Chemiluminescent restriction fragment length polymorphism analysis (DNA fingerprinting) to identify keratinocytes from two donors in co-culture.
The transplantation of allogeneic cells such as pancreatic islet cells, bone marrow, and keratinocytes for skin replacement is in growing use clinically. Present techniques which are used to distinguish transplanted allogeneic cells from phenotypically identical host cells in chimeric tissue have serious limitations. This study assesses the feasibility of using chemiluminescent restriction fragment length polymorphism (RFLP) analysis to distinguish between keratinocyte cell lines from 2 different donors grown in co-culture. We evaluated the sensitivity of this technique in tissue cultured cells in order to define its usefulness in clinical situations. RFLP analysis readily detects heterologous DNA comprising only 10% of a 5 micrograms sample. This was a detection threshold of 500 ng of high molecular weight DNA. With standard extraction methods, this represents the DNA obtained from 4000 cells. This new chemiluminescent technique is as sensitive as older techniques which employ radioactive probes, but avoids the hazards of handling radioactive material. Using this RFLP analysis, the presence of transplanted allogeneic cells can be readily detected in small biopsy specimens. This technique has wide potential application in allogeneic cellular transplantation investigations.
Young
D M
DM
Section of Plastic and Reconstructive Surgery, Yale University School of Medicine, New Haven, Connecticut.
Summers
W C
WC
Cuono
C B
CB
D002460:Cell Line; D017690:Cell Transplantation / Q000473:pathology / Q000502:physiology; D016172:DNA Fingerprinting / Q000379:methods* / Q000706:statistics & numerical data; D004274:DNA, Recombinant / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry* / Q000166:cytology / Q000637:transplantation*; D008163:Luminescent Measurements; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D016038:Skin Transplantation / Q000473:pathology / Q000502:physiology; D014019:Tissue Donors
D004274:DNA, Recombinant