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Shielding of sleeping beauty DNA transposon-delivered transgene cassettes by heterologous insulators in early embryonal cells.

ABSTRACT: The Sleeping Beauty (SB) transposon system represents an important alternative to viral integrating vector systems but may, as its viral counterparts, be subject to transcriptional silencing. To investigate shielding of SB-delivered transgene cassettes against transcriptional repression, we establish silencing assays in which SB vector-containing F9 murine teratocarcinoma cell clones are identified by strategies that include or exclude selection for transgene expression. Among clones carrying one or more SB transposon vectors, more than one-third are immediately silenced, and most of the remaining clones move toward silencing during prolonged passage. In line with the lack of an intrinsic ability of SB to resist silencing, we show that the stable transfection rate of SB vectors in F9 cells is significantly improved by flanking the transgene with heterologous 5'-HS4 chicken beta-globin (cHS4) insulators. In approaches based on drug selection and subsequent flow-cytometric detection of transgene expression, clones containing cHS4-insulated vectors are to a much higher degree protected against transcriptional silencing, resulting in long-term expression of the fluorescent marker. Our findings demonstrate that SB vectors, prone for transcriptional silencing by positional effects in F9 cells, are protected by insulators. We believe that insulated SB-derived vectors will become useful tools in transposon-based transgenesis and therapeutic gene transfer.

SUBMITTER: Dalsgaard T

PROVIDER: S-EPMC2834987 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers.

Project description:BACKGROUND:The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The Sleeping Beauty (SB) system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI) method is able to determine SB transposon copy numbers regardless of the genetic cargo. RESULTS:We designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene RPPH1 and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle. CONCLUSIONS:We have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy.

| S-EPMC3060107 | biostudies-literature

Effective gene trapping mediated by Sleeping Beauty transposon.

Project description:Gene trapping is a high-throughput approach to elucidate gene functions by disrupting and recapitulating expression of genes in a target genome. A number of transposon-based gene-trapping systems are developed for mutagenesis in cells and model organisms, but there is still much room for the improvement of their efficiency in gene disruption and mutation. Herein, a gene-trapping system mediated by Sleeping Beauty (SB) transposon was developed by inclusion of three functional cassettes. The mutation cassette can abrogate the splice of trapped genes and terminate their translation. Once an endogenous gene is captured, the finding cassette independently drives the translation of reporter gene in HeLa cells and zebrafish embryos. The efficiency cassette controls the remobilization of integrated traps through inducible expression of SB gene. Analysis of transposon-genome junctions indicate that most of trap cassettes are integrated into an intron without an obvious 3' bias. The transcription of trapped genes was abrogated by alternative splicing of the mutation cassette. In addition, integrated transposons can be induced to excise from their original insertion sites. Furthermore, the Cre/LoxP system was introduced to delete the efficiency cassette for stabilization of gene interruption and bio-safety. Thus, this gene-trap vector is an alternative and effective tool for the capture and disruption of endogenous genes in vitro and in vivo.

| S-EPMC3432063 | biostudies-literature

Sleeping Beauty transposon integrates into non-TA dinucleotides.

Project description:BackgroundSleeping Beauty transposon (SB) has become an increasingly important genetic tool for generating mutations in vertebrate cells. It is widely thought that SB exclusively integrates into TA dinucleotides. However, this strict TA-preference has not been rigorously tested in large numbers of insertion sites that now can be detected with next generation sequencing. Li et al. found 71 SB insertions in non-TA dinucleotides in 2013, suggesting that TA dinucleotides are not the only sites of SB integration, yet further studies on this topic have not been carried out.ResultsIn this study, we re-analyzed 600 million pairs of Illumina sequence reads from a high-throughput SB mutagenesis screen and identified 28 thousand SB insertions in non-TA sites. We recovered some of these non-TA sites using PCR and confirmed that at least a subset of the insertions at non-TA sites are real integrations. The consensus sequence of these non-TA sites shows an asymmetric pattern distinct from the symmetric pattern of the canonical TA sites. Perfect similarity between the downstream flanking sequence and SB transposon ends indicates there may be interaction between the transposon DNA binding domain of transposase and the target DNA.ConclusionThe TA-preference of SB transposon is not as strict as what people had thought. And the SB integrations at non-TA sites might be guided by the interaction between the transposon DNA binding domain of SB transposase and the target DNA.

| S-EPMC5801840 | biostudies-literature

The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells.

