Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis
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Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4 %; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3 %, 46/117) and IVF embryos (39.6 %, 44/111). Pregnancy rate was detected in 50.0 % (6/12) of recipient ewes with TG embryos, in 46.7 % (7/15) with IVF embryos, and in 65.0 % (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9 %) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs.
KeywordsGenetic modification Transgenic Micromanipulation Ovine Green fluorescent protein Lentivirus
The authors wish to thank Alison Creneguy for the technical labor in the production of recombinant lentivirus, Robert Wijma for technical help during in vitro embryo production, Sofía Ramírez for technical assistance during fetal measurement, and Sabrina Olmos, Fatima Rodríguez and Santiago Machado for nursing and handling of newborns. This project was financially supported by ANII PR_FSA_2009_1_1333 grant, FOCEM (MERCOSUR Structural Convergence Fund), COF 03/11 and received additional support by PEDECIBA (UdelaR) and Union Agriculture Group. MC, MV, IA and AM are fellows of Sistema Nacional de Investigadores (SNI).
All procedures that include animal handling were approved by the Animal Care Committee of the Fundación IRAUy and certified by the National Council of Animal Care of Uruguay and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All persons gave their informed consent prior to their inclusion in the study.
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