A whole-genome radiation hybrid panel for bovine gene mapping
A whole-genome radiation hybrid panel for bovine gene mapping
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DOI：
10.1007/s003359900593
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发表时间：
1997-11-01
期刊：
影响因子：
2.5
通讯作者：
Yang, YP
中科院分区：
文献类型：
--
作者：
Womack, JE;Johnson, JS;Yang, YP
虽然辐射和融合基因转移的想法在20多年前就发表了(Goss和Harris 1975)，并在10年后用于孤立的定位实验(Willard等人)。1985年)，直到Cox等人(1990)为构建人类Chr 21的高分辨率图谱而复活，它才被系统地用作人类基因图谱工具。利用辐照的二倍体人类细胞而不是单染色体杂交细胞的全基因组辐射杂交(WG-RH)图谱由Walter和他的同事们(1994)复兴，并随后成为高分辨率人类基因组图谱的主要工具。正如McCarthy(1996)所回顾的，人类辐射杂交体(RH)的面板已经被有效地用于整合连锁和物理图谱，锚定或排序大的插入重叠群，以及构建表达序列标签(EST)图谱，该图谱已经包含超过12,000个条目并且正在迅速增长。RH图谱还没有被有效地用于构建其他哺乳动物物种的图谱，除了小鼠(Schmitt等人。1996年)。然而，由于将链接和物理地图相结合的技术的潜力，这种情况注定会改变。它是比较基因定位的一个特别强大的工具，因为可以为表达的基因建立染色体顺序，这些基因通常在物种之间保守，但由于缺乏等位基因变异而往往不愿进行连锁定位。用于构建该面板的牛供体细胞是从安格斯公牛JEW38建立的正常二倍体成纤维细胞培养物。几乎融合的烧瓶被胰酶消化并悬浮在Gibco DMEM中，不添加任何补充剂。大约107个细胞在室温下，在T75瓶中加入10ml悬浮液进行照射。钴60源提供185拉德/分钟，总剂量为5000拉德。用胰酶去除贴壁细胞，悬浮于无钙/镁的Hank‘s平衡盐溶液(HBSS)中，pH 8.0,106cell/ml，取出0.5ml(5×105个细胞)作为对照，4.5ml用于融合。受体中国仓鼠TK-成纤维细胞系A23由David Cox(斯坦福大学医学院)提供。将这些细胞以106/ml的浓度悬浮在HBSS中，取出0.7ml作为对照，9ml用于融合。将JEW38(4.5×106个细胞)和A23(9×106个细胞)充分混合，制粒，再悬浮。缓慢加入0.5毫升的聚乙二醇(勃林格曼海姆聚乙二醇1500在50%无菌溶液中)，并保持混合。2分钟后，加入10mlHBSS，pH 8.0，轻轻搅拌。细胞制粒，再悬浮于5mlHBSS，pH 8.0，37℃，15min，每一对照系按相同的过程暴露于聚乙二醇组。将融合悬液混合到Gibco DMEM中，加入10%FBS+HAT(Sigma)+5×10-7M哇巴因，总体积为90毫升。将10毫升的混合物分配到9个100 mm板(约1.5×105个细胞/皿)中。对照组在相同的溶液中混合，并进行相同的电镀。所有JEW38对照细胞在37~5%CO2中孵育，第7天全部死亡，而一个A23菌落明显为TK回复突变体。
Although the idea of irradiation and fusion gene transfer was published more than 20 years ago (Goss and Harris 1975) and employed in an isolated mapping experiment ten years later (Willard et al. 1985), it was not systematically employed as a human gene mapping tool until resurrected by Cox and associates (1990) for constructing a high-resolution map of human Chr 21. Wholegenome radiation hybrid (WG-RH) mapping utilizing irradiated diploid human cells rather than single chromosome hybrids was revived by Walter and colleagues (1994) and has subsequently become a major tool for high-resolution mapping of the human genome. As reviewed by McCarthy (1996), panels of human radiation hybrids (RH) have been effectively utilized to integrate linkage and physical maps, to anchor or order large insert contigs, and to construct expressed sequence tag (EST) maps that already contain more than 12,000 entries and are growing rapidly. RH mapping has not been effectively utilized in constructing maps of other mammalian species, with the exception of the mouse (Schmitt et al. 1996). This situation is destined to change, however, owing to the potential of the technique for integrating linkage and physical maps. It is an especially powerful tool for comparative gene mapping, since chromosomal order can be established for expressed genes that are usually conserved between species but often recalcitrant to linkage mapping for lack of allelic variation. The bovine donor celll used in constructing this panel were a normal diploid fibroblast culture established from an Angus bull, JEW38. Nearly confluent flasks were trypsinized and suspended in Gibco DMEM without supplements. Approximately 10 7 cells were irradiated at room temperature in 10 ml suspension medium in a T75 flask. A cobalt 60 source delivered 185 rad/min for a total dose of 5000 rad. Attached cells were removed with trypsin, and all cells were suspended in ca/mg-free Hank's balanced salt solution (HBSS), pH 8.0, at 106 cells/ml. One-half ml (5 x 105 cells) was removed as control and 4.5 ml used for fusion. The recipient Chinese hamster TK-fibroblast line A23 was kindly provided by David Cox (Stanford University School of Medicine). These cells were also suspended in HBSS at 106/ml, and 0.7 ml of this suspension was removed as control and 9 ml used for fusion. JEW38 (4.5 x 10 6 cells) and A23 (9 x 10 6 cells) were thoroughly mixed, pelleted, and resuspended. One-half ml PEG (Boehringer Mannheim polyethylene glycol 1500 in 50% sterile solution) was slowly added with constant mixing. After 2 min, 10 ml HBSS, pH 8.0, was also added slowly with gentle mixing. Cells were pelleted, then resuspended in 5 ml HBSS, pH 8.0, for 15 min at 37~ Each control line was exposed to PEG by the same process. The fusion suspension was mixed into Gibco DMEM to 10% FBS plus HAT (Sigma) plus 5 x 10-7 M ouabain to a total volume of 90 ml. Ten ml of this mixture was dispensed to each of nine 100-mm plates (approximately 1.5 x 105 cells/plate). Controls were mixed in the same solution and plated identically. All were incubated at 37~ in 5% CO 2. All JEW38 control cells were dead on day 7, while one A23 colony, apparently a TK revertant, sur-