation but not Colonization of Fresh Produce.

Listeria monocytogenes has been extensively studied as a model facultative intracellular pathogen. While the roles of major virulence factors in host-pathogen interactions have been extensively characterized, recent work suggests that some of these factors can also contribute to environmental proliferation of this pathogen. In this study, we characterized two non-hemolytic transposon mutants of strain 2011L-2858 (serotype 1/2b), implicated in the 2011 listeriosis outbreak via whole cantaloupe, for their capacity to form biofilms on polystyrene, aggregate, and colonize cantaloupe rind. One mutant harbored a single mariner-based transposon insertion in hly, encoding the hemolysin Listeriolysin O, while the other harbored a single insertion in prfA, encoding PrfA, a master regulator for hly and numerous other virulence genes. Biofilm formation was significantly reduced in the prfA mutant, and to a lesser extent, in the hly mutant. Inactivation of either hly or prfA significantly reduced L. monocytogenes aggregation. However, both mutants adhered similarly to the wildtype parental strain on cantaloupe rind at either 25 or 37°C. Furthermore, growth and competitive fitness of the mutants on cantaloupe rind was not significantly impacted at either temperature. The findings suggest that, in spite of their involvement in biofilm formation and aggregation, these key virulence determinants may not be required for the ability of L. monocytogenes to colonize fresh produce.

Analysis of metal tolerance in Rhizobium leguminosarum strains isolated from an ultramafic soil.

Natural habitats containing high amounts of heavy metals provide a valuable source of bacteria adapted to deal with metal toxicity. A functional analysis of the population of legume endosymbiotic bacteria in an ultramafic soil was undertaken by studying a collection of Rhizobium leguminosarum bv viciae (Rlv) isolates obtained using pea as trap plant. One of the isolates, Rlv UPM1137, was selected on the basis of its higher tolerance to nickel and cobalt and presence of inducible mechanisms for such tolerance. A random transposon mutagenesis of Rlv UPM1137 allowed the generation of 14 transposant derivatives with increased nickel sensitivity; five of these transposants were also more sensitive to cobalt. Sequencing of the insertion sites revealed that one of the transposants (D2250) was affected in a gene homologous to the cation diffusion facilitator gene dmeF first identified in the metal-resistant bacterium Cupriavidus metallidurans CH34. The symbiotic performance of D2250 and two other transposants bearing single transposon insertions was unaffected under high-metal conditions, suggesting that, in contrast to previous observations in other Rlv strain, metal tolerance in UPM1137 under symbiotic conditions might be supported by functional redundancy between several mechanisms.

Novel Cadmium Resistance Determinant in Listeria monocytogenes

Listeria monocytogenes is a foodborne pathogen that can cause severe disease (listeriosis) in susceptible individuals. It is ubiquitous in the environment and often exhibits resistance to heavy metals. One of the determinants that enables Listeria to tolerate exposure to cadmium is the cadAC efflux system, with CadA being a P-type ATPase. Three different cadA genes (designated cadA1 to cadA3) were previously characterized in L. monocytogenes A novel putative cadmium resistance gene (cadA4) was recently identified through whole-genome sequencing, but experimental confirmation for its involvement in cadmium resistance is lacking. In this study, we characterized cadA4 in L. monocytogenes strain F8027, a cadmium-resistant strain of serotype 4b. By screening a mariner-based transposon library of this strain, we identified a mutant with reduced tolerance to cadmium and that harbored a single transposon insertion in cadA4 The tolerance to cadmium was restored by genetic complementation with the cadmium resistance cassette (cadA4C), and enhanced cadmium tolerance was conferred to two unrelated cadmium-sensitive strains via heterologous complementation with cadA4C Cadmium exposure induced cadA4 expression, even at noninhibitory levels. Virulence assessments in the Galleria mellonella model suggested that a functional cadA4 suppressed virulence, potentially promoting commensal colonization of the insect larvae. Biofilm assays suggested that cadA4 inactivation reduced biofilm formation. These data not only confirm cadA4 as a novel cadmium resistance determinant in L. monocytogenes but also provide evidence for roles in virulence and biofilm formation.IMPORTANCEListeria monocytogenes is an intracellular foodborne pathogen causing the disease listeriosis, which is responsible for numerous hospitalizations and deaths every year. Among the adaptations that enable the survival of Listeria in the environment are the abilities to persist in biofilms, grow in the cold, and tolerate toxic compounds, such as heavy metals. Here, we characterized a novel determinant that was recently identified on a larger mobile genetic island through whole-genome sequencing. This gene (cadA4) was found to be responsible for cadmium detoxification and to be a divergent member of the Cad family of cadmium efflux pumps. Virulence assessments in a Galleria mellonella model suggested that cadA4 may suppress virulence. Additionally, cadA4 may be involved in the ability of Listeria to form biofilms. Beyond the role in cadmium detoxification, the involvement of cadA4 in other cellular functions potentially explains its retention and wide distribution in L. monocytogenes.

