Methods of Non-Human Cell Line Authentication and Identification
Non-human Cell
Line Authentication
Methods to Authenticate
Non-human Cells
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Identity Testing of
Non-human Cell Lines
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Karyotyping
DNA barcoding
Public database available
Standard methods in place
Multiplex PCR using species-specific primers
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Short Tandem Repeat (STR) genotyping
Commercial services available for mouse
Public database available (limited # entries)
Standard methods not currently available
Commercial services available for rat (strain level identification only)
Karyotyping
The counting of modal chromosome number and observing specific
chromosomal markers of viable cells during metaphase. Each species has a
unique chromosomal karyotype characterized by the size, shape, and
number of chromosomes.
Attention is paid to
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Length
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Position of the centromeres
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Banding pattern
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Any notable differences
between the sex
chromosomes
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Any other physical
characteristics
www.pathology.washington.edu/galleries/cytogallery
Each species has a
unique karyotype
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Species ID
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Chromosomal
rearrangements
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Index of genomic
stability
Karyotyping Cell Lines
www.coriell.org/research-services/cytogenetics/karyotyping
DNA Barcoding
A DNA based approach that is used to
identify vertebrate and invertebrate
animal cells at the species level. This
method targets 648 bp of the 5’ region
of the mitochondrial cytochrome c
oxidase subunit 1 (CO1 or COX1) gene.
The target sequence is compared to a
reference library of sequences to
determine the species of the sample
in question.
Written consensus standard for DNA Barcoding:
Designation: ASN-0003 (published in 2015)
Species-Level Identification of Animal Cells through Mitochondrial Cytochrome c
Oxidase Subunit 1 (CO1) DNA Barcodes
Picture created by Emmanuel Douzery
Human Mitochondrial
Genome
DNA Barcoding – Flow Chart
R
= purines (A/G)
Y
= pyrimidines (T/C)
Primers are tailed with M13 sequence (commonly used
sequencing primers)
ANSI/ASN-0003-2015
Primers used to amplify the 648 bp CO1 barcode
region are shown in the table below.
Degenerate universal primers (VF1d and VR1d)
are used as sequencing primers.
LepF1 and LepR1 primers are used for insect cell
lines.
Barcode of Life Data Systems (BOLD)
The reference database contains verified sequences derived from voucher
(reference) specimens. Such authenticated cultures/tissues can serve as
standard reference materials or as controls of authenticated animal cells for
tissue culture, regulatory, and taxonomic applications.
GenBank
(no voucher specimens)
To meet BARCODE standards in GenBank, records must contain:
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Complete species name
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Unique identifier for voucher specimen (archived in museum or biological
repository)
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Country of origin
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Primer sequences used
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Bi-directional high-quality sequencing trace files (cover at least 75% of CO1
barcode region)
DNA Barcoding - Databases
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Some closely related species cannot be readily distinguished
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Rates of evolution of mitochondrial genes may vary between species,
which can lead to overlap of species variation
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This method is unable to resolve cell lines from different
individuals within the same species
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DNA barcoding may or may not flag samples that are mixtures of
two different species
DNA Barcoding - Limitations
This is a useful method to determine species-level identification:
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Simple
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Rapid
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Inexpensive
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Adapted for high throughput analysis
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Importantly, universal primers are used (other methods rely on specific primers for
specific species)
Species-Specific Multiplex
PCR Primers
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An ATCC SDO working group is currently working on a written
consensus standard for this method (ANSI/ASN-0004)
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The assay described by Cooper et al. covers 14 of the most
common species found in tissue culture
•
Primers are based on mitochondrial cytochrome c oxidase 1
(CO1) and cytochrome b genes
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Tests for cross contamination of other species
•
Visualize PCR products using gel electrophoresis where
amplicon size is species-specific
Species-Specific Multiplex Primers
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Only capable of detecting species that are represented in the
primer multiplex (14).
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Is unable to discriminate between cell lines from the same
species (ex: 2 different mouse cell lines)
Species-Specific Multiplex Primers
This is an easy method to determine species-level identification:
•
Fast (same day results)
•
Inexpensive
•
Easily shows contamination of cell lines from different species
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Any lab equipped with a thermal cycler and gel electrophoresis apparatus can perform
this assay
Identification at the
Individual Level
STR genotyping
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Simple repeats
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Di (
CA
CACA)
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Tri (
CAT
CAT)
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Tetra (
CATG
CATG)
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Penta (
CATGA
CATGA)
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Complex repeats
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(CATG)R(TA)(TAGA)
Human
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Written consensus standard
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Commercial services available
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Public Databases
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Commercial kits
Mouse
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Commercial services available
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Mouse Cell Line Authentication Consortium
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Validated 18 mouse STR markers
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Public database (NCBI BioSample and Cellosaurus)
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Allelic ladder development (in progress)
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Written consensus standard (needed)
Rat
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Commercial service available for rat using STR markers
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Identity of rat strain only, not individual resolution
STR Genotyping Services
Limitations of STR genotyping
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Cell lines that are derived from the same parent line will most
likely have matching STR genotypes (they are from the same
individual)
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Inter-species contamination will not be detected
Summary
How to authenticate non-human cell lines?
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Identity testing is a part of authentication
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Species-level identification
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Karyotyping
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DNA barcoding
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Species-specific multiplex PCR
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Individual level identification
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STR genotyping
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Human
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Mouse
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Rat
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Authentication involves fully characterizing the cell line
(morphology, mycoplasma testing, protein expression, genomic
stability)
Welcome to the training module for non-human cell line authentication.
