CULTURE MEDIA FOR PREIMPLANTATION DEVELOPMENT

植入前发育的培养基

基本信息

批准号：
3552548
负责人：
JOHN D BIGGERS
金额：
$ 43.81万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-09-01 至 1996-08-31
项目状态：
已结题

项目摘要

Two major areas are addressed in this proposal. The first concerns the development of better methods for the culture of mammalian preimplantation embryos, particularly methods which support normal differentiation of the blastocyst in vitro. The inability to successfully culture blastocysts is one of the remaining gaps in our methodology. This methodology is needed to experimentally analyze the early stages of organogenesis in mammals. The main technique to be used will be simplex optimization to find the optimum concentrations of the components of culture solution. The specific aims are: (1) To test whether optimum concentrations of components in a medium for the culture of preimplantation embryos depend on the response parameter, (2) To devise methods for the optimization of media with large numbers of components, such as 20 amino acids, (3) To obtain evidence that preimplantation embryos contain small organic molecules that act as osmolytes, and (4) To examine whether the response to growth factors is dependent on the background provided by other components of the medium. The second area concerns the construction of efficient ways of producing non-human mature oocytes in vitro. Studies of preimplantation development in primates are limited by the small number of embryos that can be produced by conventional means. In biotechnology the production of transgenic primates will be severely hampered by this problem. An alternative approach is to tap the large store of oocytes in the ovary by culture methods to produce large numbers of mature oocytes for fertilization in vitro. The specific aims are: (1) To assemble a database on relevant properties of viable rhesus and tamarin follicles and oocytes that can be recovered from animals of different ages, (2) To construct culture conditions which support the development of granulosa cell enclosed oocytes obtained from rhesus and tamarin antral follicles to the fertilizable mataphase II state, and (3) To construct culture conditions which support the development of oocytes in preantral follicles obtained from rhesus and tamarin ovaries to the fertilizable metaphase II state.

该提案中解决了两个主要领域。第一个涉及开发更好的哺乳动物培养方法植入前胚胎，尤其是支持正常的方法体外胚泡的分化。无法成功培养胚泡是我们的剩余差距之一方法论。需要这种方法来实验分析哺乳动物中器官发生的早期阶段。要使用的主要技术将是单纯形优化，以找到最佳浓度培养解决方案的组成部分。具体目的是：（1）测试培养基中的最佳组成部分是否适用于培养物植入前胚胎的依赖于响应参数，（2）设计方法，以优化大量的媒体成分，例如20个氨基酸，（3）获得证据表明植入前胚胎包含小的有机分子，充当渗透压和（4）检查对生长因素的反应是否是取决于介质其他组件提供的背景。第二个领域涉及产生有效方法的构建非人类成熟的卵母细胞体外。植入前的研究灵长类动物的发展受到少量胚胎的限制可以通过常规手段产生。在生物技术中生产该问题将严重阻碍转基因灵长类动物的束缚。一个替代方法是点击卵巢中的大量卵母细胞通过培养方法，生产大量成熟的卵母细胞体外受精。具体目的是：（1）组装有关可行的恒河类和罗龙蛋白卵泡的相关特性的数据库和可以从不同年龄的动物中回收的卵母细胞，（2）至构建支持颗粒发展的文化条件从恒河猴和罗氨酸腹腔卵泡获得的细胞封闭的卵母细胞到可肥大的Mataphase II状态，（3）建造文化支持卵母细胞在培养前的条件从恒河猴和塔玛蛋白卵巢获得的卵泡到可肥的中期II状态。