Species identification and authentification of human and rodent cell cultures using polymerase chain reaction analysis of vomeronasal receptor genes

Abstract

Cell culture and the use of cell lines are routinely used in basic scientific research. It is therefore imperative for researchers to ensure the origin of the cell lines used and that they are routinely re-analysed for contamination and misidentification. Inter-species contamination is relatively frequent, and the most commonly used cell lines are of human, mouse and rat derivation. We have developed simple species specific primer assays based on genomic sequence differences in vomeronasal receptor gene family members to discriminate between human, mouse and rat DNA using standard agarose gel electrophoresis. Furthermore, these PCR assays are able to identify the species composition within an inter-species mixed population. This approach therefore provides a valuable tool to enable a rapid, simple and relatively inexpensive determination of the authentication and contamination of cell cultures.

Fig. 1

PCR amplification with species specific primer pairs. PCR analysis of genomic DNA from rat, mouse and human cell lines and primary tissue using species specific primer pairs. PCRs assays used and source of input DNA (50 ng each) are shown. Amplified products generated at 32 cycles were detected by ethidium bromide staining of 1.5% agarose gels and UV transillumination. Blood refers to human peripheral white blood cells. RNase free water was used as negative control. Arrows indicate the DNA molecular weight marker of 500 bp (Hyperladder IV; Bioline)

Fig. 2

Verification of the ability of the assay to detect interspecies cross contamination. DNA from each species was mixed at a ratio of 5 ng:45 ng (order as labelled) or used as 50 ng input from a single species. DNA used was: WRC-256 (rat); 3T3 (mouse); H400 (human). PCRs were performed with the primer assays indicated. Amplified products generated at 32 cycles were detected by ethidium bromide staining of 1.5% agarose gels and UV transillumination. RNase free water was used as negative control. Arrows indicate the DNA molecular weight marker of 500 bp (Hyperladder IV; Bioline)