Identification of novel virulence-related genes in Aeromonas hydrophila by screening transposon mutants in a Tetrahymena infection model

Outbreaks of motile Aeromonad septicemia (MAS) in fish caused by sequence type (ST) 251 Aeromonas hydrophila have become a prominent problem for the aquaculture industry. The pathogenesis of A. hydrophila is very complicated, and some virulence factors remain to be identified. In this study, to identify novel virulence-related factors, ST251 A. hydrophila strain NJ-35 was used as the parental strain to construct a mutant library comprising 1030 mutant strains by transposon insertion mutagenesis. Subsequently, 33 virulence-attenuated transposon insertion mutants were identified using Tetrahymena and zebrafish as model hosts in sequence. Thermal asymmetric interlaced (Tail)-PCR and Southern blot analysis identified 21 single transposon insertion sites. Seven of the insertion sites are located in non-coding regions, whereas the other 14 insertion sites are located in genes, including aroA, rmlA, rtxA, chiA and plc. All insertion mutants exhibited attenuated virulence in Tetrahymena and zebrafish. Furthermore, the relationship of two genes, chiA and trkH, to virulence was confirmed by gene inactivation and subsequent restoration assays. This study provides new information about the genetic determinants of A. hydrophila pathogenicity and validates the Aeromonas-Tetrahymena co-culture model for high-throughput screening of A. hydrophila virulence factors.

Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis.

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm(2) relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.

Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations

Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.

DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors

DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.

Abstract 048: Identifying Genetic Modifiers for Cardiomyopathy via Mutagenesis Screening

Rationale: The generation of animal models for human cardiomyopathy creates opportunities to uncover mechanisms of diseases’ pathogenesis and to identify genes that modify the progression of pathogenesis. In contrast to candidate gene-based approaches, the phenotype-based forward genetic screening approach is less effectively employed in a vertebrate, mainly due to the prohibitively enormous colony management efforts. Objective: To identify genetic modifiers of cardiomyopathy through developing an unbiased forward genetic approach in the vertebrate Danio rerio. Methods and Results: Using an expression-based insertional mutagenesis screening strategy, we identified 49 Zebrafish Insertional Cardiac (ZIC) mutants from a collection of ~300 gene break-transposon (GBT) lines. By applying doxorubicin (DOX) stress to these heterozygous ZIC mutants, we initially identified four GBT lines that either enhanced or suppressed DOX-induced cardiomyopathy phenotypes. Here, we focus on the GBT0411 line which significantly enhances DOX-induced cardiomyopathy phenotypes. We cloned the gene and identified a single transposon insertion that disrupts the long nuclei isoform of dnajb6b in this GBT0411 mutant. Consistent with its cardiomyopathy modifier function, we detected a predominant expression of dnajb6b in the heart. While mutations affecting the cytoplasmic functions of DNAJB6 were recently reported to cause limb-girdle muscular dystrophy type 1D, our study suggested that the impaired balance between Dnajb6b long (nuclei) and short (cytoplasmic) isoform accounted for the cardiomyopathy modifier effect. Conclusions: Our results demonstrated the feasibility of identifying genetic modifiers of cardiomyopathy using a forward genetic approach in a vertebrate. Specifically, our data implicate DNAJB6 as a new genetic risk factor for DOX-induced cardiomyopathy. Without any presumption, this forward genetic strategy opens the door to systematically identify genetic factors that modify the progression of cardiomyopathy, which will facilitate the practice of personalized medicine and the development of novel therapies.

Transposon Mutagenesis in Bifidobacterium breve: Construction and Characterization of a Tn5 Transposon Mutant Library for Bifidobacterium breve UCC2003

Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.

MalF is essential for persistence of Mycoplasma gallisepticum in vivo

There is limited understanding of the molecular basis of virulence in the important avian pathogen Mycoplasma gallisepticum. To define genes that may be involved in colonization of chickens, a collection of mutants of the virulent Ap3AS strain of M. gallisepticum were generated by signature-tagged transposon mutagenesis. The collection included mutants with single insertions in the genes encoding the adhesin GapA and the cytadherence-related protein CrmA, and Western blotting confirmed that these mutants did not express these proteins. In two separate in vivo screenings, two GapA-deficient mutants (ST mutants 02-1 and 06-1) were occasionally recovered from birds, suggesting that GapA expression may not always be essential for persistence of strain Ap3AS. CrmA-deficient ST mutant 33-1 colonized birds poorly and had reduced virulence, indicating that CrmA was a significant virulence factor, but was not absolutely essential for colonization. ST mutant 04-1 contained a single transposon insertion in malF, a predicted ABC sugar transport permease, and could not be reisolated even when inoculated by itself into a group of birds, suggesting that expression of MalF was essential for persistence of M. galliseptium strain Ap3AS in infected birds.