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PiggyBac mediated multiplex gene transfer in mouse embryonic stem cell.

ABSTRACT: 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.

SUBMITTER: Lu X

PROVIDER: S-EPMC4269400 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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PiggyBac mediated multiplex gene transfer in mouse embryonic stem cell.

Lu Xibin X Huang Wei W

PloS one 20141217 12

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, whic ...[more]

PMID: 25517991

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Project description:BackgroundLittle is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC) differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5) undergoing undirected differentiation into embryoid bodies (EBs) over a period of two weeks.ResultsWe identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1), our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2) that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set.ConclusionOur analysis profiles for the first time gene expression at a very early stage of mESC differentiation, and identifies a functional and phylogenetic signature for the genes involved. The data generated constitute a valuable resource for further studies. All DNA microarray data used in this study are available in the StemBase database of stem cell gene expression data 1 and in the NCBI's GEO database.

Project description:BackgroundUp to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability.ResultsThe mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window.ConclusionOur results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

Project description:AimTo compare gene targeting efficiencies, expression profiles, and Ca(2+) handling potentials in two widely used mouse embryonic stem cell lines.MethodsThe two widely used mouse embryonic stem cell lines, R1 and HM-1, were cultured and maintained on Mitomycin C treated mouse embryonic fibroblast feeder cell layers, following standard culture procedures. Cells were incubated with primary and secondary antibodies before fluorescence activated cell sorting analysis to compare known pluripotency markers. Moreover, cells were harvested by trypsinization and transfected with a kinase-inactive murine Tyk2 targeting construct, following the BioRad and Amaxa transfection procedures. Subsequently, the cells were cultured and neomycin-resistant cells were picked after 13 d of selection. Surviving clones were screened twice by polymerase chain reaction (PCR) and finally confirmed by Southern blot analysis before comparison. Global gene expression profiles of more than 20 400 probes were also compared and significantly regulated genes were confirmed by real time PCR analysis. Calcium handling potentials of these cell lines were also compared using various agonists.ResultsWe found significant differences in transfection efficiencies of the two cell lines (91% ± 6.1% vs 75% ± 4.2%, P = 0.01). Differences in the targeting efficiencies were also significant whether the Amaxa or BioRad platforms were used for comparison. We did not observe significant differences in the levels of many known pluripotency markers. However, our genome-wide expression analysis using more than 20 400 spotted cDNA arrays identified 55 differentially regulated transcripts (P < 0.05) implicated in various important biological processes, including binding molecular functions (particularly Ca(2+) binding roles). Subsequently, we measured Ca(2+) signals in these cell lines in response to various calcium agonists, both in high and low Ca(2+) solutions, and found significant differences (P < 0.05) in the regulation of Ca(2+) homeostasis between the investigated cell lines. Then we further compared the detection and expression of various membrane and intracellular Ca(2+) receptors and similarly found significant (P < 0.05) variations in a number of calcium receptors between these cell lines.ConclusionResults of this study emphasize the importance of considering intrinsic cellular variations, during selection of cell lines for experiments and interpretations of experimental results.

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PiggyBac mediated multiplex gene transfer in mouse embryonic stem cell.

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PiggyBac mediated multiplex gene transfer in mouse embryonic stem cell.

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