Kliot-Fields T, Finney D A, Wiseman A
Somatic Cell Genet. 1983 May;9(3):375-89. doi: 10.1007/BF01539145.
A general method to isolate and purify substantial numbers of viable cybrids from cultured mammalian cells immediately following cytoplast-cell fusion is described. This method uses cytoplasts whose mitochondria are selectively stained in vivo by the cationic fluorescent rhodamine dye, rhodamine 123. Large numbers of highly purified, rhodamine-stained cytoplasts are fused to appropriate recipient cell lines and then the fusion mixture is sorted based on forward angle scatter and fluorescence parameters. Plating the positively sorted population in culture for as short as 12 h eliminates contaminating cytoplasts which, lacking a nucleus, are unable to adhere or survive. The resultant population, based on an analysis of genetic markers, is 75-100% cybrids, an enrichment of 1000- to 10,000-fold over the initial fusion mixture. Cybrids purified by cell sorting may be useful for detailed molecular studies of mitochondrial DNA gene expression and in the specific induction of new mitochondrial DNA mutants.
本文描述了一种在细胞质体 - 细胞融合后立即从培养的哺乳动物细胞中分离和纯化大量有活力的胞质杂种的通用方法。该方法使用的细胞质体，其线粒体在体内被阳离子荧光罗丹明染料罗丹明123选择性染色。大量高度纯化的、罗丹明染色的细胞质体与合适的受体细胞系融合，然后根据前向角散射和荧光参数对融合混合物进行分选。将正向分选的群体在培养中培养短短12小时，就可消除缺乏细胞核而无法附着或存活的污染性细胞质体。基于遗传标记分析，所得群体中胞质杂种占75% - 100%，比初始融合混合物富集了1000至10000倍。通过细胞分选纯化的胞质杂种可能有助于线粒体DNA基因表达的详细分子研究以及新线粒体DNA突变体的特异性诱导。
