The Pig-a Gene Mutation Assay is an in vivo assay primarily performed in rats. Gene mutation assays, such as the Pig-a assay, provide an advantage for product development and regulatory decision making since they measure the induction of mutations at a specific gene as opposed to measuring overall DNA damage. Prior to the introduction of the Pig-a assay, other in vivo gene mutation assays had the disadvantages of being

expensive and time-consuming, and required the use of specialized transgenic animals.

What is the Pig-a Gene Mutation Assay?The Pig-a assay is an in vivo mutation assay based on the X-linked Pig-a gene (phosphatidylinositol N-acetylglucosaminyltransferase, subunit A) which is involved in the production of glycosylohosphatidylino-sitol (GPI) anchor proteins for surface markers and antigens (Figure 1). Single-step forward mutations result in loss of the GPI anchors and the surface marker phenotype, and these neutral mutations can be detected early in immature red blood cells (reticulocytes) by flow cytometry (Figure 2).

Figure 1.

Figure 2.

Why consider the Pig-a Gene Mutation Assay?Pig-a is a highly conserved gene present in humans, rats, mice, and monkeys. Thus, this assay can help bridge the gap between preclinical models and humans. The technology is relevant for both pharmaceutical develop-

BioReliance Toxicology Services

Clear advantages of the Pig-a Gene Mutation Assay:

• Easilyintegratesintostandardtoxicity studies

• Reducesanimaluse

•Replacesexpensivetransgenicrodentmutationassays

• Fast,quantifiableresultswithflowcytometry

Pig-a In Vivo Gene Mutation Assay

Fully Qualified, Commercial AssayBioReliancewasanintegralmemberoftheinternationalPig-a validation effort and has officially qualifiedthisassay,underGLPconditions,accordingtomethodsand designs set forth in the collaborativetrialsofthatgroup.

Membrane

Protein

GPI-anchor

Phosphoethanolamine

Mannose

Glucosamine

Inositolphosphate

GlcNAc transferase

UDP-N-acetyl-glucosamine

Mannose transferase

Dolicol-phospate-mannose

Phosphoethanolamine

(deacetylation)Dol-P-Man syntase

NH2

CO

Acyl-CoA

Transamidase

Phosphatidylinositol

PIG-L

GPI-anchored Protein

(transfer of ethanolamine)

PIG-A

PIG-CPIG-HGPI1

PIG-B

PIG-F

GP18GAA1Protein

DPM1DPM2

EA

EA

P

P

EAP

P

ManMan

Man

ManGLcN

GLcN

I

P

I

GPIFluorescently labeled antibodies against GPI-anchored proteins

FCM analysis FCM analysis

Fluorescent negative

Pig-A gene

Pig-A Mutant CellWild-type Cell

GPI-anchored protein gene

Genotoxin xFluorescent postive

www.bioreliance.comToll Free: 800 553 5372 Tel: 301 738 1000

Email: toxicology@bioreliance.com

©2012 Sigma-Aldrich Co. LLC. All rights reserved. BioReliance and SAFC are trademarks of Sigma-Aldrich Co. LLC or its Affiliates, registered in the US and other countries. F-1031112

BioRelianceToxicology Services

Ordering InformationThe Pig-a Gene Mutation Assay can be performed under GLP on non-GLP conditions. Below is an example of a standard experimental design, but please consult a BioReliance representative for guidance and ordering.

Selected ReferencesIn vivoassessmentofPig-agenemutation–recentdevelopmentsandassayvalidation. Dertinger SD. and Heflich RH. Environmental & Molecular Mutagenesis. 52(9):681–4, 2011 Dec.

NeedandpotentialvalueofthePig-a in vivomutationassay–AHESIperspective.Schuler, M., Gollapudi, B. B., Thybaud, V. and Kim, J. H. Environmental & Molecular Mutagenesis. 52(9):685-9, 2011 Dec.

