Chromosomal transposition of PiggyBac in mouse embryonic stem cells.
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ABSTRACT:
SUBMITTER: Wang W
PROVIDER: S-EPMC2440425 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PiggyBac system has been shown to have a high efficiency to mediate gene transfer. However, there are no reports on its efficiency to mediate multiplex transgenes in mouse embryonic stem cells. Here we first established an immortalized feeder cell line by introducing four antibiotic resistance genes simultaneously into the original SNL 76/7 feeder cell line utilizing the PiggyBac system. This is the feeder cell line with the most diverse types of antibiotic resistance genes reported so far, which will enable researchers to perform simultaneous multiplex gene transfer or gene targeting experiments in ES cells. With such feeder cell line, we were able to quantitatively characterize the transposition efficiency of PiggyBac system in mouse ES cells using five transposons carrying different inducible fluorescence proteins and antibiotic resistance genes, and the efficiency ranged from about 2% for one transposon to 0.5% for five transposons. The highly efficient multiplex gene transfer mediated by PiggyBac will no doubt provide researchers with more choices in biomedical research and development.
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Project description:Permanent and reversible genetic modifications are important approaches to study gene function in different cell types. They are also important for stem cell researchers to explore and test the therapeutic potential of stem cells. The piggyBac transposon from insects is a rising nonviral system that efficiently mutagenizes and mediates gene transfer into the mammalian genome. It is also characterized by its precise excision, leaving no trace sequence behind so that the genomic integrity of the mutated cell can be restored. Here, we use an optimized piggyBac transposon system to mediate gene transfer and expression of a bifunctional fluorescent reporter in human embryonic stem (ES) cells. We provide molecular evidence for transposase-mediated piggyBac integration events and functional evidence for successful expression of a transferred fluorescent protein genes in human ES cells and their in vitro differentiated derivatives. We also demonstrate that the integrated piggyBac transposon can be removed and an undisrupted insertion site can be restored, which implies potential applications for its use in gene therapy and genetics studies.
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Project description:The most stringent criterion for evaluating pluripotency is generation of chimeric animals with germline transmission ability. Because the quality of induced pluripotent stem cell (iPSC) lines is heterogeneous, an easy and accurate system to evaluate these abilities would be useful. In this study, we describe a simple but comprehensive system for generating and evaluating iPSCs by single transfection of multiple piggyBac (PB) plasmid vectors encoding Tet-inducible polycistronic reprogramming factors, a pluripotent-cell-specific reporter, a constitutively active reporter, and a sperm-specific reporter. Using this system, we reprogrammed 129 and NOD mouse embryonic fibroblasts into iPSCs, and then evaluated the molecular and functional properties of the resultant iPSCs by quantitative RT-PCR analysis and chimera formation assays. The iPSCs contributed extensively to chimeras, as indicated by the constitutively active TagRFP reporter, and also differentiated into sperm, as indicated by the late-spermatogenesis-specific Acr (acrosin)-EGFP reporter. Next, we established secondary MEFs from E13.5 chimeric embryos and efficiently generated secondary iPSCs by simple addition of doxycycline. Finally, we applied this system to establishment and evaluation of rat iPSCs and production of rat sperm in mouse-rat interspecific chimeras. By monitoring the fluorescence of Acr-EGFP reporter, we could easily detect seminiferous tubules containing rat iPSC-derived spermatids and sperm. And, we succeeded to obtain viable offspring by intracytoplasmic sperm injection (ICSI) using these haploid male germ cells. We propose that this system will enable robust strategies for induction and evaluation of iPSCs, not only in rodents but also in other mammals. Such strategies will be especially valuable in non-rodent species, in which verification of germline transmission by mating is inefficient and time-consuming.
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Project description:Safer and more efficient methods for directing therapeutic genes to specific sequences could increase the repertoire of treatable conditions. Many current approaches act passively, first initiating a double-stranded break, then relying on host repair to uptake donor DNA. Alternatively, we delivered an actively integrating transposase to the target sequence to initiate gene insertion. We fused the hyperactive piggyBac transposase to the highly specific, catalytically dead SpCas9-HF1 (dCas9) and designed guide RNAs (gRNAs) to the CCR5 safe harbor sequence. We introduced mutations to the native DNA-binding domain of piggyBac to reduce non-specific binding of the transposase and cause the fusion protein to favor binding by dCas9. This strategy enabled us, for the first time, to direct transposition to the genome using RNA. We showed that increasing the number of gRNAs improved targeting efficiency. Interestingly, over half of the recovered insertions were found at a single TTAA hotspot. We also found that the fusion increased the error rate at the genome-transposon junction. We isolated clonal cell lines containing a single insertion at CCR5 and demonstrated long-term expression from this locus. These vectors expand the utility of the piggyBac system for applications in targeted gene addition for biomedical research and gene therapy.
