Non-human Cell Line Authentication - PowerPoint Presentation

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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 placeMultiplex 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 chromosomesAny other physical characteristicswww.pathology.washington.edu/galleries/cytogallery

Each species has a unique karyotype

Species IDChromosomal rearrangements

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:Complete species name

Unique identifier for voucher specimen (archived in museum or biological repository)Country of originPrimer sequences used Bi-directional high-quality sequencing trace files (cover at least 75% of CO1 barcode region)

DNA Barcoding - Databases

Some closely related species cannot be readily distinguishedRates 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 speciesDNA 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:

Simple

Rapid

InexpensiveAdapted for high throughput analysisImportantly, 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)

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

Any lab equipped with a thermal cycler and gel electrophoresis apparatus can perform this assay

Identification at the

Individual Level

[GATA]

8

=

STR genotyping

Simple repeats

Di (

CA

CACA)

Tri (

CAT

CAT)

Tetra (

CATG

CATG)

Penta (

CATGA

CATGA)

Complex repeats

(CATG)R(TA)(TAGA)

HumanWritten consensus standard

Commercial services availablePublic DatabasesCommercial kitsMouse

Commercial services availableMouse Cell Line Authentication ConsortiumValidated 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 markersIdentity of rat strain only, not individual resolution

STR Genotyping Services

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 authenticationSpecies-level identificationKaryotyping

DNA barcodingSpecies-specific multiplex PCRIndividual level identification

STR genotypingHumanMouseRat

Authentication involves fully characterizing the cell line (morphology, mycoplasma testing, protein expression, genomic stability)