Authentication of African green monkey cell lines using human short tandem repeat markers

Abstract

Background: Tools for authenticating cell lines are critical for quality control in cell-based biological experiments. Currently there are methods to authenticate human cell lines using short tandem repeat (STR) markers based on the technology and procedures successfully used in the forensic community for human identification, but there are no STR based methods for authenticating nonhuman cell lines to date. There is significant homology between the human and vervet monkey genome and we utilized these similarities to design the first multiplex assay based on human STR markers for vervet cell line identification.

Results: The following STR markers were incorporated into the vervet multiplex PCR assay: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389. The eight markers were successful in uniquely identifying sixty-two vervet monkey DNA samples and confirmed that Vero76 cells and COS-7 cells were derived from Vero and CV-1 cells, respectively. The multiplex assay shows specificity for vervet DNA within the determined allele range for vervet monkeys; however, the primers will also amplify human DNA for each marker resulting in amplicons outside the vervet allele range in several of the loci. The STR markers showed genetic stability in over sixty-nine passages of Vero cells, suggesting low mutation rates in the targeted STR sequences in the Vero cell line.

Conclusions: A functional vervet multiplex assay consisting of eight human STR markers with heterozygosity values ranging from 0.53-0.79 was successful in uniquely identifying sixty-two vervet monkey samples. The probability of a random match using these eight markers between any two vervet samples is approximately 1 in 1.9 million. While authenticating a vervet cell line, the multiplex assay may also be a useful indicator for human cell line contamination since the assay is based on human STR markers.

Figure 1

Genetic profile of the Vero cell line using the vervet multiplex assay and bins and panels developed in GeneMapper ID-X. The blue, green, black, and red peaks in the electropherogram correspond to the fluorescently labeled forward primers for that STR marker (FAM, VIC, NED, and PET, respectively). Relative fluorescent units (RFUs) are depicted on the y-axis and fragment length on the x-axis. The number(s) below each peak represent the number of repeats at that locus. Vero peaks appear in the shaded bins and panels designated for each STR marker. The bins represent individual alleles for a specific marker.

Figure 2

Electropherogram representing a mixture of Vero and HeLa cell DNA (10:1 ratio, respectively). Genetic profiles were analysed using bins and panels developed in GeneMapper ID-X. Relative fluorescent units (RFUs) are depicted on the y-axis and fragment length on the x-axis in the electropherogram. Arrows depict HeLa alleles which may or may not appear in the bins and panels designated for Vero samples. Vero alleles appear in the shaded bins and panels designated for each STR marker. The number(s) below each peak represent the number of repeats at that locus. At the D4S2408, D5S4167, and D19S245 markers, HeLa DNA amplifies outside the observed vervet allele range and those values are represented as fragment lengths (bp) instead of repeat numbers. HeLa cells do not amplify at the DYS389 locus because they are of female origin. Human allele ranges that differ from vervet monkeys are illustrated in yellow boxes.