We describe a quantitative PCR based on the unique Nsi 1 restriction site generated by the W(v) mutation (C to T substitution at position 2007 of c-kit) to quantify the percentage of engraftment of wild-type stem cells in W/W(v) mice. Primers flanking this mutation specifically amplify c-kit sequences from either genomic DNA or cDNA. After Nsi 1 digestion, wild-type products were identified by electrophoresis as a single 104 bp (genomic DNA) or 118 bp (cDNA) band whereas W/W(v) products migrated as two bands: the 104- 118 bp band (corresponding to the W allele) and the 85 bp band (corresponding to the W(v) allele). The relationship between the ratio/W(v)/total c-kit product versus the ratio W/W(v) cells/total cells obtained in PCR amplification of artificial cell mixtures was expressed by a statistically significant linear regression. This indicated that the W(v)/total c-kit ratio depends mainly upon the ratio between the two cell types in the preparation to be analysed. Therefore it is possible to determine the percentage of donor cells in tissues of W/W(v) transplanted with normal stem cells by comparing the amplification ratio obtained with DNA extracted from the tissues with the ratio obtained in standard calibration curves. As applications of the technique, we determined the percentage of donor-derived cells in mononuclear blood fractions, in preparations of splenic B. T and myelomonocytic cells and in GM colonies cultured from the marrow of W/W(v) mice transplanted with wild-type stem cells.
|ジャーナル||British Journal of Haematology|
|出版ステータス||Published - 1997|
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