INSITU HYBRIDIZATION OF IMMUNOGLOBULIN-SPECIFIC RNA IN SINGLE CELLS OF THE LYMPHOCYTE-B LINEAGE WITH RADIOLABELED DNA PROBES
INSITU HYBRIDIZATION OF IMMUNOGLOBULIN-SPECIFIC RNA IN SINGLE CELLS OF THE LYMPHOCYTE-B LINEAGE WITH RADIOLABELED DNA PROBES
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DOI：
10.1002/j.1460-2075.1986.tb04181.x
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发表时间：
1986-01-01
期刊：
影响因子：
11.4
通讯作者：
BERGER, CN
中科院分区：
文献类型：
--
作者：
BERGER, CN
已经开发出一种原位杂交方法，该方法使用放射性标记的免疫球蛋白基因特异性单链 DNA 探针检测单个鼠 B 淋巴细胞中的免疫球蛋白特异性 mRNA 转录物。该方法已应用于骨髓瘤和杂交瘤细胞，以及处于不同突变阶段的 B 淋巴细胞（从小型静息 B 细胞到分泌 Ig 的浆细胞）。该过程的关键步骤是用链霉蛋白酶处理细胞。已发现各种细胞类型对这种治疗的敏感性不同。杂交中使用了不同长度（即 26 至 1000 bp）的单链 DNA 探针。细胞上银颗粒的数量随着探针的长度及其在杂交反应中的浓度成比例地增加。在用脂多糖刺激鼠B细胞后，显示了单细胞中μ-重链特异性RNA分子增加的动力学以及“转换的”、γ-重链表达细胞的出现。
A method for in situ hybridization has been developed which detects immunoglobulin-specific mRNA transcripts in single murine B lymphocytes with radiolabelled, immunoglobulin gene-specific single-stranded DNA probes. The method has been applied to myeloma and hybridoma cells and to B lymphocytes at various stages of their muturation from small, resting B cells to Ig-secreting plasma cells. A critical step in the procedure is the treatment of the cells with pronase. The various cell types have been found to be differently susceptible to this treatment. Single-stranded DNA probes of different lengths, i.e., between 26 and 1000 bp, have been employed in the hybridization. The number of silver grains over a cell increase proportionally with the length of the probe and with its concentration in the hybridization reaction. The kinetics of the increase of .mu.-heavy chain-specific RNA molecules in single cells and the appearance of ''switched'', .gamma.-heavy chain-expressing cells are shown after stimulation of murine B cells with lipopolysaccharide.