A “Real Time” PCR Assay to Detect Transplanted Human Liver Cells in RAG-1-/- Mice

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Abstract:

Xenotransplantation of human liver cells is an expanding field in need of new and precise quantitative techniques. “Real time” PCR is a sensitive and accurate method of quantifying picogram quantities of DNA. We used “real time” PCR with primers complementary to the human α-1-antitrypsin gene to assess the efficiency of engraftment of human liver cells transplanted into immunotolerant RAG-1-/- mice. Standard curves were created by mixing known proportions of human and mouse cells. There was a linear relationship between the PCR cycle at which DNA was amplified [threshold cycle (CT)] and the percent human cells (linear regression, p < 0.00009). Results were reliable, with a maximum 1.27-fold variation in the slopes of repeated standard curves. Linearity was maintained from 100% to as low as 0.01%. Therefore, 1 in 10,000 mouse cells could be detected in a 100 ng DNA sample. We measured the percent engraftment of human liver cells transplanted into the spleen of RAG-1-/- mice. By “real time” PCR assay, 0.23% human cells could be detected at 1 day after human cell transplantation. These results show that “real time” PCR assay is highly sensitive, reproducible, and accurate for detecting human cells in xenotransplanted mice.

Keywords: Key words: Hepatocyte transplantation; Xenotransplantation; “Real; time” RCR; Human liver cells; RAG-1-/- mice

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/000000001783986990

Affiliations: 1: *University of Chicago, Department of Pediatrics, University of Chicago Children's Hospital, Chicago, IL 60637 2: †Northwestern University, Department of Pediatrics, Children's Memorial Hospital, Chicago, IL 60614

Publication date: February 1, 2001

More about this publication?
  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.
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