Elimination of non-viable 6-thioguanine-sensitive T cells from viable T cells prior to PCR analysis.
The study of T cell clones at the genomic level is expanding our understanding of their role in diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). We have been carrying out genotypic analysis by PCR of hypoxanthine phosphoribosyltransferase (hprt) mutations in these cells. Mutant T cells in the population can be cloned on the basis of their resistance to the cytotoxic drug, 6-thioguanine-(6-TG). A difficulty is that the majority of primary human T cells are capable of only limited growth ex vivo, even in the presence of 'feeder' cells. PCR analysis of DNA from such clones is made difficult by the limited number of viable mutant (drug-resistant) T cells and the large number of dead (drug-sensitive) mononuclear cells and feeder cells. DNA from the 'dead' cells remains sufficiently intact for many weeks in culture and can represent a significant source of background in PCR analysis. Here we describe a method employing hypotonic shock and micrococcal nuclease that reliably eliminates non-viable 6-TG-sensitive cells, allowing the study of the hprt gene in < 200 T cells by PCR.
Grant
D D
DD
Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada.
Goldstein
R
R
Karsh
J
J
Birnboim
H C
HC
D001172:Arthritis, Rheumatoid / Q000097:blood; D002470:Cell Survival / Q000502:physiology; D004252:DNA Mutational Analysis; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D009103:Multiple Sclerosis / Q000097:blood; D016133:Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000737:chemistry* / Q000166:cytology; D013866:Thioguanine / Q000032:analysis
D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine