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Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos

Published online by Cambridge University Press:  06 May 2010

Marcella Pecora Milazzotto*
Affiliation:
Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Reprodução Animal. Av. Orlando Marques de Paiva, 87, Laboratório de Fecundação In Vitro, Clonagem e Transgenia Animal. 05508-270 – São Paulo, SP, Brazil.
Marcelo Demarchi Goissis
Affiliation:
Department of Animal Reproduction, School of Veterinary Medicine, University of São Paulo, São Paulo, Brazil.
Weber Beringui Feitosa
Affiliation:
Department of Animal Reproduction, School of Veterinary Medicine, University of São Paulo, São Paulo, Brazil.
Leydson Ferreira Martins
Affiliation:
Department of Animal Reproduction, School of Veterinary Medicine, University of São Paulo, São Paulo, Brazil.
Bryan Eric Strauss
Affiliation:
Heart Institute, InCor, School of Medicine, University of São Paulo, São Paulo, Brazil.
Marcio Chaim Bajgelman
Affiliation:
Heart Institute, InCor, School of Medicine, University of São Paulo, São Paulo, Brazil.
Mayra Elena Ortiz D'Ávila Assumpção
Affiliation:
Department of Animal Reproduction, School of Veterinary Medicine, University of São Paulo, São Paulo, Brazil.
Jose Antonio Visintin
Affiliation:
Department of Animal Reproduction, School of Veterinary Medicine, University of São Paulo, São Paulo, Brazil.
*
All correspondence to: Marcella Pecora Milazzotto. Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Reprodução Animal. Av. Orlando Marques de Paiva, 87, Laboratório de Fecundação In Vitro, Clonagem e Transgenia Animal. 05508-270 – São Paulo, SP, Brazil. Tel: +55 11 3091 7916. Fax: +55 11 3091 7412. e-mail: mazamila@hotmail.com

Summary

Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.

Type
Research Article
Information
Zygote , Volume 18 , Issue 4 , November 2010 , pp. 339 - 344
Copyright
Copyright © Cambridge University Press 2010

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