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Bevacqua R.J.,University of Buenos Aires | Fernandez-Martin R.,University of Buenos Aires | Canel N.G.,University of Buenos Aires | Gibbons A.,Instituto Nacional de Tecnologia Agropecuaria | And 12 more authors.
PLoS ONE | Year: 2017

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. © 2017 Bevacqua et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Moura R.R.,Laboratorio Of Fisiologia E Controle Da Reproducao | Souza-Fabjan,Laboratorio Of Fisiologia E Controle Da Reproducao | Fonseca J.F.,Embrapa Caprinos e Ovinos | Melo C.H.S.,Laboratorio Of Fisiologia E Controle Da Reproducao | And 9 more authors.
Animal Reproduction | Year: 2014

This study aimed to monitor estrous cycle parameters of a human granulocyte colony-stimulating factor (hG-CSF)-transgenic founder female goat and to perform superovulation and embryo recovery (surgical or transcervical method) for further transfer to recipients to quickly obtain offspring. Two experiments were performed using a transgenic (TF) and a non-transgenic (NTF) female. In experiment 1, three estrous cycles were monitored for the following parameters: estrus behavior, progesterone concentration and ovarian activity. In experiment 2, two superovulation/embryo recovery sessions were performed and the recovered embryos were transferred to previously prepared recipients. Data were compared by either t test or Fisher's exact test. The mean interval between natural estrus was 20.7 ± 0.6 and 19.7 ± 0.6 (P > 0.05) days for the TF and NTF, respectively. Progesterone concentrations and ovarian activity were normal and similar between goats. The ovulation rate was similar between TF and NTF (12.0 ± 1.4 vs. 18.0 ± 4.2 CL; P > 0.05). No significant differences in embryo recovery rate (P > 0.05) were observed between the surgical and transcervical methods for TF (69.2 vs. 72.7%) or NTF (100.0 vs. 86.7%). Sixteen embryos from the TF were transferred to recipients, and eight kids were born. Among these kids, the transgene was identified in three (two males and one female), resulting in a transgenesis rate of 37.5%. In summary, the TF is a true founder, since she proved fertility and capacity of transmitting the hG-CSF transgene to progeny, suggesting that the analyzed reproductive traits were not compromised by the presence of the transgene.

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