Ovarian granulosa cell tumor characterization identifies ...
CELL LINE CHARACTERIZATION - BioReliance€¦ · CELL LINE CHARACTERIZATION DNA BARCODE ASSAY For...
2
CELL LINE CHARACTERIZATION DNA BARCODE ASSAY For cells that are to be used in GMP biomanufacturing processes, confirmation of cell identity in a Master Cell Bank (MCB) is required 1,2 . The identification of species though genotypic testing is an acceptable confirmatory test, with species identification by “DNA analysis to detect a genomic polymorphism pattern” being specifically called out in the applicable regulations 1 . The desire to rapidly identify and discriminate cellular species de novo by genetic markers originated in the 1980’s with the development of DNA fingerprinting 3 . However, it was not until the technology and scientific understanding had progressed sufficiently that a standardized approach to animal species classification was proposed in 2003 4 . The cytochrome c oxidase subunit 1 (CO1) gene is an ideal target for species discrimination as this gene is conserved across higher eukaryotes, including insects, fish, birds and mam- mals. CO1 is located in the mitochondrial DNA, meaning it has a relatively fast mutation rate that aligns with speciation timescales. The absence of intronic regions on mitochondrial DNA also makes this a favorable target for near universal species identification. BioReliance offers two cell identity test services based on the CO1 barcode methodology. To perform this assay, a ~650bp region of CO1, known as the Folmer (or barcode) region 5 , is amplified with degenerate primers, followed by DNA sequence analysis of the amplification product. This sequence is then compared with known (and validated), species-specific CO1, reference sequences to identify the sample’s origin. For insect species, a smaller region of the CO1 gene (~450bp) is targeted. The CO1 barcode assay workflow is outlined in Figure 1. BIOLOGICS SAFETY TESTING SERVICES Figure 1 CO1 Barcode Assay Workflow. Universal Primer + Adaptor Sequencing Primer Extract DNA Sequencing Analysis 1 2 3 4 CO1 gene Amplify Barcode Region • CO1 barcoding is a well documented state-of-the- art technique for cell line identification. • The use of CO1 barcode technique for species identification is well understood by the regulators and used widely by the large cell repositories. • Availability of this assay in both the UK and USA reduces the burden of obtaining shipping permits. • GMP assays enable successful regulatory submission.
Transcript of CELL LINE CHARACTERIZATION - BioReliance€¦ · CELL LINE CHARACTERIZATION DNA BARCODE ASSAY For...
CELL LINE CHARACTERIZATIONDNA BARCODE ASSAY
For cells that are to be used in GMP biomanufacturing processes, confirmation of cell identity in a Master Cell Bank (MCB) is required1,2. The identification of species though genotypic testing is an acceptable confirmatory test, with species identification by “DNA analysis to detect a genomic polymorphism pattern” being specifically called out in the applicable regulations1. The desire to rapidly identify and discriminate cellular species de novo by genetic markers originated in the 1980’s with the development of DNA fingerprinting3. However, it was not until the technology and scientific understanding had progressed sufficiently that a standardized approach to animal species classification was proposed in 20034.
The cytochrome c oxidase subunit 1 (CO1) gene is an ideal target for species discrimination as this gene is conserved across higher eukaryotes, including insects, fish, birds and mam-mals. CO1 is located in the mitochondrial DNA, meaning it has a relatively fast mutation rate that aligns with speciation timescales. The absence of intronic regions on mitochondrial DNA also makes this a favorable target for near universal species identification. BioReliance offers two cell identity test services based on the CO1 barcode methodology. To perform this assay, a ~650bp region of CO1, known as the Folmer (or barcode) region5, is amplified with degenerate primers, followed by DNA sequence analysis of the amplification product. This sequence is then compared with known (and validated), species-specific CO1, reference sequences to identify the sample’s origin. For insect species, a smaller region of the CO1 gene (~450bp) is targeted. The CO1 barcode assay workflow is outlined in Figure 1.
BIOLOGICS SAFETY TESTING SERVICES
Figure 1 CO1 Barcode Assay Workflow.
Universal Primer+ Adaptor
SequencingPrimer
Extract DNA
Sequencing
Analysis
1
2
3
4
CO1 gene
Amplify Barcode Region
• CO1 barcoding is a well documented state-of-the-art technique for cell line identification.
• The use of CO1 barcode technique for species identification is well understood by the regulators and used widely by the large cell repositories.
• Availability of this assay in both the UK and USA reduces the burden of obtaining shipping permits.
• GMP assays enable successful regulatory submission.
Contact Us
North America Toll Free: 800 553 5372 Tel: +1 301 738 1000
Europe & International Tel: +44 (0)141 946 9999
Japan Tel: +81 (0)3 5796 7430
Email: [email protected]
DNA BARCODE ASSAY
CO1 Barcode Assay for Cell Line IdentificationDegenerate primers with adaptor sections (see Figure 1) were designed, optimized and validated to target the ~650bp barcode region of the CO1 gene. After amplification of the extracted DNA, the sample is sequenced (Sanger method) prior to being compared to a known reference sequence. Current validated reference sequences are shown below. Additional species coverage will follow:
• Human (e.g., HEK293)
• Mouse (Mus musculus e.g., SP2/0)
• Chinese Hamster (e.g., CHO)
• Syrian Hamster (e.g., BHK)
• African Green Monkey (e.g., Vero)
CO1 Barcode Assay for Insect Cell Line Identification
Similar to the above assay, the CO1 barcode assay for insect cell identification uses degenerate primers with adaptor sections designed, optimized and validated to target the ~450bp barcode region for insect species identification. Sanger sequencing followed by comparison to the validated reference sequence provides confirmation. The current validated species coverage is shown below, with additional insect species to follow:
• Spodoptera frugiperda (e.g., Sf9)
Conclusion
The CO1 Barcode assay is ideal for cell line identification, as it is able to confirm the identity of either mammalian or insect species. This methodology is known to the regulators6 and is already widely adopted by the major cell repositories as the method of choice for species confirmation.
These assays are available from BioReliance's Glasgow, Scotland and Rockville, MD facilities.
References1. Notes for Guidance on Quality of Biotechnological Products: Derivation
and Characterisation of Cell Substrates Used for Production of Biotechnological/Biological Products. (1988) CPMP/ICH/294/95. ICH Harmonised Tripartite Guideline: Topic Q5D
2. Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals. (1993). CBER/FDA
3. Jeffreys AJ, Wilson V and Thein SL. (1985). Hypervariable 'minisatellite' regions in human DNA. Nature, 314, 67-73.
4. Herbert PD, Cywinska A, Ball SL, and deWaard JR. (2003). Biological identifications through DNA barcodes. Proc. Biol. Sci., 270(1512), 313-21.
5. Folmer, O et al. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294-299
6. Handy, SM et al. (2011). A single laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance. J. AOAC. 94(1), 201-210.
For more information about BioReliance services, visit bioreliance.com
Ordering Information
Assay Number Assay DescriptionSample
RequirementsRegulatory Compliance
108601GMP.BSV (US Labs)108601GMP.BUK (UK Labs)
CO1 Barcode Assay for Cell Line Identification 1 vial of 2×106 cells GMP
108602GMP.BSV (US Labs)108602GMP.BUK (UK Labs)
CO1 Barcode Assay for Insect Cell Line Identification 1 vial of 2×106 cells GMP
BIOLOGICS SAFETY TESTING SERVICES
RUBF-1490815
0815
©2015 Sigma-Aldrich Co. LLC. All rights reserved. BioReliance is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other countries.
