Development of C57BL/6 ES cell technology for high thoughput use.

描述：(由申请人提供)C57BL/6(B6)近交系小鼠是小鼠遗传学家的首选品系，然而，到目前为止，高通量的基因靶向/捕获只有129个品系的胚胎干细胞(ES)。129个ES细胞系即使在没有小鼠胚胎成纤维细胞(MEF)饲养层的情况下，在补充骨形态发生蛋白(BMP)和白血病抑制因子(LIF)的情况下，也能在广泛的条件下保持自我更新。相反，B6 ES细胞的自我更新依赖于MEF的存在。在初步数据中，我们已经表明129条件培养液显著改善了B6 ES细胞的生长特性。我们将利用这一发现减少B6细胞对饲养层细胞的依赖，如果可能的话，使其完全独立于饲养层细胞。重要的是，我们的方法没有预先假设129和B6细胞系之间生长特性差异的生物学性质。我们将通过五个具体目标来实现我们的目标：在目标1中，我们将评估18个雄性B6 ES细胞系和6个雄性白化B6系的细胞遗传学稳定性、倍增时间和生殖系传播。在目标2中，我们将使用已公布的添加剂以及新的细胞因子和/或生长因子来优化B6 ES细胞系的无饲养层生长条件。这些新的添加剂将通过129和B6 ES细胞系的表达谱来鉴定，因为来自129而不是B6 ES细胞的条件培养液促进了B6 ES细胞的生长和饲养层独立性。在目标3中，我们将利用B6 ES细胞无饲养层生长的优化条件，通过靶向每个细胞系中的5个基因来最终评估基因打靶能力。在目标4中，我们将开发一种新的具有成本效益的高通量测试，该测试可以快速预测ES系的生殖系传播，并具有高度的准确性。我们将利用表达谱来识别96个基因的子集，这些基因的表达水平最能指示自我更新和生殖系传播。然后，这些基因将被用来开发一种基于qPCR的筛查，以预测比传统细胞遗传学更快速、更准确、更便宜的生殖系传播。在目标5中，我们将使用袋子重组工程和我们的优化生长条件来获得KOMP指导委员会选择的50个小鼠基因的零突变。总之，响应RFA-DA-06-009，我们将开发高通量的B6 ES细胞靶向基因，并推导出一种新的分子检测方法，以经济高效和快速地监测B6 ES细胞系及其靶向衍生物的生殖系能力。

DESCRIPTION: (provided by applicant) The C57BL/6 (B6) inbred strain of mice is the preferred strain of mouse geneticists, however, high throughput gene targeting/trapping has been possible to date only with 129 strain embryonic stem (ES) cells. 129 ES lines maintain self-renewal under a wide range of conditions even in absence of mouse embryonic fibroblast (MEF) feeders if supplied with Bone Morphogenetic Protein (BMP) and Leukemia Inhibitory Factor (LIF). In contrast, self-renewal of B6 ES cells is dependent on the presence of MEFs. In preliminary data, we have shown that 129-conditioned medium dramatically improves the growth characteristics of B6 ES cells. We will exploit this discovery to make B6 cells less dependent on feeder cells and, if possible completely feeder-cell independent. Importantly, our approaches make no prior assumption of the biological nature of the difference in growth characteristics between 129 and B6 cell lines. We will pursue our goals through five specific aims: In Aim 1, we will evaluate 18 male B6 ES-cell lines and 6 male albino B6 lines for cytogenetic stability, doubling time, and germ line transmission. In Aim 2, we will optimize the growth conditions for feeder-less growth of B6 ES-cell lines using both published additives as well as novel cytokines and/or growth factors. These new additives will be identified through expression profiling of 129 vs. the B6 ES lines, because conditioned medium from 129, but not B6 ES cells promotes growth and feeder-independence of B6 ES-cells. In Aim 3 we will utilize the optimized conditions of feeder-less growth of B6 ES-cells for a final assessment of gene targeting capability by targeting five genes in each cell line. In Aim 4, we will develop a novel cost-effective high throughput test that rapidly predicts germ line transmission of ES lines and with a high degree of accuracy. We will exploit expression profiling to identify a subset of 96 genes whose expression level is most indicative of self-renewal and germ line transmission. These genes will then be used to develop a qPCR-based screen to predict germ line transmission that is more rapid, accurate and less expensive than conventional cytogenetics. In Aim 5, we will use BAG recombineering and our optimize growth conditions to derive null mutations in 50 mouse genes as selected by the KOMP steering committee. In summary, in reponse to RFA-DA-06-009 we will develop high throughput gene targeting with B6 ES-cells and derive a novel molecular assay to cost-effectively and rapidly monitor B6 ES cell lines and their targeted derivatives for germ-line competence.