Establishment of a quantitative pcr analysis to assess engraftment of normal stem cells in w/wv mice

F. Kajumo, Ryuji Tanosaki, A. R. Migliaccio

Research output: Contribution to journalArticle

Abstract

Reconstitution models in which wild type stem cells are transplanted in stem eel! defective hosts, such as W/W% mice, are useful tools to understand hematopoietic engraftmenl into nonablated microenvironments The wide use of these models is hampered by the paucity of markers available to document reconstitution in the absence of sex- or Ly-5-antigen mismatched transplants. The W mutation is a nucleotide substitution (C to T) at position 2006 of the first kinase domain, near the 3' end of exon 13, of the c-Kit gene and generates a unique Nsi I restriction site. The existence of this restriction site, not present in the wild type gene, was used by us to design a quantitative PCR method which measures engraftment of normal cells in W/WV mice. Amplification primers flanking the Wv mutation were designed in exon 13 (5 primers) and in the 13 exon-intron junction (3' primers). Genomic DNA amplification with this pair of primers generates a major 105 bp product which specifically hybridizes with a probe spanning the c-Kit region internal to the primers. As predicted, products amplified from wild type genomic DNA are not cut by Nsi 1. On the other hand, since the W mutation does not involve the first kinase domain, products amplified from W/W genomic DNA generate, after Nsi 1 digestion, two bands: a SOSbp and a 85 bp band (in a 5;2 ratio) which correspond to the W and the W1" ailele. respectively. Since a) the same primers are used to amplify both bands, b)theNsi I digestion reaction reaches very quickly ( I hour) high levels (>98% of product digested) of saturation, and c) the autoradiographic detection system is linear in the range of products to be analyzed, DNA extracted from a given mixture of wild type and W/V/1 cells will generate amplified products in a ratio which will depend solely on the ratio between wild type and W/WV cells in the mixture. In preliminary experiments, the ratio of Wv to WW was measured using genomic DNA extracted from progressive dilutions of wild type cells into W/WV cells (0 -100% of W/WV). The corresponding regression curve was linear (in the range of 5 - 85% of wild type cells) with no statistically significant difference detected in more than 10 separate individual regression curves measured. This amplification technique has the potential to quantify the percent of engraftmcnt of normal stem cells in W/WV mice even in ihe case where very few cells are available for analysis.

Original languageEnglish
Pages (from-to)1029
Number of pages1
JournalExperimental Hematology
Volume24
Issue number9
Publication statusPublished - 1996
Externally publishedYes

Fingerprint

Stem Cells
DNA
Exons
Mutation
Digestion
Ly Antigens
Phosphotransferases
Eels
Introns
Genes
Nucleotides
Transplants
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

Establishment of a quantitative pcr analysis to assess engraftment of normal stem cells in w/wv mice. / Kajumo, F.; Tanosaki, Ryuji; Migliaccio, A. R.

In: Experimental Hematology, Vol. 24, No. 9, 1996, p. 1029.

Research output: Contribution to journalArticle

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title = "Establishment of a quantitative pcr analysis to assess engraftment of normal stem cells in w/wv mice",
abstract = "Reconstitution models in which wild type stem cells are transplanted in stem eel! defective hosts, such as W/W{\%} mice, are useful tools to understand hematopoietic engraftmenl into nonablated microenvironments The wide use of these models is hampered by the paucity of markers available to document reconstitution in the absence of sex- or Ly-5-antigen mismatched transplants. The W mutation is a nucleotide substitution (C to T) at position 2006 of the first kinase domain, near the 3' end of exon 13, of the c-Kit gene and generates a unique Nsi I restriction site. The existence of this restriction site, not present in the wild type gene, was used by us to design a quantitative PCR method which measures engraftment of normal cells in W/WV mice. Amplification primers flanking the Wv mutation were designed in exon 13 (5 primers) and in the 13 exon-intron junction (3' primers). Genomic DNA amplification with this pair of primers generates a major 105 bp product which specifically hybridizes with a probe spanning the c-Kit region internal to the primers. As predicted, products amplified from wild type genomic DNA are not cut by Nsi 1. On the other hand, since the W mutation does not involve the first kinase domain, products amplified from W/W genomic DNA generate, after Nsi 1 digestion, two bands: a SOSbp and a 85 bp band (in a 5;2 ratio) which correspond to the W and the W1{"} ailele. respectively. Since a) the same primers are used to amplify both bands, b)theNsi I digestion reaction reaches very quickly ( I hour) high levels (>98{\%} of product digested) of saturation, and c) the autoradiographic detection system is linear in the range of products to be analyzed, DNA extracted from a given mixture of wild type and W/V/1 cells will generate amplified products in a ratio which will depend solely on the ratio between wild type and W/WV cells in the mixture. In preliminary experiments, the ratio of Wv to WW was measured using genomic DNA extracted from progressive dilutions of wild type cells into W/WV cells (0 -100{\%} of W/WV). The corresponding regression curve was linear (in the range of 5 - 85{\%} of wild type cells) with no statistically significant difference detected in more than 10 separate individual regression curves measured. This amplification technique has the potential to quantify the percent of engraftmcnt of normal stem cells in W/WV mice even in ihe case where very few cells are available for analysis.",
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