Gravel R A, Leung A, Saunders M, Hösli P
Proc Natl Acad Sci U S A. 1979 Dec;76(12):6520-4. doi: 10.1073/pnas.76.12.6520.
A whole-cell microtechnique for the determination of complementation of human metabolic disorders is presented. This procedure permits the isolation of individual multinucleate cells produced by cell fusion for the quantitative evaluation of complementation. Mutant fibroblasts with a deficiency of propionyl-CoA carboxylase activity (EC 6.4.1.3) that had been mapped to complementation groups pcc and bio were used to evaluate the microtechnique. Complementation was monitored by the determination of [14C]propionate incorporation into cellular macromolecules. Single cells or a small number of cells were isolated from plastic film dishes after radioactive incubation by cutting out the portion of the plastic film holding the desired cells. Isotope incorporation was linear in 10-50 unfused cells and in 10-50 fused normal cells containing five or more nuclei. There was also a direct correlation between the nuclear content of cells and the amount of isotope incorporated. Three pcc and two bio mutants were fused in pairwise combinations by means of polyethylene glycol and complementation was determined by isotope incorporation in sets of 50 multinucleate cells, each cell isolated individually. The results agreed with autoradiographic data for both complementing and noncomplementing strains. The method is quantitative and gives severalfold higher sensitivity than current procedures. The method can be applied to the complementation analysis of a wide variety of inherited disorders of intermediary metabolism.
本文介绍了一种用于测定人类代谢紊乱互补性的全细胞微技术。该方法允许分离细胞融合产生的单个多核细胞，用于互补性的定量评估。利用已定位到互补组pcc和bio的丙酰辅酶A羧化酶活性（EC 6.4.1.3）缺乏的突变成纤维细胞来评估该微技术。通过测定[14C]丙酸掺入细胞大分子中来监测互补性。放射性孵育后，通过切下含有所需细胞的塑料薄膜部分，从塑料薄膜培养皿中分离单个细胞或少量细胞。在10 - 50个未融合细胞以及10 - 50个含有五个或更多细胞核的融合正常细胞中，同位素掺入呈线性关系。细胞中的核含量与掺入的同位素量之间也存在直接相关性。通过聚乙二醇将三个pcc突变体和两个bio突变体两两融合，并通过对每组50个单独分离的多核细胞进行同位素掺入来测定互补性。结果与互补和非互补菌株的放射自显影数据一致。该方法是定量的，并且比目前的方法灵敏度高几倍。该方法可应用于多种中间代谢遗传性疾病的互补分析。
