title: Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos.
abstract: 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.
authors.affiliation: Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Reprodução Animal, São Paulo, SP, Brazil. mazamila@hotmail.com
authors.forename: Marcella Pecora
authors.initials: MP
authors.lastname: Milazzotto
authors.forename: Marcelo Demarchi
authors.initials: MD
authors.lastname: Goissis
authors.forename: Weber Beringui
authors.initials: WB
authors.lastname: Feitosa
authors.forename: Leydson Ferreira
authors.initials: LF
authors.lastname: Martins
authors.forename: Bryan Eric
authors.initials: BE
authors.lastname: Strauss
authors.forename: Marcio Chaim
authors.initials: MC
authors.lastname: Bajgelman
authors.forename: Mayra Elena Ortiz D'Avila
authors.initials: ME
authors.lastname: Assumpção
authors.forename: Jose Antonio
authors.initials: JA
authors.lastname: Visintin
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle; D002460:Cell Line; D004622:Embryo, Mammalian / Q000166:cytology*; D005240:Feasibility Studies; D055785:Gene Knockdown Techniques / Q000379:methods*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D055435:Myostatin / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D055435:Myostatin; D034741:RNA, Small Interfering