During the tumor progression, micrometastases at their earliest stages have been difficult to analyze qualitatively or quantitatively because of lacking suitable sensitive markers to discriminate small numbers of tumor cells from cell populations of normal tissue. To identify the distribution of micrometastases and tracing of F9 teratocarcinoma stem cells in viva, we used chick embryos and the polymerase chain reaction(PCR) as simple, sensitive, and quantitative assay system. Hence, F9 cells which metastasized in the liver of chick embryo were detected by PCR using specific primers for mouse B-globin gene. In addition, another assay method was developed for detecting micrometastases of F9 cells with E.coli lacZ gene as a genetical and histochemical marker. After the subsequent transfection of the lacZ gene into F9 cells, an intensely blue-staining clone harboring lacZ gene was isolated by using chromogenic substrate, 5-bromo-4-chloro-3-indoyl-B-D-galacto-pyranoside (X-Gal). The lacZ-bearing F9 cells at primary tumor sites as well as at secondary organs can be stained intensely blue spots and easily distinguished from the host tissue cells after hours, days, or weeks postinjection.
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