Techniques such as chromosomal karyotyping, DNA barcoding, and STR genotyping are used for authenticating and identifying non-human cell lines to ensure accurate research outcomes. Species-level identification, karyotyping, and commercial services for mouse and rat cell lines are discussed, highlighting the importance of maintaining authenticity in cell line studies.
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Presentation Transcript
Non-human Cell Line Authentication
Methods to Authenticate Non-human Cells Identification Contamination testing Chromosomal karyotype Protein expression
Identity Testing of Non-human Cell Lines Species-level identification Karyotyping DNA barcoding Public database available Standard methods in place Multiplex PCR using species-specific primers Individual level identification Short Tandem Repeat (STR) genotyping Commercial services available for mouse Public database available (limited # entries) Standard methods not currently available Commercial services available for rat (strain level identification only)
Karyotyping The counting of modal chromosome number and observing specific chromosomal markers of viable cells during metaphase. Each species has a unique chromosomal karyotype characterized by the size, shape, and number of chromosomes. Attention is paid to Length Position of the centromeres Banding pattern Any notable differences between the sex chromosomes Any other physical characteristics www.pathology.washington.edu/galleries/cytogallery
Karyotyping Cell Lines Each species has a unique karyotype Species ID Chromosomal rearrangements Index of genomic stability www.coriell.org/research-services/cytogenetics/karyotyping
DNA Barcoding A DNA based approach that is used to identify vertebrate and invertebrate animal cells at the species level. This method targets 648 bp of the 5 region of the mitochondrial cytochrome c oxidase subunit 1 (CO1 or COX1) gene. The target sequence is compared to a reference library of sequences to determine the species of the sample in question. Human Mitochondrial Genome Picturecreated by Emmanuel Douzery Written consensus standard for DNA Barcoding: Designation: ASN-0003 (published in 2015) Species-Level Identification of Animal Cells through Mitochondrial Cytochrome c Oxidase Subunit 1 (CO1) DNA Barcodes
DNA Barcoding Flow Chart ANSI/ASN-0003-2015 Primers used to amplify the 648 bp CO1 barcode region are shown in the table below. Degenerate universal primers (VF1d and VR1d) are used as sequencing primers. LepF1 and LepR1 primers are used for insect cell lines. R = purines (A/G) Y = pyrimidines (T/C) Primers are tailed with M13 sequence (commonly used sequencing primers)
DNA Barcoding - Databases Barcode of Life Data Systems (BOLD) The reference database contains verified sequences derived from voucher (reference) specimens. Such authenticated cultures/tissues can serve as standard reference materials or as controls of authenticated animal cells for tissue culture, regulatory, and taxonomic applications. GenBank (no voucher specimens) To meet BARCODE standards in GenBank, records must contain: Complete species name Unique identifier for voucher specimen (archived in museum or biological repository) Country of origin Primer sequences used Bi-directional high-quality sequencing trace files (cover at least 75% of CO1 barcode region)
DNA Barcoding - Limitations Some closely related species cannot be readily distinguished Rates of evolution of mitochondrial genes may vary between species, which can lead to overlap of species variation This method is unable to resolve cell lines from different individuals within the same species DNA barcoding may or may not flag samples that are mixtures of two different species This is a useful method to determine species-level identification: Simple Rapid Inexpensive Adapted for high throughput analysis Importantly, universal primers are used (other methods rely on specific primers for specific species)
Species-Specific Multiplex PCR Primers An ATCC SDO working group is currently working on a written consensus standard for this method (ANSI/ASN-0004) The assay described by Cooper et al. covers 14 of the most common species found in tissue culture Primers are based on mitochondrial cytochrome c oxidase 1 (CO1) and cytochrome b genes Tests for cross contamination of other species Visualize PCR products using gel electrophoresis where amplicon size is species-specific
Species-Specific Multiplex Primers Limitations Only capable of detecting species that are represented in the primer multiplex (14). Is unable to discriminate between cell lines from the same species (ex: 2 different mouse cell lines) This is an easy method to determine species-level identification: Fast (same day results) Inexpensive Easily shows contamination of cell lines from different species Any lab equipped with a thermal cycler and gel electrophoresis apparatus can perform this assay
Identification at the Individual Level STR genotyping Simple repeats Di (CACACA) Tri (CATCAT) Tetra (CATGCATG) Penta (CATGACATGA) Complex repeats (CATG)R(TA)(TAGA) [GATA]8 =
STR Genotyping Services Human Written consensus standard Commercial services available Public Databases Commercial kits Mouse Commercial services available Mouse Cell Line Authentication Consortium Validated 18 mouse STR markers Public database (NCBI BioSample and Cellosaurus) Allelic ladder development (in progress) Written consensus standard (needed) Rat Commercial service available for rat using STR markers Identity of rat strain only, not individual resolution
Limitations of STR genotyping Cell lines that are derived from the same parent line will most likely have matching STR genotypes (they are from the same individual) Inter-species contamination will not be detected
Summary How to authenticate non-human cell lines? Identity testing is a part of authentication Species-level identification Karyotyping DNA barcoding Species-specific multiplex PCR Individual level identification STR genotyping Human Mouse Rat Authentication involves fully characterizing the cell line (morphology, mycoplasma testing, protein expression, genomic stability)
