Terminal Inverted Repeat Sequences Research Articles

A self-complementary AAV proviral plasmid system to reduce aberrant cross-packaging and ITR promoter activity in AAV vector preparations

Adeno-associated viral vectors (AAV) are a leading delivery system for gene therapy in animal models and humans. With several FDA-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences; (2) increases correctly packaged AAV payloads; and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.

Characterization of miniature inverted repeat transposable elements inserted in the CitRWP gene controlling nucellar embryony and development of molecular markers for reliable genotyping of CitRWP in Citrus species

Nucellar embryony, a form of apomixis, is one of the most important traits in citrus breeding. Transposition of a miniature inverted repeat transposable element (MITE) in the promoter of CitRWP gene has been previously reported as the causal mutation. However, features of the MITE such as sequences of terminal inverted repeats (TIRs) and target site duplications (TSDs) remain unclear. An MITE family, DTM10, showing the highest homology was identified from 110 citrus MITE families. At least 80 DTM10 elements were identified from each of 10 genome sequences of Citrinae species. Analysis of 114 sweet orange elements showed that DTM10 contained 56-bp TIRs and mostly 9-bp TSDs. Phylogenetic analyses of DTM10 elements identified from five Citrinae species showed that MITEs in CitRWP might have transposed in a common ancestor of Citrus species. Two inverted MITEs were identified from three mutant alleles analyzed. In addition, three tandem MITEs were identified in the mutant allele of Fortunella CitRWP. A strong DNA secondary structure formed between MITEs was predicted. Due to a strong stem-loop structure, a phenomenon of polymerase jumping was observed during PCR amplification. To develop reliable molecular markers, sequences of mutant and wild-type CitRWP alleles were obtained from diverse citrus accessions. Phylogenetic analysis of CitRWP alleles showed that mutant alleles might have originated from a common ancestor of Citrus species. Based on MITE insertion and a single nucleotide polymorphism in the genic region, three complementary molecular markers were developed. Genotyping results of 241 citrus accessions were found to be identical among three molecular markers.

Structural and genome-wide analyses suggest that transposon-derived protein SETMAR alters transcription and splicing

Extensive portions of the human genome have unknown function, including those derived from transposable elements. One such element, the DNA transposon Hsmar1, entered the primate lineage approximately 50 million years ago leaving behind terminal inverted repeat (TIR) sequences and a single intact copy of the Hsmar1 transposase, which retains its ancestral TIR-DNA-binding activity, and is fused with a lysine methyltransferase SET domain to constitute the chimeric SETMAR gene. Here, we provide a structural basis for recognition of TIRs by SETMAR and investigate the function of SETMAR through genome-wide approaches. As elucidated in our 2.37 Å crystal structure, SETMAR forms a dimeric complex with each DNA-binding domain bound specifically to TIR-DNA through the formation of 32 hydrogen bonds. We found that SETMAR recognizes primarily TIR sequences (∼5000 sites) within the human genome as assessed by chromatin immunoprecipitation sequencing analysis. In two SETMAR KO cell lines, we identified 163 shared differentially expressed genes and 233 shared alternative splicing events. Among these genes are several pre–mRNA-splicing factors, transcription factors, and genes associated with neuronal function, and one alternatively spliced primate-specific gene, TMEM14B, which has been identified as a marker for neocortex expansion associated with brain evolution. Taken together, our results suggest a model in which SETMAR impacts differential expression and alternative splicing of genes associated with transcription and neuronal function, potentially through both its TIR-specific DNA-binding and lysine methyltransferase activities, consistent with a role for SETMAR in simian primate development.

Sequence specific integration by the family 1 casposase from CandidatusNitrosopumilus koreensis AR1

Casposase, a homolog of Cas1 integrase, is encoded by a superfamily of mobile genetic elements known as casposons. While family 2 casposase has been well documented in both function and structure, little is known about the other three casposase families. Here, we studied the family 1 casposase lacking the helix-turn-helix (HTH) domain from Candidatus Nitrosopumilus koreensis AR1 (Ca. N. koreensis). The determinants for integration by Ca. N. koreensis casposase were extensively investigated, and it was found that a 13-bp target site duplication (TSD) sequence, a minimal 3-bp leader and three different nucleotides of the TSD sequences are indispensable for target specific integration. Significantly, the casposase can site-specifically integrate a broad range of terminal inverted repeat (TIR)-derived oligonucleotides ranging from 7-nt to ∼4000-bp, and various oligonucleotides lacking the 5′-TTCTA-3′ motif at the 3′ end of TIR sequence can be integrated efficiently. Furthermore, similar to some Cas1 homologs, the casposase utilizes a 5′-ATAA-3′ motif in the TSD as a molecular ruler to dictate nucleophilic attack at 9-bp downstream of the end of the ruler during the spacer-side integration. By characterizing the family 1 Ca. N. koreensis casposase, we have extended our understanding on mechanistic similarities and evolutionary connections between casposons and the adaptation elements of CRISPR-Cas immunity.

