Manzini Stefano, Vargiolu Alessia, Stehle Isa M, Bacci Maria Laura, Cerrito Maria Grazia, Giovannoni Roberto, Zannoni Augusta, Bianco Maria Rosaria, Forni Monica, Donini Pierluigi, Papa Michele, Lipps Hans J, Lavitrano Marialuisa
Department of Surgical Sciences, University of Milano-Bicocca, 20052 Milan,Italy.
Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17672-7. doi: 10.1073/pnas.0604938103. Epub 2006 Nov 13.
Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.
细胞和动物的基因改造是生物技术和生物医学中一项非常宝贵的工具。目前，整合载体被用于此目的。然而，这些载体可能导致插入诱变和转基因表达的变化，并可能发生沉默。基于支架/基质附着区域的载体是非病毒表达系统，可在哺乳动物细胞中自主复制，从而使对高等真核细胞和生物体进行安全可靠的基因改造成为可能。在本研究中，利用基于支架/基质附着区域的载体pEPI通过精子介导的基因转移方法将其导入胚胎，从而产生了基因改造的猪胎儿。在分析的不同组织中，18个胎儿中的12个检测到了pEPI载体，并且显示其作为附加体保留。pEPI载体编码的报告基因在12个基因改造胎儿中的9个中表达。在阳性动物中，所有分析的组织均表达报告基因；此外，在这些组织中，阳性细胞平均占79%。高比例的表达EGFP的细胞以及不存在嵌合体现象对于生物技术和生物医学应用具有重要意义。这些结果是动物转基因研究向前迈出的重要一步，可为未来生殖系基因治疗的发展提供基础。
