The main goal of husbandry and beef cattle production is to enhance performance rates, for example, weight gain. Myostatin is referred to as a negative regulator of skeletal muscle growth. Genetic engineering of this character in order to produce double muscling animals that can transmit to future progeny will enhance its usefulness. The present research aimed to analyze myostatin inhibition through lentiviral-mediated delivery of shRNA in mouse myoblast culture and the feasibility of the lentiviral-mediated delivery of shRNA into in vitro-produced transgenic bovine embryos. In order to achieve knockdown of myostatin in cell and embryo culture, a lentiviral vector was constructed with ubiquitin C promoter-driven GFP gene (green fluorescent protein) and shRNA to suppress myostatin gene expression driven by the U6 promoter. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR of myostatin and GAPDH genes. Later, bovine oocytes were in vitro-matured and the lentiviral vector was microinjected into the oocyte perivitelline space (2.5 � 106 IU mL-1) after mechanical and chemical cumulus cell removal. Non-microinjected mature oocytes were considered as control. After microinjection, oocytes were fertilized and cultured in vitro. After 4 and 9 days of culture, embryos were evaluated by epifluorescence microscopy. The GFP-positive embryos were green under fluorescence. Cell morphology and embryo development rate data were analyzed by Minitab Release 14 Statistical Software (Minitab, Inc., State College, PA, USA), submitted to ANOVA, and compared by Tukey test (P d 0.05). Real-time PCR data were analyzed by Pair-Wise Fixed Reallocation Randomization Test using REST2005 software. Cell morphology results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells as the transducted group progressed less to myotubes than in the control group. A lower amount of myostatin mRNA after 72 h of differentiation indicated an inhibition tendency by real-time PCR. In relation to the transgenic embryo production, 96.9 � 0.34% (62.65) developed to cleavage, 80.24 � 4.38% (51/65) were GFP-positive, and 50.95 � 3.37% (26/65) achieved blastocyst stage. After hatching, 3.07% (2/65) of GFP-positive embryos maintained fluorescence. In relation to the control group, the cleavage rate was 93.81 � 0.68% (61/65); the blastocyst rate 38.34 � 2.36% (25/65), and none were fluorescent. In conclusion, myostatin gene knockdown was effectively performed by lentiviral vector-mediated delivery of shRNA. Thus, novel studies about the efficiency of this vector on transgenic embryo production can be performed.
This work was supported financially by FAPESP 03/0156-9.
Analysis of two putative regulatory elements of Myostatin gene in C2C12 myoblasts