In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells.
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.
Embleton
M J
MJ
MRC Centre for Protein Engineering, Cambridge, UK.
Gorochov
G
G
Jones
P T
PT
Winter
G
G
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005434:Flow Cytometry; D015321:Gene Rearrangement / Q000235:genetics; D005803:Genes, Immunoglobulin; D006801:Humans; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics
D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins