Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes

PLOS ONE, Dec 2019

Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest.

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Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes

March Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes R. J. Bevacqua 0 1 R. Fernandez-Martin 0 1 N. G. Canel 0 1 A. Gibbons 1 D. Texeira 1 F. Lange 1 G. Vans Landschoot 0 1 V. Savy 0 1 O. Briski 0 1 M. I. Hiriart 0 1 E. Grueso 1 Z. Ivics 1 O. Taboga 1 2 W. A. Kues 1 S. Ferraris 1 D. F. Salamone 0 1 0 Animal Biotechnology Laboratory, Facultad de Agronomia. INPA-CONICET, Buenos Aires University , Buenos Aires, Argentina, 2 Experimental Station Bariloche, INTA, Bariloche , Argentina , 3 Laboratorio de Fisiologia e Controle da ReproducËão, FAVET, UECE, Cear a State, Brasil, 4 Cloning and Transgenesis Laboratory, Maim oÂnides University , Buenos Aires, Argentina, 5 Paul-Ehrlich-Institute, Langen , Germany 1 Editor: Xiuchun Tian, University of Connecticut , UNITED STATES 2 CICVyA Biotechnology Institute, INTA Castelar , Buenos Aires , Argentina , 7 Friedrich-Loeffler-Institut , Neustadt , Germany Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest. - Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by Grant: Agencia Nacional de PromocioÂn CientÂõfica y TecnoloÂgica PICT-2013-2375 received by Daniel Salamone (DS); http://www.agencia.mincyt.gob.ar/ . The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Transgenic animals constitute an important tool for basic and applied research. In particular, transgenic farm animals could provide more accurate biomedical models than rodents [ 1,2 ] and also represent an alternative to current bioreactors for large-scale production of biopharmaceuticals [ 3,4 ]. The feasibility of using domestic species for the generation of highly complex proteins, which cannot be efficiently manufactured by conventional microorganisms or animal cell bioreactors, has been demonstrated for the past two decades [4±9]. However, the techniques available for transgenic animals production remain inefficient. Pronuclear microinjection and blastocyst injection with embryonic stem cells (ES) are the techniques most commonly used to produce transgenic mice [ 10,11 ]. However, their application to domestic species requires proper pronuclei visualization and species-specific ES cells maintenance, respectively, which up to date are not efficiently achieved in livestock [ 12,13 ]. For these reasons, somatic cell nuclear transfer (SCNT) soon turned into the technique of choice for the production of transgenic domestic species, and it has allowed the production of transgenic cattle [4;14], sheep [ 3 ] and pigs [ 15 ]. Nevertheless, the efficiency of SCNT remains low [ 16,17 ], and only a fraction of the transgenic offspring produced by any of these methods shows the desired transgene expression. This is mainly consequence of the random transgene integration as concatamers that can lead to epigenetic silencing of the transgene [18±20]. In recent years, active transgenesis approaches were introduced, based on the use of enzymes that actively promote the integration of the transgene, in opposition to traditional transgenesis techniques that rely on the introduction of transgenes into existing double stranded breaks in the genome. One of the first active transgenesis techniques introduced was lentivirus-mediated transgenesis [ 21 ]. Despite its high efficiency, this technique shows many drawbacks in (...truncated)


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R. J. Bevacqua, R. Fernandez-Martin, N. G. Canel, A. Gibbons, D. Texeira, F. Lange, G. Vans Landschoot, V. Savy, O. Briski, M. I. Hiriart, E. Grueso, Z. Ivics, O. Taboga, W. A. Kues, S. Ferraris, D. F. Salamone. Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes, PLOS ONE, 2017, Volume 12, Issue 3, DOI: 10.1371/journal.pone.0174025