Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos
Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos
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DOI：
10.1017/s0967199410000031
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发表时间：
2010-11-01
期刊：
影响因子：
1.7
通讯作者：
Visintin, Jose Antonio
中科院分区：
文献类型：
--
作者：
Milazzotto, Marcella Pecora;Goissis, Marcelo Demarchi;Visintin, Jose Antonio
肌生长抑制素被描述为骨骼肌生长的负调节剂。基因工程可能是有用的，因为它可以生产出肌肉质量加倍的动物，并将这种特征遗传给未来的后代。在这种情况下，本研究的目的是分析的可行性，慢病毒介导的交付短发夹RNA（shRNA）靶向肌肉生长抑制素到体外生产的转基因牛胚胎。慢病毒载体用于递送表达绿色荧光蛋白（GFP）的转基因和靶向肌生长抑制素的shRNA。通过诱导分化后的体外鼠成肌细胞（C2C12）形态学和通过实时PCR验证载体效率。将慢病毒载体显微注射到体外成熟卵母细胞的卵黄周间隙中。未显微注射的卵母细胞用作对照。注射后，卵母细胞受精并体外培养。通过落射荧光显微镜评价囊胚。结果表明，载体能够抑制C2C12细胞中的肌生长抑制素mRNA，因为转导组在分化72小时后具有更少量的肌生长抑制素mRNA（p < 0.05），并且具有比未转导组更少的肌管形成（p < 0.05）。显微注射组和对照组之间的卵裂率和囊胚率没有差异。孵化后，3.07%的胚胎表现出GFP表达，表明它们表达靶向肌肉生长抑制素的shRNA。总之，我们证明了慢病毒载体有效地进行shRNA肌肉生长抑制素基因敲低和基因递送到体外产生的牛胚胎。因此，该技术可以被认为是生产转基因胚胎和双肌肉质量动物的新选择。
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.