Nelson D L, Weis J H, Przyborski M J, Mulligan R C, Seidman J G, Housman D E
J Mol Appl Genet. 1984;2(6):563-77.
A general method for creating somatic cell hybrids which maintain chromosome segments by selection is described. To extend the technique of metaphase chromosome transfer to regions of the genome which do not carry a selectable marker, a retroviral vector has been employed to introduce the dominant selectable neo gene at many genomic locations in a population of murine cells. We have used such cells as donors in metaphase chromosome transfer experiments in which hamster and monkey cells were used as recipients. Cells acquiring a transferred chromosomal segment containing the neo gene were selected by growth in the presence of the drug G418. Hybridization to cloned interspersed repeat DNA sequences of the mouse was used to estimate the proportion of mouse DNA in each transferent. These experiments indicate that transferents produced in this manner contain 0.01 to 1.0% of the mouse genome. To analyze the organization of the DNA transferred to each recipient, we used Southern transfer hybridization of DNA from each transferent to a cloned mouse interspersed repeated DNA probe which did not cross-hybridize to hamster or monkey DNA. We found that each primary transferent gave a unique pattern of restriction fragments hybridizing to this probe. Secondary transferents from two independent primary transferents were compared by this technique. Each set of secondary transferents exhibited a hybridization pattern which resembled closely that of the primary transferent from which it was derived. However, a number of hybridizing DNA segments present in each primary transferent were absent in some of the secondary transferents. These results are most compatible with the view that an intact segment of the mouse chromosome surrounding the integration site of the retroviral vector can be transferred by the techniques used in this study. We believe this technique will be generally applicable to cells from many species, and will allow the study of chromosome regions previously refractory to analysis by chromosome transfer techniques.
本文描述了一种通过选择来创建维持染色体片段的体细胞杂种的通用方法。为了将中期染色体转移技术扩展到基因组中不携带选择标记的区域，已使用逆转录病毒载体在一群鼠细胞的许多基因组位置引入显性选择neo基因。我们已将此类细胞用作中期染色体转移实验的供体，其中仓鼠和猴细胞用作受体。通过在药物G418存在下生长来选择获得含有neo基因的转移染色体片段的细胞。与小鼠的克隆散布重复DNA序列杂交用于估计每个转移体中小鼠DNA的比例。这些实验表明，以这种方式产生的转移体包含0.01%至1.0%的小鼠基因组。为了分析转移到每个受体的DNA的组织，我们使用每个转移体的DNA与克隆的小鼠散布重复DNA探针进行Southern转移杂交，该探针与仓鼠或猴DNA不交叉杂交。我们发现每个初级转移体都给出了与该探针杂交的独特限制性片段模式。通过该技术比较了来自两个独立初级转移体的次级转移体。每组次级转移体都表现出与它所衍生的初级转移体非常相似的杂交模式。然而，每个初级转移体中存在的一些杂交DNA片段在一些次级转移体中不存在。这些结果与以下观点最相符，即围绕逆转录病毒载体整合位点的小鼠染色体完整片段可以通过本研究中使用的技术转移。我们相信该技术将普遍适用于许多物种的细胞，并将允许对以前难以通过染色体转移技术进行分析的染色体区域进行研究。