Novel Plasmid-Borne Fimbriae-Associated Gene Cluster Participates in Biofilm Formation in Escherichia coli.

This study reported the involvement of a gene cluster from a conjugative plasmid in the biofilm formation of Escherichia coli. We used a novel EZ-Tn5 transposon technique to generate a transposon library and used arbitrarily primed PCR to detect the insertion sites in biofilm formation-deficient mutants. To validate the function of candidate biofilm formation genes, the genes were cloned into plasmid pBluescript II SK (+) and transformed into E. coil DH5α. Biofilm production from the transformants was then assessed by phenotypic biofilm formation using Crystal Violet staining and microscopy. A total of 3,000 transposon mutants of E. coli DH5α-p253 were screened, of which 28 were found to be deficient in biofilm formation. Further characterization revealed that 24/28 mutations were detected with their insertions in chromosome, while the remaining 4 mutations were evidenced that the functional genes for biofilm formation were harbored in the plasmid. Interestingly, the plasmid sequencing showed that these four transposon mutations were all inserted into a fimbriae-associated gene cluster (fim-cluster). This fim-cluster is a hybrid segment spanning a 7,949 bp sequence, with a terminal inverted repeat sequence and two coding regions. In summary, we performed a high-efficiency screening to a library constructed with the EZ-Tn5-based transposon approach and identified the gene clusters responsible for the biofilm production of E. coli, especially the genes harbored in the plasmid. Further studies are needed to understand the spread of this novel plasmid-mediated biofilm formation gene in clinical E. coli isolates and the clinical impacts.

Disruptors, a new class of oligonucleotide reagents, significantly improved PCR performance on templates containing stable intramolecular secondary structures

Intramolecular secondary structures within templates have been shown to lower PCR performance. Whereas many approaches have been developed to mitigate such impairment on PCR, their effects can vary greatly depending on template sequences. Here we present a novel, universally effective approach to improve PCR performance involving specifically designed oligonucleotides called disruptors. A disruptor contained three functional components, an anchor designed to initiate template binding, an effector to disrupt intramolecular secondary structure, and a 3’ blocker to prevent its elongation by DNA polymerase. A functional mechanism for a disruptor to improve PCR efficiency was proposed where anchor first binds to template followed by effector-mediated strand displacement to unwind intramolecular secondary structure. Such a mechanism was consistent with the observation that anchor played a more critical role for disruptor function. As an example of potential disruptor applications, inverted terminal repeat sequences of recombinant adeno-associated virus vectors were successfully amplified in the presence of disruptors despite their well-known reputation as some of the most difficult templates for PCR amplification and Sanger sequencing due to their ultra-stable T-shaped hairpin structures. In stark contrast, both DMSO and betaine, two PCR additives routinely used to facilitate PCR amplification and Sanger sequencing of GC-rich templates, did not demonstrate any improving effect.

Detecting Genetic Mobility Using a Transposon-Based Marker System in Gamma-Ray Irradiated Soybean Mutants.

Transposable elements (TEs)—major components of eukaryotic genomes—have the ability to change location within a genome. Because of their mobility, TEs are important for genome diversification and evolution. Here, a simple rapid method, using the consensus terminal inverted repeat sequences of PONG, miniature inverted-repeat transposable element (MITE)-Tourist (M-t) and MITE-Stowaway (M-s) as target region amplification polymorphism (TE-TRAP) markers, was employed to investigate the mobility of TEs in a gamma-irradiated soybean mutant pool. Among the different TE-TRAP primer combinations, the average polymorphism level and polymorphism information content value were 57.98% and 0.14, respectively. Only the PONG sequence separated the mutant population into three major groups. The inter-mutant population variance, determined using the PONG marker (3.151 and 29%) was greater than that of the M-t (2.209 and 20%) and M-s (2.766 and 18%) markers, whereas the reverse was true for the intra-mutant population variations, with M-t and M-s values, being 15.151 (82%) and 8.895 (80%), respectively, compared with the PONG marker (7.646 and 71%). Thus, the MITE markers revealed more dynamic and active mobility levels than the PONG marker in gamma-ray irradiated soybean mutant lines. The TE-TRAP technique associated with sensitive MITEs is useful for investigating genetic diversity and TE mobilization, providing tools for mutant selection in soybean mutation breeding.

