Excision Efficiency Is Not Strongly Coupled to Transgenic Rate: Cell Type-Dependent Transposition Efficiency of<i>Sleeping Beauty</i>and<i>piggyBac</i>DNA Transposons

Kolacsek, Orsolya; Erdei, Zsuzsa; Apáti, Ágota; Sándor, Sára; Izsvák, Zsuzsanna; Ivics, Zoltán; Sarkadi, Balázs; Orbán, Tamás I.

doi:10.1089/hgtb.2013.149

Cited by 21 publications

(25 citation statements)

References 53 publications

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“…A previous study reports that excision efficiency and re-integration rates of SB transposition are not coupled (47). Our capture assays showed the presence of twenty ETFs after SB excision (Figure 7B).…”

Section: Discussionmentioning

confidence: 99%

“…This refined model accounts for the following observations and predictions: 1) The presence of multiple SB-mediated ETFs (48, 52) that may be generated through asymmetric cleavage of transposon termini. 2) Rates of excision and reintegration are not equal (21,47,53,54). Many ETFs are not integrated at detectable levels although there does appear to be a pretty constant ratio of integration/excision that might allow the prediction of a rough level of integration events based on determination of excision products.…”

Section: Discussionmentioning

confidence: 99%

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Asymmetrical cleavages ofSleeping Beautytransposons generate multiple excised transposon fragments during transposition

Yang

et al. 2019

Preprint

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1Although the Sleeping Beauty (SB) transposon is the most validated DNA transposon used as a 2 gene delivery vehicle in vertebrates, many details of the excision and integration steps in the transposition 3 process are unclear. We have probed in detail the products of the excision step and apparent selective 4 integration of a subset of those products during transposition. The standard model of SB transposase-5 mediated transposition includes symmetrical cleavages at both ends of the transposon for excision and 6 re-integration in another DNA sequence. In our analysis of excised transposon fragments (ETFs), we 7 found evidence for the requirement of certain flanking sequences for efficient cleavage and a significant 8 rate of asymmetrical cleavage during the excision process that generates multiple ETFs. Our results 9 suggest that the cleavage step by SB transposase is not as precise as indicated in most models. Repair 10 of the donor ends can produce eight footprint sequences (TACTGTA, TACAGTA, TACATA, TACGTA, 11 TATGTA, TACTA, TAGTA and TATA). Our data also suggest that mismatch repair (MMR) is not an 12 essential requirement for footprint formation. Among the twenty liberated ETFs, only eight appear to 13 effectively re-integrate into TA sites distributed across the genome, supporting earlier findings of unequal 14 rates of excision and reintegration during SB transposition. These findings may be important in 15 considerations of efficiency of SB transposon remobilization, selection of TA integration sites and 16 detection of SB excision and integration loci, all of which may be important in human gene therapy. 17 18 20 31 poorly understood because i) in vitro SB transposition assays remain unsuccessful; ii) the crystal structure 32 of full-length SB transposase has not been resolved, although the NMR structure of the SB11 DNA-33 binding domain (33) and crystal structure of the SB100X catalytic domain (11) have been reported; and iii) 34 compared to other Tc1/mariner transposons, the inverted terminal repeat (ITR) sequences that comprise 35 the termini of SB transposons are considerably longer and each has two transposase-binding, direct 36 4 repeat (DR) sequences (Figure 1A) (7). Hence, the ITRs of SB transposons are called IR/DRs for their 1 unusual structure. This last feature introduces several questions about the coordination of transposase 2 enzymes in both the cleavage and integration reactions; e.g., whether dimers or tetramers form to affect 3 either or both steps. 4In the standard model, SB transposon excision results from staggered cuts at both ends of the 5 transposon that leaves a 3-bp single stranded overhanging sequence in both the donor DNA and the 6 excised transposon. When the wounded DNA is repaired, either of two 7-bp canonical footprints, 7 TACAGTA and TACTGTA, that differ in the fourth position (underlined) are left due to ambiguous repair of 8 the mismatch at this position ( Figure 1B) (20). The model suggests that SB transposase precisely cleaves 9 the donor DNA and the targeted integra...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Asymmetrical cleavages ofSleeping Beautytransposons generate multiple excised transposon fragments during transposition

Yang

et al. 2019

Preprint

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“…Human embryonic kidney cells (HEK-293) and HeLa cells were maintained as described previously (Kolacsek et al, 2014a); the HUES9 embryonic stem cell line was cultured as described earlier (Apati et al, 2008). The establishment and maintenance of the MSCL-2 mesenchymal-like cell line was described in detail previously (Varga et al, 2011).…”

Section: Cell Lines and Culturingmentioning

confidence: 99%

Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications

Kolacsek

Pergel

Varga

et al. 2017

Gene

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There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the ΔΔCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies.

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“…the epichromosomal cellular uptake of plasmids-is sufficient, the stable integration of linearized plasmid DNA into the host cell genome is a rare event. Depending on the host cell type, the donor organism and the transfection protocol used, most stably transfected cells only harbor low vector copy numbers (VCN) ranging from single to about five plasmid molecules randomly integrated into the host cell genome (Chusainow et al 2009;Kolacsek et al 2014). The integrated plasmid sequences do not only contain the expression cassette entailing a promoter/ enhancer required to drive the expression of the gene of interest (GOI) encoding the POI and a polyadenylation signal (p(A)), but also long bacteria-derived sequences.…”

Section: Introductionmentioning

confidence: 99%

Transposon vector-mediated stable gene transfer for the accelerated establishment of recombinant mammalian cell pools allowing for high-yield production of biologics

2020

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Stable recombinant mammalian cells are of growing importance in pharmaceutical biotechnology production scenarios for biologics such as monoclonal antibodies, growth and blood factors, cytokines and subunit vaccines. However, the establishment of recombinant producer cells using classical stable transfection of plasmid DNA is hampered by low stable gene transfer efficiencies. Consequently, subsequent selection of transgenic cells and the screening of clonal cell populations are time-and thus cost-intensive. To overcome these limitations, expression cassettes were embedded into transposon-derived donor vectors. Upon the co-transfection with transposase-encoding constructs, elevated vector copy numbers stably integrated into the genomes of the host cells are readily achieved facilitating under stringent selection pressure the establishment of cell pools characterized by sustained and high-yield recombinant protein production. Here, we discuss some aspects of transposon vector technologies, which render these vectors promising candidates for their further utilization in the production of biologics.

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Excision Efficiency Is Not Strongly Coupled to Transgenic Rate: Cell Type-Dependent Transposition Efficiency ofSleeping BeautyandpiggyBacDNA Transposons

Cited by 21 publications

References 53 publications

Asymmetrical cleavages ofSleeping Beautytransposons generate multiple excised transposon fragments during transposition

Asymmetrical cleavages ofSleeping Beautytransposons generate multiple excised transposon fragments during transposition

Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications

Transposon vector-mediated stable gene transfer for the accelerated establishment of recombinant mammalian cell pools allowing for high-yield production of biologics

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