Species Identification and Confirmation of Human and Animal Cell Lines: A PCR-Based Method

Abstract

Misidentification and cross-contamination of cell lines are major problems of cell cultures that can make scientific results and their reproducibility unreliable. This paper describes a PCR-based method for easily identifying or confirming the species of origin of cell lines by using a panel of oligonucleotides specific for the nine animal species most common in cell culture laboratories. A panel of 35 human and animal cell lines, whose species of origin were previously confirmed by isoenzyme assay, was studied with nine species-specific primer pairs that specifically anneal to DNA sequences codifying for human, cat, dog, mouse, rat, horse, rabbit, African Green monkey cytochrome c oxidase subunit I (cox I), and one primer pair specific for the cytochrome b gene of Chinese hamster. The amplified fragments were analyzed by electrophoresis in ethidium bromide-stained 2% agarose gels. The method is simple, rapid, highly sensitive, and useful for routinely monitoring the species identity of cell cultures.

Human RNA Polymerase I-Driven Reverse Genetics for Influenza A Virus in Canine Cells

“…To confirm that the MDCK cells were of canine origin and free of contamination with human cells, we used previously described multiplex PCR-based assays to verify the species identity of the cultures (4,14). We extracted DNA from the cell cultures and amplified two mitochondrial genes for cytochrome c oxidase subunit I (cox I) and cytochrome b, using mixtures of human-specific and canine-specific primer pairs.…”

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

“…RNase free water was used as negative control. Arrows indicate the DNA molecular weight marker of 500 bp (Hyperladder IV; Bioline) sensitivity or ability to detect contamination (Liu et al 2003;Ono et al 2007;Ramya et al 2009;Higgins et al 2010;Parodi et al 2002). Inexpensive assays for the identification and authentication of cell lines are therefore clearly necessary and should be relatively easily developed using the vast DNA databases as is demonstrated here.…”

“…Yet despite the burgeoning information present in the genome databases for these species only a handful of PCR-based technologies for cell line authentification have been developed (Stacey et al 1997;Parodi et al 2002;Liu et al 2003;López-Andreo et al 2005;Steube et al 2003Steube et al , 2008. Whilst these approaches may be relatively inexpensive many of these assays utilise multiple primers or require interpretation of relatively complex gel banding patterns.…”

Establishment and Biological characterization of a Large Yorkshire Pig fibroblast line

“…They are species-specific and evolutionarily conserved, but the subtypes and activities differ among species, providing a biochemical indicator of species classification by chromatography and electrophoresis. LDH and MDH were therefore selected to determine the species origin of the cells and to detect cross-contamination [27][28]. Biochemical analysis of isoenzyme polymorphism is currently considered the standard method for quality control of cell line identification and interspecies contamination, as demonstrated when Arai et al measured the LDH isoenzyme patterns in horse leucocytes and plasma, and in Debao pony fibroblasts [29].…”