Genome-wide mapping of binding sites of the transposase-derived SETMAR protein in the human genome.

Throughout evolution, DNA transposons provide a recurrent supply of genetic information to give rise to novel gene functions by fusion of their transposase domain to various domains of host-encoded proteins. One of these "domesticated", transposase-derived factors is SETMAR/Metnase which is a naturally occurring fusion protein that consists of a histone-lysine methyltransferase domain and an HsMar1 transposase. To elucidate the biological role of SETMAR, it is crucial to identify genomic targets to which SETMAR specifically binds and link these sites to the regulation of gene expression. Herein, we mapped the genomic landscape of SETMAR binding in a near-haploid human leukemia cell line (HAP1) in order to identify on-target and off-target binding sites at high resolution and to elucidate their role in terms of gene expression. Our analysis revealed a perfect correlation between SETMAR and inverted terminal repeats (ITRs) of HsMar1 transposon remnants, which are considered as natural target sites for SETMAR binding. However, we did not detect any untargeted events at non-ITR sequences, calling into question previously proposed off-target binding sites. We identified sequence fidelity of the ITR motif as a key factor for determining the binding affinity of SETMAR for chromosomes, as higher conservation of ITR sequences resulted in increased affinity for chromatin and stronger repression of SETMAR-bound gene loci. These associations highlight how SETMAR's chromatin binding fine-tune gene regulatory networks in human tumour cells.

AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity

The gene therapy field has been galvanized by two technologies that have revolutionized treating genetic diseases: vectors based on adeno-associated viruses (AAVs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-editing tools. When combined into one platform, these safe and broadly tropic biotherapies can be engineered to target any region in the human genome to correct genetic flaws. Unfortunately, few investigations into the design compatibility of CRISPR components in AAV vectors exist. Using AAV-genome population sequencing (AAV-GPseq), we previously found that self-complementary AAV vector designs with strong DNA secondary structures can cause a high degree of truncation events, impacting production and vector efficacy. We hypothesized that the single-guide RNA (sgRNA) scaffold, which contains several loop regions, may also compromise vector integrity. We have therefore advanced the AAV-GPseq method to also interrogate single-strand AAV vectors to investigate whether vector genomes carrying Cas9-sgRNA cassettes can cause truncation events. We found that on their own, sgRNA sequences do not produce a high degree of truncation events. However, we demonstrate that vector genome designs that carry dual sgRNA expression cassettes in tail-to-tail configurations lead to truncations. In addition, we revealed that heterogeneity in inverted terminal repeat sequences in the form of regional deletions inherent to certain AAV vector plasmids can be interrogated.

Molecular characterization of precise in vivo targeted gene integration in human cells using AAVHSC15.

Targeted gene integration via precise homologous recombination (HR)-based gene editing has the potential to correct genetic diseases. AAV (adeno-associated virus) can mediate nuclease-free gene integration at a disease-causing locus. Therapeutic application of AAV gene integration requires quantitative molecular characterization of the edited sequence that overcome technical obstacles such as excess episomal vector genomes and lengthy homology arms. Here we describe a novel molecular methodology that utilizes quantitative next-generation sequencing to characterize AAV-mediated targeted insertion and detects the presence of unintended mutations. The methods described here quantify targeted insertion and query the entirety of the target locus for the presence of insertions, deletions, single nucleotide variants (SNVs) and integration of viral components such as inverted terminal repeats (ITR). Using a humanized liver murine model, we demonstrate that hematopoietic stem-cell derived AAVHSC15 mediates in vivo targeted gene integration into human chromosome 12 at the PAH (phenylalanine hydroxylase) locus at 6% frequency, with no sign of co-incident random mutations at or above a lower limit of detection of 0.5% and no ITR sequences at the integration sites. Furthermore, analysis of heterozygous variants across the targeted locus using the methods described shows a pattern of strand cross-over, supportive of an HR mechanism of gene integration with similar efficiencies across two different haplotypes. Rapid advances in the application of AAV-mediated nuclease-free target integration, or gene editing, as a new therapeutic modality requires precise understanding of the efficiency and the nature of the changes being introduced to the target genome at the molecular level. This work provides a framework to be applied to homologous recombination gene editing platforms for assessment of introduced and natural sequence variation across a target site.