InternationalPig-agenemutationassaytrial:Evaluationoftransferabilityacross14laboratories. Dertinger SD. Phonethepswath S. Weller P. Nicolette J. Murray J. Sonders P. Vohr H-W. Shi J. Krsmanovic L. Gleason C. Custer L. Henwood A. Sweder K. Stankowski LF. Roberts DJ. Giddings A. Kenny J. Lynch AM. Defrain C. Nesslany F. van der Leede B-jM. Van Doninck T. Schuermans A. Tanaka K. Hiwata Y. Tajima O. Wilde E. Elhajouji A. Gunther WC. Thiffeault CJ. Shutsky TJ. Fiedler RD. Kimoto T. Bhalli JA. Heflich RH. and MacGregor JT. Environmental & Molecular Mutagenesis. 52(9):690-8, 2011 Dec.

Assessmentofgenotoxicityinducedby7,12-dimethylbenz(a)anthraceneordiethyl-nitrosamineinthePig-a,micronucleusandCometassaysintegratedinto28-dayrepeatdose studies. Shi J. Krsmanovic L. Bruce S. Kelly T. Paranjpe M. Szabo K. Arevalo M. Atta-Safoh S. Debelie F. LaForce MK. Sly J. Springer S. Environmental & Molecular Mutagenesis. 52(9):711–20, 2011 Dec.

IntegrationofPig-a,micronucleus,chromosomeaberration,andcometassayendpointsina28-dayrodenttoxicitystudywith4-nitroquinoline-1-oxide. Stankowski LF. Roberts DJ. Chen H. Lawlor T. McKeon M. Murli H. Thakur A. Xu Y. Environmental & Molecular Mutagenesis. 52(9):738–47, 2011 Dec.

The in vivo Pig-agenemutationassay,apotentialtoolforregulatorysafetyassessment. Dobrovolsky VN. Miura D. Heflich RH. Dertinger SD. Environmental & Molecular Mutagenesis. 51(8-9):825-35, 2010 Oct-Dec.

In vivomutationassaybasedontheendogenousPig-a locus. Bryce SM. Bemis JC. Dertinger SD. Environmental & Molecular Mutagenesis. 49(4):256-64, 2008 May.

Assay NameProtocolNumber

Assay Format GXP Test System Assay DesignTAT (Weeks to Verbal)

Sample Required

Tier II (in vivo) In Vivo Cytogenetics

Pig-a Gene Mutation Assay

460.BTLFlow cytometric

scoringGLP

Rat Peripheral Blood

28 day in-life, 2 doses, 1 harvest on day 29. 6 male rats per grroup, 3 dose groups plus vehicle control. 20,000 cells scored per sample.

6Please Inquire

Pig-a Gene Mutation Assay

460NGLP.BTLFlow cytometric

scoringNon-GLP

Rat Peripheral Blood

28 day in-life, 2 doses, 1 harvest on day 29. 6 male rats per grroup, 3 dose groups plus vehicle control. 20,000 cells scored per sample.

6Please Inquire

Pig-a analysis may also be ordered as an endpoint to a 28-day toxicity study. Entire study can be performed at BioReliance, or samples can be sent for analysis that can be performed alone or conjunction with other endpoints (Micronucleus Assay, Comet Assay, Metaphase Analysis, etc.).

Assay NameProtocolNumber

Assay Format GXP Test System Assay DesignTAT (Weeks to Verbal)

Sample Required

GeneTox EndpointsFlow Cytometric Evaluation from In Vivo StudiesPig-a Study Options

Pig-a Analysis (Flow Cytometry)

160FlowWBGLPFlow cytometric

scoringGLP

Samples (WB)

Flow cytometric evaluation of prepared samples (Sponsor-provided whole blood). 20,000 cells scored per sample. Includes summary report

1 week 100 µL

Pig-a Analysis (Flow Cytometry)

160FlowWBNGLPFlow cytometric

scoringNon-GLP

Samples (WB)

Flow cytometric evaluation of prepared samples (Sponsor-provided whole blood). 20,000 cells scored per sample. Includes summary report

1 week 100 µL

ment and chemical safety, and can easily be integrated into standard toxicity studies to provide additional and valuable data. This assay offers significant value as a secondary tier assay to understand a compound’s mode of action and to follow up a positive in vitro gene tox assay. Demonstration that mutations accumulate with repeat dosing, and improvements in assay design and power, make the assay well qualified to look at low dose responses and threshold effects.