One-step Multiplex Transgenesis via Sleeping Beauty Transposition in Cattle

Garrels, Wiebke; Talluri, Thirumala Rao; Apfelbaum, Ronja; Carratalá, Yanet Prieto; Bosch, Pablo; Pötzsch, Kerstin; Grueso, Esther; Ivics, Zoltán; Kues, Wilfried A.

doi:10.1038/srep21953

Cited by 35 publications

(20 citation statements)

References 22 publications

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“…Our results indicate that the non-viral plasmid-based approach to VP1 -shRNA cassette delivery is more efficient with the SB transposon system than without it as to generate transgenic animals, The transgenic positive offspring with SB system was 13.04% (3/23) compared to the others without SB system 3.57% (1/28) and 7.14% (1/14), respectively, P < 0.05 (Table 1 ). The transgenic positive rate was also higher than that previously reported 36 .…”

Section: Discussioncontrasting

confidence: 69%

RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells

Deng

Tian

et al. 2017

Sci Rep

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Foot and mouth disease, which is induced by the foot and mouth disease virus (FMDV), takes its toll on the cloven-hoofed domestic animals. The VP1 gene in FMDV genome encodes the viral capsid, a vital element for FMDV replication. Sleeping Beauty (SB) is an active DNA-transposon system responsible for genetic transformation and insertional mutagenesis in vertebrates. In this study, a conserved VP1-shRNA which specifically targets the ovine FMDV-VP1 gene was constructed and combined with SB transposase and transposon. Then, they were microinjected into pronuclear embryos to breed transgenic sheep. Ninety-two lambs were born and the VP1-shRNA was positively integrated into eight of them. The rate of transgenic sheep production in SB transposon system was significantly higher than that in controls (13.04% vs. 3.57% and 7.14%, P < 0.05). The ear fibroblasts of the transgenic lambs transfected with the PsiCheck2-VP1 vector had a significant inhibitory effect on the VP1 gene of the FMDV. In conclusion, the VP1-shRNA transgenic sheep were successfully generated by the current new method. The ear fibroblasts from these transgenic sheep possess a great resistance to FMDV. The result indicated that RNAi technology combining the “Sleeping Beauty” transposon system is an efficient method to produce transgenic animals.

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Section: Discussioncontrasting

confidence: 69%

RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells

Deng

Tian

et al. 2017

Sci Rep

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“…The most hyperactive SB transposase version currently available, SB100X, displays a $100fold hyperactivity compared with the originally resurrected transposase [50]. The SB100X transposase enables highly efficient germline transgenesis in relevant mammalian models, including mice, rats, rabbits, pigs, sheep, and cattle [51][52][53][54][55][56]. Moreover, the use of the SB100X system yielded robust gene transfer efficiencies into human HSCs [50,57], mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), induced pluripotent stem cells (iPSCs) [58], and T cells [59], which are relevant targets for regenerative medicine and gene-and cell-based therapies of complex genetic diseases.…”

Section: Glossarymentioning

confidence: 98%

Gene Therapy with the Sleeping Beauty Transposon System

et al. 2017

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“…Till date, few data are available describing the optimization of electroporation conditions for bovine fetal fibroblasts (BFFs). Cattle is an economically important livestock [9], and increasingly used as a model species for research in artificial reproduction [10,11]. The establishment of somatic cell nuclear transfer (SCNT) [12] allowed the generation of transgenic and knock-out cattle via the use of genetically modified fibroblast donor cells [13,14].…”

Section: Introductionmentioning

confidence: 99%

Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts

Hyder

Eghbalsaied

2020

BMC Mol and Cell Biol

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Background: Gene transfer by electroporation is an established method for the non-viral mediated transfection of mammalian cells. Primary cells pose a particular challenge for electroporation-mediated gene transfer, since they are more vulnerable than immortalized cells, and have a limited proliferative capacity. Improving the gene transfer by using square wave electroporation in difficult to transfect cells, like bovine fetal fibroblasts, is a prerequisite for transgenic and further downstream experiments. Results: Here, bovine fetal fibroblasts were used for square-wave electroporation experiments in which the following parameters were systematically tested: electroporation buffer, electroporation temperature, pulse voltage, pulse duration, pulse number, cuvette type and plasmid DNA amount. For the experiments a commercially available square-wave generator was applied. Post electroporation, the bovine fetal fibroblasts were observed after 24 h for viability and reporter expression. The best results were obtained with a single 10 millisecond square-wave pulse of 400 V using 10 μg supercoiled plasmid DNA and 0.3 × 10 6 cells in 100 μl of Opti-MEM medium in 4 mm cuvettes. Importantly, the electroporation at room temperature was considerably better than with pre-cooled conditions. Conclusions: The optimized electroporation conditions will be relevant for gene transfer experiments in bovine fetal fibroblasts to obtain genetically engineered donor cells for somatic cell nuclear transfer and for reprogramming experiments in this species.

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One-step Multiplex Transgenesis via Sleeping Beauty Transposition in Cattle

Cited by 35 publications

References 22 publications

RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells

RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells

Gene Therapy with the Sleeping Beauty Transposon System

Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts

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