Characterization of blaCTX-M-14 transposition from plasmid to chromosome in Escherichia coli experimental strain

Mostly, blaCTX-M is found on transferable plasmids as a component of the blaCTX-M transposition unit containing an insertion sequence, ISEcp1, which exists on the upstream region of blaCTX-Ms. Several recent studies conducted in clinical and community settings have reported the presence of chromosomally located blaCTX-M in extended spectrum β-lactamase (ESBL)-producing bacterial isolates. In this study, we observed the frequency and molecular nature of the ISEcp1-mediated transposition of blaCTX-M-14 from a plasmid to a chromosome by using an experimental strain of Escherichia coli. We determined 102 different chromosomal transposition sites of blaCTX-M-14 in 126 E. coli isolates following five independent screening procedures. The characterization of the 102 different chromosomal transposition sites of blaCTX-M-14 observed in this study revealed the presence of 5-bp direct repeat (DR) sequences and identical left terminal inverted sequences in 80 E. coli isolates. However, 5′-flanking sequences of the right terminal DR sequences in the 80 E. coli isolates were highly diverse, and consensus sequences of the right terminal inverted repeat sequences were not observed. In case of our E. coli experimental strain, the frequency of the ISEcp1-mediated transposition of blaCTX-M-14 from a plasmid to a chromosome was determined to be 0.51% (SD = 0.37). Collectively, the molecular nature of ISEcp1 could plausibly be a factor contributing to the high detection rates of E. coli possessing chromosomally located blaCTX-M-14 in both clinical and community settings.

Functional requirement of terminal inverted repeats for efficient ProtoRAG activity reveals the early evolution of V(D)J recombination.

The discovery of ProtoRAG in amphioxus indicated that vertebrate RAG recombinases originated from an ancient transposon. However, the sequences of ProtoRAG terminal inverted repeats (TIRs) were obviously dissimilar to the consensus sequence of mouse 12/23RSS and recombination mediated by ProtoRAG or RAG made them incompatible with each other. Thus, it is difficult to determine whether or how 12/23RSS persisted in the vertebrate RAG system that evolved from the TIRs of ancient RAG transposons. Here, we found that the activity of ProtoRAG is highly dependent on its asymmetric 5′TIR and 3′TIR, which are composed of conserved TR1 and TR5 elements and a partially conserved TRsp element of 27/31 bp to separate them. Similar to the requirements for the recombination signal sequences (RSSs) of RAG recombinase, the first CAC in TR1, the three dinucleotides in TR5 and the specific length of the partially conserved TRsp are important for the efficient recombination activity of ProtoRAG. In addition, the homologous sequences flanking the signal sequences facilitate ProtoRAG- but not RAG-mediated recombination. In addition to the diverged TIRs, two differentiated functional domains in BbRAG1L were defined to coordinate with the divergence between TIRs and RSSs. One of these is the CTT* domain, which facilitates the specific TIR recognition of the BbRAGL complex, and the other is NBD*, which is responsible for DNA binding and the protein stabilization of the BbRAGL complex. Thus, our findings reveal that the functional requirement for ProtoRAG TIRs is similar to that for RSS in RAG-mediated recombination, which not only supports the common origin of ProtoRAG TIRs and RSSs from the asymmetric TIRs of ancient RAG transposons, but also reveals the development of RAG and RAG-like machineries during chordate evolution.

Systemic delivery of Eg5 shRNA-expressing plasmids using PEGylated DC-Chol/DOPE cationic liposome: Long-term silencing and anticancer effects in vivo

Duration of gene silencing due to the short-term silencing effects induced by exogenous siRNA have limited the therapeutic applications of RNAi and the development of RNAi-based therapeutics. We here generated Eg5 shRNA-expressing plasmids using the inverted terminal repeats (ITRs) sequences to produce Eg5 hairpin RNA under the control of U6 promoter. Using PEGylated DC-Chol/DOPE cationic liposomes, we demonstrated that a single systemic administration of Eg5 shRNA-expressing plasmid/liposome lipoplexes induced the long-term Eg5 silencing in the tumor sites of tumor-bearing mice, and ultimately lead to more sustained anticancer effects than standard synthetic siEg5/liposome lipoplexes. This non-viral Eg5 shRNA expression system had no risk of immunogenicity anticipated in the use of viral vectors, and could reduce the potential of off-target effects by scaling down the administration dose of RNAi therapeutics in patient. Therefore, the sustainable shRNA expression properties in the tumor sites suggest an efficient strategy to overcome the limitations caused by chemically synthesized siRNA methods such as short-term silencing effects and off-target effects. Herein, this study provides a non-viral silencing strategy for inducing long-term Eg5 silencing in vivo and suggests the great potential of Eg5 shRNA-expressing lipoplexes as a DNA-based RNAi therapeutics for cancer treatment.

