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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.
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In the citing paper
“confirmed that HIBEC cells were originally from rat and C201441 cells were from mouse”
From Investigation of Cross-Contamination and Misidentification of 278 Widely Used Tumor Cell Lines (2017) · cites this paper as [7]
In the citing paper
“confirmed that HIBEC cells were originally from rat and C201441 cells were from mouse”
From Investigation of Cross-Contamination and Misidentification of 278 Widely Used Tumor Cell Lines (2017) · cites this paper as [7]
In the citing paper
“Cooper and colleagues implemented a two-pronged molecular approach in which they combined a multiplex PCR-based assay to identify the most common cell culture species and a protocol for sequencing of a 648 bp region located in the cytochrome C oxidase I (COI) gene, i.e., the “barcode region”, that accurately determines the species of cell lines [69].”
From Genetic Insights into the Economic Toll of Cell Line Misidentification: A Comprehensive Review (2026) · cites this paper as [69]
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Zandieh, S., Lotfi, M., Kamalzadeh, M., Shiri, N., Parmour, E., Es-haghi, A., Masoudi, S., Hablolvarid, M., Shoushtari, A., Goudarzi, H., et al. (2017) · Archives of Razi Institute · doi:10.22092/ari.2017.111601 · Cited by 1
Cell Line Authentication and Characterisation
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“CO1 possesses a suitable balance of interspecific sequence variation and intraspecific conservation, making it a commonly employed marker for species discrimination and taxonomic verification (Cooper et al. 2007; Kim et al. 2010).”
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“The cox1 gene widely adopted as a universal barcode for animal species identification [100], exhibits relatively low variability within the Polycelis genus.”
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The mitochondrial genomes of Panorpidae: sequence, structure and phylogenetic analysis
Hua, Y., Li, N., Chen, J., Hua, B.-Z., & Tao, S.-H. (2020) · doi:10.21203/rs.3.rs-17210/v1
“Although fragment of 648 bp of the gene cox1 is often considered as a universal barcode for species identification in animals [50], it is the least variable gene in Panorpidae and has a relatively lower ratio of Ka/Ks among the PCGs in these 19 mitogenomes.”
BackgroundAnalysis of the dystrophin interactome
Thorley, M. (2016) · theses.fr (ABES) · doi:10.70675/f6582854zdd32z4484zae7fz22c8ecd45969
The support of undifferentiated human embryonic stem cell lines by different matrices
Khadun, S. (2014) · University of Hertfordshire Research Archive (University of Hertfordshire) · doi:10.18745/th.14447
Rezvani-Boroujeni, E., Hosseini, S., Sharifi, Z., Latifi, H., Mirshafiee, H., & Jamshidi-Seresht, M. (2013) · doi:10.21859/isv.7.4.14
“In this way, it is important to retain tissue or DNA from the donor and to do comparative DNA STR profiling or DNA bar-coding (27, 28) between the new cell line and its presumed source as soon as it becomes established.”
Metadata
- Authors
- Cooper, J. K., Sykes, G., King, S., Cottrill, K., Ivanova, N. V., Hanner, R., & Ikonomi, P.
- Affiliations
- American Type Culture Collection (US)
- University of Guelph (CA)
- Corresponding author
- Ikonomi, Pranvera
- ORCID
- Hanner, Robert
- Venue
- In Vitro Cellular & Developmental Biology - Animal
- Published
- 2007-10-13
- Volume
- 43
- Issue
- 10
- Pages
- 344-351
- ISSN
- 1071-2690 (ISSN-L)
- 1543-706X
- OpenAlex
- W1907982143
- Cited by
- 174
- References
- 0
- Citations by year
- 2026: 7 · 2025: 7 · 2024: 7 · 2023: 11 · 2022: 10 · 2021: 10 · 2020: 8 · 2019: 10 · 2018: 10 · 2017: 13 · 2016: 8 · 2015: 7 · 2014: 11 · 2013: 14 · 2012: 4
- FWCI
- 2.64
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- In Vitro Cellular & Developmental Biology - Animal (publishedVersion)
- PubMed (publishedVersion)
- BioOne Complete (BioOne) (submittedVersion)
- Funders
- Genome Canada
- Natural Sciences and Engineering Research Council of Canada
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Identification and Quantification in Food
- Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- cross-contamination detection, mitochondrial genome, cytochrome c oxidase I, short tandem repeat profiling, multiplex PCR, cell culture contamination, research reproducibility
- MeSH terms
- Animals, Electron Transport Complex IV, Humans, Phylogeny, Species Specificity, Cell Culture Techniques, K562 Cells, Protein Subunits
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