最新基因工程原理动物的基因操作.ppt

其他的哺乳动物和鸟类 * 对其他脊椎动物进行DNA转移 非洲爪蟾的基因转移 鱼类的基因转移 * 无脊椎动物的DNA转移 果蝇的P元件 P元件是果蝇的转座元件，在特定的条件下，可在果蝇的生殖细胞中高度转移 P-M杂种不育 P元件和侧翼序列克隆到pBR322载体上，用于转座 * 果蝇的早期胚胎发生 * 作为载体系统的P元件衍生物 * 对动物进行遗传操作，可以在动物个体的水平上对基因的表达和功能进行检测，从根本上改变我们对生物学的理解。 转基因动物每个细胞都携带有新的遗传信息，和那些通过对基因组进行特意修饰而筛选到的突变体一样。 完整动物是最终的测定系统，通过此系统可以研究基因功能，特别是像发育这样的复杂的生物过程。 * 对小鼠生殖细胞系进行DNA导入是20世纪取得的最大成就之一，它为其他哺乳动物的转化铺平了道路。 * Several human genetic diseases are caused by mitochondrial mutations, and to model such diseases it is necessary to introduce DNA into the mitochondrial genome of suitable animals. This cannot be achieved using standard transgenesis techniques and a novel method has been developed in which donor cell lines carrying mitochondrial mutations are enucleated and then fused with recipient mouse zygotes. The resulting transmitochondrial mice carry the mutation in every cell and pass the mutation through the female germline, since mitochondria in the sperm only rarely contribute to the embryo. The procedure for producing transmitochondrial mice is shown in Fig. B13.1. This involves five distinct steps. First, a cell line must be produced carrying mitochondria containing the appropriate transgene or mitochondrial mutation. There are many ways to achieve this, but one of the most efficient is fusion between cells depleted in mitochondrial DNA (mtDNA) and synaptosomes from aged mice which often contain mtDNA deletions and other mutations. The resulting cell lines are called cybrids because their cytoplasm is a hybrid of two cell lines. The next step is to remove the nucleus from the cybrid cells, so that they can act as mitochondrial donors without introducing additional nuclei into the egg. This is generally achieved by centrifugation in the presence of cytochalasin B. After washing the enucleated cybrids, zygotes are collected from donor females and a micromanipulator is used to drill a hole through the zona pellucida and place the cybrids in the perivitelline space. After a brief recovery period in culture, embr