Abstract

Biomedical research, especially in the area of gene therapy, often requires the development of animal models in order to test the efficiency and safety of experimental methods ultimately intended for use on human subjects. One such model may be created by implanting human stem cells into animals to determine the ability of the human cell line to proliferate. In many cases in which researchers have developed an animal model in this fashion, proliferation of implanted cells must be quantified. To do this, researchers have developed assays to monitor cell proliferation. One method involves tracking human cells by testing for the presence of specific genetic sequences. Current methods of gene-based detection use a human-specific gene with only one copy in each cell's genome. This constrains the system's ability to detect human cells at low concentrations. The purpose of this project was to develop an assay with greater detection sensitivity for human cells by exploiting a multi-locus genetic sequence specific to human cells. This system used a technique called real-time quantitative polymerase chain reaction to amplify the target sequences and signal their presence with a fluorescent probe. With this system, human DNA was detected at concentrations 10 to 100 times lower than the lowest detectable concentrations using the current, single locus method. Though this system successfully met the goal of increasing the sensitivity of human DNA detection, unidentified contamination produced false positives in non-human samples.