Species identification in cell culture: a two-pronged molecular approach

Abstract

Species identification of cell lines and detection of cross-contamination are crucial for scientific research accuracy and reproducibility. Whereas short tandem repeat profiling offers a solution for a limited number of species, primarily human and mouse, the standard method for species identification of cell lines is enzyme polymorphism. Isoezymology, however, has its own drawbacks; it is cumbersome and the data interpretation is often difficult. Furthermore, the detection sensitivity for cross-contamination is low; it requires large amounts of the contaminant present and cross-contamination within closely related species may go undetected. In this paper, we describe a two-pronged molecular approach that addresses these issues by targeting the mitochondrial genome. First, we developed a multiplex PCR-based assay to rapidly identify the most common cell culture species and quickly detect cross-contaminations among these species. Second, for speciation and identification of a wider variety of cell lines, we amplified and sequenced a 648-bp region, often described as the "barcode region" by using a universal primer mix targeted at conserved sequences of the cytochrome C oxidase I gene (COI). This method was challenged with a panel of 67 cell lines from 45 diverse species. Implementation of these assays will accurately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts.

Phenotypic Variation among Culex pipiens Complex (Diptera: Culicidae) Populations from the Sacramento Valley, California: Horizontal and Vertical Transmission of West Nile Virus, Diapause Potential, Autogeny, and Host Selection

“…First, primers flanking COI were used to amplify the entire gene, and then the 658-bp "barcoding" region was amplified using vertebratespecific primers. 50,51 Host DNA was identified by sequencing the COI gene and using the "Identify Specimen" feature of the Barcode of Life Data Systems (BOLD; www.boldsystems.org).…”

DNA barcoding discriminates freshwater fishes from southeastern Nigeria and provides river system-level phylogeographic resolution within some species

“…Furthermore, DNA barcoding has gained wide application in forensic analysis to investigate cases of illegal poaching (Eaton et al 2009), separation of species (Wilson-Wilde et al 2010), gut content analysis in ecological studies (Smith et al 2005;Berry et al 2007;Clare et al 2009), food product analysis and market substitution (Wong and Hanner 2008;Cohen et al 2009), and Asian medicine trade regulation (Peppin et al 2008). DNA barcoding has also been employed to validate the identity of biomaterial collections and cell lines (Lorenz et al 2005;Cooper et al 2007). A sufficient accumulation of DNA barcodes can also help conservation managers to identify interim priority areas for conservation efforts in the absence of species data.…”

Development and characterization of two cell lines PDF and PDH from Puntius denisonii (Day 1865)

“…Hebert et al (2003) have demonstrated the utility of COI gene as a universal barcode, referred as "DNA barcoding" for the genetic identification of animal life. Recently, Cooper et al (2007) used COI region for identification of 67 cell lines used for barcode analysis. Our analysis also proves the utility of COI gene in the identification of the cell lines from P. denisonii.…”