IN-CELL PCR FROM MESSENGER-RNA - AMPLIFYING AND LINKING THE REARRANGED IMMUNOGLOBULIN HEAVY AND LIGHT CHAIN V-GENES WITHIN SINGLE CELLS
IN-CELL PCR FROM MESSENGER-RNA - AMPLIFYING AND LINKING THE REARRANGED IMMUNOGLOBULIN HEAVY AND LIGHT CHAIN V-GENES WITHIN SINGLE CELLS
复制标题
DOI：
10.1093/nar/20.15.3831
复制
发表时间：
1992-08-11
影响因子：
14.9
通讯作者：
WINTER, G
中科院分区：
文献类型：
--
作者：
EMBLETON, MJ;GOROCHOV, G;WINTER, G
我们描述了一个过程中的单细胞内的mRNA的识别，证明与免疫球蛋白（IG）可变区（V）基因的两个小鼠杂交瘤细胞系和bcr-abl融合基因的人K562髓性白血病线。将细胞固定并透化，将mRNA逆转录为cDNA，并通过聚合酶链反应（PCR）扩增cDNA。使用荧光PCR引物后，扩增的DNA可以在细胞内检测，如通过共聚焦荧光显微镜和流式细胞仪所示。此外，扩增的IG VH和VL DNA可以使用合适的PCR引物在同一细胞内组装。我们没有检测到细胞间扩增DNA的交叉污染：从经细胞内PCR和组装处理的两种杂交瘤细胞系（B1-8和NQ 10/12.5）的混合物中分离的DNA通过克隆显示对应于亲本杂交瘤的VH和VL基因的组合。我们预见了通过PCR的细胞内组装的多种应用，特别是用于分析细胞群体中重排的IG或T细胞受体（TCR）V基因的链的组合，以及从免疫B淋巴细胞的V基因构建人抗体。
We describe a process for the identification of mRNAs within single cells, as demonstrated with the immunoglobulin (Ig) variable region (V) genes of two mouse hybridoma cell lines and the bcr-abl fusion gene of the human K562 myeloid leukaemia line. The cells were fixed and permeabilised, the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction (PCR). After using fluorescent PCR primers, the amplified DNA could be detected within the cells as demonstrated by confocal fluorescence microscopy and flow cytometry. Furthermore the amplified Ig VH and VL DNA could be assembled within the same cell using suitable PCR primers. We detected no cross-contamination of amplified DNA between cells: the DNA isolated from mixtures of two hybridoma cell lines (B1-8 and NQ10/12.5) treated to in-cell PCR and assembly, was shown by cloning to correspond to the combinations of VH and VL genes of the parent hybridomas. We forsee diverse applications of in-cell assembly by PCR, especially for the analysis of the combinations of chains of rearranged Ig or T cell receptor (TCR) V-genes in a population of cells, and the construction of human antibodies from the V-genes of immune B-lymphocytes.