To Detect Cell Cross-Contamination By Using Molecular Genetic Markers

Cell Lines are widely used in laboratory-based research and the manufacture of biological product, while cross-contamination of cell lines is a longstanding problem and a repeated and frequent cause of scientific misrepresentation. Estimates fromsome studies indicate that up to 17～36% of cell lines are of a different origin or species to that claimed.In our research, we planned to find an easy method to identify the cell lines as KMB-17, Hep-2, Vero, 2BS and MRC-5 by using molecular genetic markers.We chose 11 hypervariable STR loci as D18S1364, D13S325, D11S2368, D8S1179, D7S820, D7S3048, D6S1043, D5S818, D2S1772, D2S1338, FGA, CSF1PO and designed the primers ourselves based on the special requirement of our assay. We have done serials of PCR experiments by using the genome DNA extracted from the 5 cell lines and the 11 pairs of primers. The product of the experiments was analyzed by using polyacrylamide gel electrophoresis and agarose gel electrophoresis. Finally, we screened 3 pairs of primers of the loci as D18S1364, D6S1043 and D5S818 to identify the 5 cell lines. We made the standard patterns by using AGE and defined a standard way to identify the 5 cell lines. To identify Cell Cross Contamination (CCC) of the KMB-17 cell line, we find that using the primer of loci D18S1364 to do the PCR assay and using AGE to analyze the product can identify the CCC from Vero cell line, and by using the primer of loci D6S1043 can identify the CCC from Hep-2, 2BS and MRC-5 cell lines. We made the standard patterns by using AGE and defined a standard way to identify the CCC of KMB-17 cell line. Also, to guarantee availability and reliability of the ways, we studied the sensitivity of the method and did a double blind assay to identify given cell cultures. The method we defined of identifying the 5 cell lines is low cost, easy to use and reproducible, maybe it can provide an opportunity for mass screening of CCC and detection of cross-contaminated cultures in every laboratory dealing with cell cultures.