A "real time" PCR assay to detect transplanted human liver cells in RAG-1-/- mice.
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 alpha-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 (C(T)] 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.
Funkhouser
A W
AW
University of Chicago, Department of Pediatrics, University of Chicago Children's Hospital, IL 60637, USA. afunkhou@peds.bsd.uchicago.edu
Vahed
S
S
Soriano
H E
HE
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D022781:Hepatocytes / Q000166:cytology* / Q000637:transplantation*; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014183:Transplantation, Heterologous
D017931:DNA Primers; D018398:Homeodomain Proteins; C064658:RAG-1 protein; D004247:DNA