Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Biochem Biophys Res Commun. 2011 Nov 25;415(3):429-33. doi: 10.1016/j.bbrc.2011.10.044. Epub 2011 Oct 18.
Transgenic animals have potential applications in medicine, life sciences, and biopharmacy. In this study, we developed a convenient, economic, and efficient method for gene transfer by transfection of male spermatogonial stem cells. Three fragments of the Thanatin gene, encoding an antibacterial peptide, were synthesized and amplified by overlap extension polymerase chain reaction (PCR). They were inserted into vector pIRES2-EGFP. The pIRES2-EGFP-Thanatin plasmid was mixed with liposomes and injected into the testes of male mice by a minimally invasive operation. Six weeks after injection, male mice were mated with normal female mice to produce an F1 generation. PCR and Southern blotting were performed to analyze F1 mice. Among those 52 F1 mice produced, 38.46% were found to be positive for the Thanatin gene by PCR and 30.77% by Southern blotting. Six positive mice were selected from the F1 generation and mated with normal females to an F2 generation, in which 36.36% were found to be positive for the Thanatin gene. Expression of the green fluorescence protein in the transgenic mice was confirmed by fluorescence imaging. These results showed that the Thanatin gene was integrated into the mouse genome. The study provides a useful method for the future development of disease-resistant animals and production of antibacterial peptides through transgenic animals.
转基因动物在医学、生命科学和生物制药方面具有潜在的应用。在这项研究中，我们通过转染雄性精原干细胞开发了一种方便、经济、高效的基因转移方法。合成并扩增了三个编码抗菌肽的 Thanatin 基因片段，通过重叠延伸聚合酶链反应 (PCR)。它们被插入到载体 pIRES2-EGFP 中。将 pIRES2-EGFP-Thanatin 质粒与脂质体混合，通过微创操作注入雄性小鼠的睾丸。注射 6 周后，雄性小鼠与正常雌性小鼠交配产生 F1 代。通过 PCR 和 Southern 印迹分析 F1 小鼠。在产生的 52 只 F1 小鼠中，38.46%通过 PCR 和 30.77%通过 Southern 印迹检测到 Thanatin 基因阳性。从 F1 代中选择 6 只阳性小鼠与正常雌性小鼠交配产生 F2 代，其中 36.36%检测到 Thanatin 基因阳性。通过荧光成像证实了转基因小鼠中绿色荧光蛋白的表达。这些结果表明，Thanatin 基因已整合到小鼠基因组中。该研究为未来通过转基因动物开发抗病动物和生产抗菌肽提供了一种有用的方法。