Recovery of Muscovy duck-origin goose parvovirus from an infectious clone containing an E-box motif (CACATG) deletion within the left terminal region

Muscovy duck-origin goose parvovirus (MDGPV) is a causative agent of MDGPV-associated Derzsy's disease. To evalute the role of the cis-acting element E-box (CACATG) deletion on MDGPV eplication, an infectious plasmid clone p-PTΔE287, having one E-box deletion at nucleotide (nt) 287 of the left inverted terminal repeat sequence (L-ITR), was constructed by overlap extension PCR deleting the 287CACATG292 motif from the plasmid pMDGPVPT containing the full-length genome of the virulent MDGPV strain PT. The p-PTΔE287 plasmid was transfected into 9-day-old non-immune Muscovy duck embryos via the yolk sac, resulting in successful rescue of the deletion mutant virus r-PTΔE287. Compared with its parental virus PT, the virulence and the replication ability of r-PTΔE287 were reduced. In addition, we examined the ability of r-PTΔE287 to manipulate cell cycle progression. The results showed that r-PTΔE287 replication results in G0/G1 phase accumulation of infected duck embryo liver mesenchymal stem cells (BMSCs) and that this accumulation is caused by the prevention of cell cycle entry from G0/G1 phase into S phase. Taken together, introducing 287CACATG292 element deletion into MDGPV PT genomic DNA that induced rescued mutant virus (r-PTΔE287) cell cycle arrest function at the G0/G1 phase, which might inhibit MDGPV replication and virus progeny production. This study laid the foundation for further understanding of the relationship between E-box deletion in the L-ITR and MDGPV virulence.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

Genomic characterization of two Orf virus isolates from Jilin province in China.

Orf virus (ORFV), a typical member of the Parapoxvirus genus within the family Poxviridae, which is the causative agent of Orf, a common epitheliotropic viral disease of sheep, goats, wild ruminants, and humans. In the present study, we sequenced the complete genomic sequences of two ORFV strains (ORFV-SY17, isolated from sheep, and ORFV-NA17, isolated from goat) and conducted the comparative analysis of multiple ORFVs. The complete genomic sequence of ORFV-SY17 was at length of 140,413bp, including 131 potential open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) of 4267bp at both ends. The ORFV-NA17 strain displayed the similar genome structure with ORFV-SY17. The whole genomic sequence of ORFV-NA17 strain was 139,287bp in length and contained 132 ORFs flanked by ITRs of 3974bp. The overall G+C contents of ORFV-SY17 and ORFV-NA17 genome sequences were about 63.8% and 63.7%, respectively. The ITR sequences analysis showed that ORFV-SY17 and ORFV-NA17 contained the terminal BamHI sites and conserved telomere resolution sequences at both ends of their genome. In addition, comparative analysis of ORFs among ORFV-SY17, ORFV-NA17, and other ORFV strains revealed several sequence variations caused by insertions or deletions, especially in ORFs 005 and 116, which were very likely associated with host species. Phylogenetic analysis based on the complete genome sequences revealed that ORFV-SY17 was genetically closely related to NA1/11 and HN3/12 strains derived from sheep, while ORFV-NA17 was closely related to YX strain derived from goat. The multiple alignment of deduced amino acid sequences further revealed the genetic relationship between host species and genetic variations of ORFV strains. Taken together, the availability of genomic sequences of ORFV-SY17 and ORFV-NA17 strains from Jilin Province will aid in our understanding of the genetic diversity and evolution of ORFV strains in this region and can assist in distinguishing between ORFV strains that originate in sheep and goats.