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Project description:Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naïve PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc). One type of biPSCs, cultured in medium supplemented with knockout serum replacement (KSR), FGF2, and bovine leukemia inhibitory factor (bLIF), had a flattened morphology like human PSCs; these were classified as primed-type. The other type biPSCs, cultured in KSR, bLIF, Mek/Erk inhibitor, GSK3 inhibitor and forskolin, had a compact morphology like mouse PSCs; these were classified as naïve-type. Cells could easily be switched between these two types of biPSCs by changing the culture conditions. Both types of biPSCs had strong alkaline phosphatase activity, expressed pluripotent markers (OCT3/4, NANOG, REX1, ESRRβ, STELLA, and SOCS3), and formed embryoid bodies that gave rise to differentiated cells from all three embryonic germ layers. However, only naïve-type biPSCs showed the hallmarks of naïve mouse PSCs, such as LIF-dependent proliferation, lack of FGF5 expression, and active XIST expression with two active X chromosomes. Furthermore, naïve-type biPSCs could contribute to the inner cell mass (ICM) of host blastocysts and most tissues within chimeric embryos. This is the first report of generation of biPSCs with several characteristics similar to those of naïve mouse PSCs and a demonstrated potential to contribute to chimeras.
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Project description:Clinical application of regenerative therapies using embryonic or induced pluripotent stem cells is within reach. Progress made during recent years has encouraged researchers to address remaining open questions in order to finally translate experimental cell replacement therapies into application in patients. To achieve this, studies in translationally relevant animal models are required to make the final step to the clinic. In this context, the baboon (Papio anubis) may represent a valuable nonhuman primate (NHP) model to test cell replacement therapies because of its close evolutionary relationship to humans and its large body size. In this study, we describe the reprogramming of adult baboon skin fibroblasts using the piggyBac transposon system. Via transposon-mediated overexpression of six reprogramming factors, we generated five baboon induced pluripotent stem cell (iPSC) lines. The iPSC lines were characterized with respect to alkaline phosphatase activity, pluripotency factor expression analysis, teratoma formation potential, and karyotype. Furthermore, after initial cocultivation with mouse embryonic fibroblasts, we were able to adapt iPSC lines to feeder-free conditions. In conclusion, we established a robust and efficient protocol for iPSC generation from adult baboon fibroblasts.
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Project description:Chromosomal integrity has been known for many years to affect the ability of mouse embryonic stem cells (mESCs) to contribute to the germline of chimeric mice. Abnormal chromosomes are generally detected by standard cytogenetic karyotyping. However, this method is expensive, time consuming, and often omitted prior to blastocyst injection, consequently reducing the frequency of mESC-derived offspring. Here, we show a fast, accurate, and inexpensive screen for identifying the two most common aneuploidies (Trisomy 8 and loss of chromosome Y) in genetically manipulated mESCs using quantitative real-time PCR (qPCR). Screening against these two aneuploidies significantly increases the fraction of normal mESC clones. Our method is extremely sensitive and can detect as low as 10% aneuploidy among a large population of mESCs. It greatly expedites the generation of mutant mice and provides a quick tool for assessing the aneuploidy percentages of any mESC line.
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Project description:Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.
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Project description:Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development. Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro. In this study, sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology, pluripotent marker expression, and differentiation ability, delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4, SOX2, KLF4, cMYC, porcine NANOG, human LIN28, SV40 large T antigen, and human TERT. Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro. A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs. This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep.
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Project description:Feeder cells like mouse embryonic fibroblasts (MEFs) have been widely applied for culture of pluripotent stem cells, but their roles remain elusive. Noticeably, ESCs cultured on the feeders display transcriptional heterogeneity. We investigated roles of feeder cells by examining the telomere maintenance. Here we show that telomere is longer in mESCs cultured with than without the feeders. mESC cultures without MEF feeders exhibit telomere loss, chromosomal fusion, and aneuploidy with increasing passages. Notably, feeders facilitate heterogeneous transcription of 2-cell genes including Zscan4 and telomere elongation. Moreover, feeders produce Fstl1 that together with BMP4 periodically activate Zscan4. Interestingly, Zscan4 is repressed in mESCs cultured in 2i (inhibitors of Mek and Gsk3β signaling) media, associated with shorter telomeres and increased chromosome instability. These data suggest the important role of feeders in maintaining telomeres for long-term stable self-renewal and developmental pluripotency of mESCs.
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