Wixler V, Klarmann B, Begemann U, Gahn G, Lorenz R, Hempel K
Institut für Medizinische Strahlenkunde, Universität Würzburg, Germany.
J Immunol Methods. 1994 May 2;171(1):121-30. doi: 10.1016/0022-1759(94)90235-6.
A method is described for determining the frequency of cells with a mutation in an autosomal gene coding for a membrane protein. Using a monoclonal antibody to H-2Kk surface antigen and magnetic cell separation (MACS) more than 10(4)-fold enrichment of the H-2Kk negative population was achieved, as tested with artificial mixtures containing a known number of antigen-negative cells. After a second magnetic sorting mutant frequencies as low as 10(-6) could be measured. The number of clonogenic mutants was evaluated by limiting dilution cloning and verification of the mutant phenotype by FACScan (flow cytometry) analysis in a representative number of clones. The spontaneous frequency of H-2Kk deficient mutants was 0.80 x 10(-6), and this increased after irradiation with 6 Gy X rays to 3.38 x 10(-6) within the next 8 weeks. About 50 mutant clones were screened for the presence of other class 1 antigens on the cell surface by FACScan analysis. All mutants continued to express other class 1 antigens.
本文描述了一种用于确定编码膜蛋白的常染色体基因突变细胞频率的方法。使用针对H-2Kk表面抗原的单克隆抗体和磁性细胞分离技术（MACS），经含有已知数量抗原阴性细胞的人工混合物检测，实现了H-2Kk阴性群体超过10^4倍的富集。经过第二次磁性分选，可检测到低至10^-6的突变频率。通过有限稀释克隆评估克隆形成突变体的数量，并通过流式细胞仪（FACScan）分析对代表性数量的克隆进行突变体表型验证。H-2Kk缺陷突变体的自发频率为0.80×10^-6，在接受6 Gy X射线照射后的接下来8周内，该频率增加至3.38×10^-6。通过流式细胞仪分析筛选了约50个突变克隆，以检测细胞表面是否存在其他I类抗原。所有突变体均继续表达其他I类抗原。