TIR-Learner, a New Ensemble Method for TIR Transposable Element Annotation, Provides Evidence for Abundant New Transposable Elements in the Maize Genome

Transposable elements (TEs) make up a large and rapidly evolving proportion of plant genomes. Among Class II DNA TEs, TIR elements are flanked by characteristic terminal inverted repeat sequences (TIRs). TIR TEs may play important roles in genome evolution, including generating allelic diversity, inducing structural variation, and regulating gene expression. However, TIR TE identification and annotation has been hampered by the lack of effective tools, resulting in erroneous TE annotations and a significant underestimation of the proportion of TIR elements in the maize genome. This problem has largely limited our understanding of the impact of TIR elements on plant genome structure and evolution. In this paper, we propose a new method of TIR element detection and annotation. This new pipeline combines the advantages of current homology-based annotation methods with powerful de novo machine-learning approaches, resulting in greatly increased efficiency and accuracy of TIR element annotation. The results show that the copy number and genome proportion of TIR elements in maize is much larger than that of current annotations. In addition, the distribution of some TIR superfamily elements is reduced in centromeric and pericentromeric positions, while others do not show a similar bias. Finally, the incorporation of machine-learning techniques has enabled the identification of large numbers of new DTA (hAT) family elements, which have all the hallmarks of bona fide TEs yet which lack high homology with currently known DTA elements. Together, these results provide new tools for TE research and new insight into the impact of TIR elements on maize genome diversity.

Evolution-guided evaluation of the inverted terminal repeats of the synthetic transposon Sleeping Beauty

Sleeping Beauty (SB) is a synthetic Tc1/mariner transposon that is widely used for genetic engineering in vertebrates, including humans. Its sequence was derived from a consensus of sequences found in fish species including the Atlantic salmon (Salmo salar). One of the functional components of SB, the transposase enzyme, has been subject to extensive mutagenesis yielding hyperactive protein variants for advanced applications. The second functional component, the transposon inverted terminal repeats (ITRs), has so far not been extensively modified, mainly due to a lack of natural sequence information. Importantly, as genome sequences become available, they can provide a rich source of information for a refined molecular definition of the functional components of these transposons. Here we have mined the Salmo salar genome for a comprehensive set of transposon sequences that were used to build a refined consensus sequence. We synthetically produced the new consensus ITR sequences and used them to build a new transposon, the performance of which has been tested in cell-based transposition assays. The consensus sequence did not support enhanced transposition, suggesting alternative mechanisms responsible for the preferential amplification of these sequence variants in the salmon genome.

Cancer Gene Discovery Utilizing Sleeping Beauty Transposon Mutagenesis.

Transposable elements are DNA sequences with the ability to move from one genomic location to another. The movement of class II transposable elements has been functionally harnessed and separated into two distinct DNA transposon components: the terminal inverted repeat sequences that flank genetic cargo to be mobilized and a transposase enzyme capable of recognizing the terminal inverted repeat sequences and catalyzing the transposition reaction. In particular, the Sleeping Beauty (SB) system was the first successful demonstration of transposon-based gene transfer in vertebrate species. Over the years, several improvements have been made to SB technology and more recent studies have demonstrated the versatility of the system for many applications including insertional mutagenesis, gene transfer, and transgenesis. These genetic engineering advances made available by SB both augment and advance large-scale efforts that have been directed toward identifying how genes and environmental factors influence human health in recent years. In the age of personalized medicine, the versatility of SB provides numerous genetic engineering avenues for answering novel questions in basic and applied research. This chapter discusses the use of SB-based insertional mutagenesis in mice for the efficient identification of candidate cancer genes across numerous types of cancers.

Construction and rescue of Muscovy duck-origin goose parvovirus from an infectious clone containing an E-box deletion within the left terminal region

To obtain a deletion mutant of Muscovy duck-origin goose parvovirus (MDGPV) and to analyze its biological characteristics, the pMDGPVPT plasmid, which contains a full-length DNA infectious clone of the MDGPV PT strain, was used in this study as the template. The E-box at nt 315 of the left inverted terminal repeat sequence (L-ITR) was deleted by overlap extension PCR to obtain the infectious recombinant plasmid p-PTΔE315. The p-PTΔE315 plasmid was transfected into 9-day-old non-immune Muscovy duck embryos via the yolk sac and the rescued deletion mutant virus r-PTΔE315 was generated. Experiments to demonstrate the novel deletion mutant virus’ biological characteristics showed that r-PTΔE315 can cause typical lesions after infection of Muscovy duck embryos. Compared with its parent strain PT, the virulence of r-PTΔE315 and its proliferation ability in Muscovy duck embryos were attenuated, but its ability to replicate in MDEF cells was enhanced. This study laid the foundation for further understanding of the relationship between E-box deletion in the L-ITR and MDGPV virulence.