Project description:The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

| S-EPMC5852330 | biostudies-literature

Engineered Sleeping Beauty transposase redirects transposon integration away from genes.

Project description:The Sleeping Beauty (SB) transposon system is a popular tool for genome engineering, but random integration into the genome carries a certain genotoxic risk in therapeutic applications. Here we investigate the role of amino acids H187, P247 and K248 in target site selection of the SB transposase. Structural modeling implicates these three amino acids located in positions analogous to amino acids with established functions in target site selection in retroviral integrases and transposases. Saturation mutagenesis of these residues in the SB transposase yielded variants with altered target site selection properties. Transposon integration profiling of several mutants reveals increased specificity of integrations into palindromic AT repeat target sequences in genomic regions characterized by high DNA bendability. The H187V and K248R mutants redirect integrations away from exons, transcriptional regulatory elements and nucleosomal DNA in the human genome, suggesting enhanced safety and thus utility of these SB variants in gene therapy applications.

| S-EPMC8934666 | biostudies-literature

Sleeping Beauty Transposon-Mediated Asparaginase Gene Delivery by a Nanoparticle Platform.

Project description:Transgenic genome integration using non-viral vehicles is a promising approach for gene therapy. Previous studies reported that asparagine is a key regulator of cancer cell amino acid homeostasis, anabolic metabolism and cell proliferation. The depletion of asparagine would inhibit the growth of many cancer cells. In this study, we develop a nanoparticle delivery system to permanently integrate the asparaginase gene into the genome of human lung adenocarcinoma cells. The asparaginase plasmid and the Sleeping Beauty plasmid were co-transfected using amine-functionalized mesoporous nanoparticles into the human lung adenocarcinoma cells. The intracellular asparaginase expression led to the cell cytotoxicity for PC9 and A549 cells. In addition, the combination of the chemotherapy and the asparaginase gene therapy additively enhanced the cell cytotoxicity of PC9 and A549 cells to 69% and 63%, respectively. Finally, we showed that the stable cell clones were successfully made by puromycin selection. The doxycycline-induced expression of asparaginase caused almost complete cell death of PC9 and A549 asparaginase-integrated stable cells. This work demonstrates that silica-based nanoparticles have great potential in gene delivery for therapeutic purposes.

| S-EPMC6686048 | biostudies-literature

Genomic analysis of Sleeping Beauty transposon integration in human somatic cells.

Project description:The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity "sandwich" (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy applications.

| S-EPMC4229213 | biostudies-literature

Sleeping Beauty Insertional Mutagenesis Reveals Important Genetic Drivers of Central Nervous System Embryonal Tumors.

Project description:Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. SIGNIFICANCE: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.

| S-EPMC6397665 | biostudies-literature

Retargeting sleeping beauty transposon insertions by engineered zinc finger DNA-binding domains.

Project description:The Sleeping Beauty (SB) transposon is a nonviral, integrating vector system with proven efficacy in preclinical animal models, and thus holds promise for future clinical applications. However, SB has a close-to-random insertion profile that could lead to genotoxic effects, thereby presenting a potential safety issue. We evaluated zinc finger (ZF) DNA-binding domains (DBDs) for their abilities to introduce a bias into SB's insertion profile. E2C, that binds a unique site in the erbB-2 gene, mediated locus-specific transposon insertions at low frequencies. A novel ZF targeting LINE1 repeats, ZF-B, showed specific binding to an 18-bp site represented by ~12,000 copies in the human genome. We mapped SB insertions using linear-amplification (LAM)-PCR and Illumina sequencing. Targeted insertions with ZF-B peaked at approximately fourfold enrichment of transposition around ZF-B binding sites yielding ~45% overall frequency of insertion into LINE1. A decrease in the ZF-B dataset with respect to transposon insertions in genes was found, suggesting that LINE1 repeats act as a sponge that "soak up" a fraction of SB insertions and thereby redirect them away from genes. Improvements in ZF technology and a careful choice of targeted genomic regions may improve the safety profile of SB for future clinical applications.

| S-EPMC3464645 | biostudies-literature

Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production.

Project description:Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.

| S-EPMC9316264 | biostudies-literature

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