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BioAssay: AID 1948

qHTS Assay for Compounds that Induce Erasure of Genomic Imprints

Genomic imprinting is the epigenetic process whereby genes are expressed in a parent of origin fashion. Imprinted genes exhibit monoallelic expression exclusively from either the paternal or maternal allele. Unlike some forms of epigenetic silencing that can be overcome by overexpression of transcription factors, genomic imprinting stably represses the expression of one allele despite potentially more ..
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 Tested Compounds
 Tested Compounds
All(112339)
 
 
Active(19)
 
 
Inactive(110417)
 
 
Inconclusive(1914)
 
 
Unspecified(32)
 
 
 Tested Substances
 Tested Substances
All(113755)
 
 
Active(20)
 
 
Inactive(111786)
 
 
Inconclusive(1917)
 
 
Unspecified(32)
 
 
 Related BioAssays
 Related BioAssays
AID: 1948
Data Source: NCGC (IMPR274)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-09-15

Data Table ( Complete ):           Active    All
BioActive Compounds: 19
Description:
qHTS Assay for Compounds that Induce Erasure of Genomic Imprints

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]

Assay Submitter (PI): JEFFRIES, SEAN, NCGC, University of Cambridge

NCGC Peg3 Imprinting Assay Overview

Genomic imprinting is the epigenetic process whereby genes are expressed in a parent of origin fashion. Imprinted genes exhibit monoallelic expression exclusively from either the paternal or maternal allele. Unlike some forms of epigenetic silencing that can be overcome by overexpression of transcription factors, genomic imprinting stably represses the expression of one allele despite potentially high levels of expression from the other allele. Imprints are highly stable in embryonic tissues and are maintained throughout development except in primordial germ cells (PGCs). During mouse embryonic day 11.5 imprints are erased in PGCs so that sex specific imprints can be reestablished during gametogenesis.

Embryonic germ (EG) cells can be derived from developing PGCs at embryonic day 11.5 and these cells often lack genomic imprints. Previous work has demonstrated that fusion of an EG cell with a somatic cell can subsequently induce imprint erasure in the somatic cell genome. Embryonic stem (ES) cells are a cell type similar to EG cells in phenotype and transcriptional profile, yet ES cells maintain genomic imprints. This similarity between ES and EG cells prompted us to use ES cells to look for factors which could induce imprint erasure.

To investigate the stability of genomic imprinting, ES cells were derived from mice with an insertion of a LacZ-Neomycin reporter construct in the Peg3 locus. Peg3 is an imprinted locus where gene expression is restricted to the paternal allele. ES cells with a maternally inherited Peg3LacZ-Neo exhibit low LacZ expression and are G418 sensitive over many passages. Using LacZ expression we are able to monitor the state of the Peg3 imprint.

In order to dissect the mechanisms involved in imprint erasure using a forward chemical genetics approach we developed a cell based phenotype assay using an endogenous silence imprinted locus. This assay utilizes maternally inherited Peg3LacZ-Neo ES cells by monitoring the LacZ expression from the normally silent Peg3 allele. To facilitate screening, high-density feeder-free ES cell culture has been optimized so that ES cells can be grown in 1536 for over four days without feeding or washes. The assay uses a luminescence based read out for LacZ activity and is a homogenous assay optimal for HTS.
Protocol
NCGC Peg3 Imprinting Assay Protocol Summary

ES cell lines were derived from blastocysts taken from Peg3LacZ-Neo x129 matings. Lines were sexed and genotyped for the Peg3LacZ-Neo allele. Female cells carrying the reporter allele were used in this study. Cells were derived on a layer of mouse embryonic fibroblasts and passaged multiple times before switching to feeder free culture. Cells were maintained feeder free on gelatin coated dishes in DMEM/F12 media supplemented with 15% FBS, 2mM L-glutamine, 100 units/ml penicillin, 100ug/ml streptomycin, 0.1mM MEM nonessential amino acids, 1mM sodium pyruvate, 0.1% sodium bicarbonate, 0.1mM 2-Mercaptoethanol, and 400 units/ml leukemia inhibitory factor (LIF) at 37C with 5% CO2. The assay was performed in 1536-well format. A single luminescence read was taken at the end of the assay and the data was normalized by plate to internal negative controls. Maximum efficacy and AC50 values were calculated from dose-response data using the standard Hill equation.


1536 well plates were filled with 0.5ul of 0.1% gelatin per well, spun down and incubated for at least 30 min at 37C. The ES cells were then trypsini

Peg3 1536-well assay protocol:
(1)Plate 0.5ul/well 0.1% gelatin in white tissue culture treated 1536-well plates, spin and incubate at 37C for at least 30 min.
(2)Add 5ul/well (300 cells) of ES cell suspension from freshly trypsinized cells and incubate at 37C, 5% CO2 for 40 hours.
(3)Add 5ul/well of freshly prepared BetaGlo solution and incubate at room temperature for 5 minutes.
(4)Read luminescence on the ViewLux plate reader.

Keywords
Genomic Imprinting, Germ Cells, Embryonic Stem Cell, DNA Methylation, Peg3, qHTS, NIH roadmap, NCGC
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0000529954 uM (5.29954e-05μM**)% Activity at given concentration.Float%
15Activity at 0.0001179724 uM (0.000117972μM**)% Activity at given concentration.Float%
16Activity at 0.0002640553 uM (0.000264055μM**)% Activity at given concentration.Float%
17Activity at 0.0005899454 uM (0.000589945μM**)% Activity at given concentration.Float%
18Activity at 0.00132 uM (0.00131965μM**)% Activity at given concentration.Float%
19Activity at 0.00300 uM (0.00300356μM**)% Activity at given concentration.Float%
20Activity at 0.00432 uM (0.00432098μM**)% Activity at given concentration.Float%
21Activity at 0.00704 uM (0.00704405μM**)% Activity at given concentration.Float%
22Activity at 0.017 uM (0.016728μM**)% Activity at given concentration.Float%
23Activity at 0.025 uM (0.0251213μM**)% Activity at given concentration.Float%
24Activity at 0.051 uM (0.0510117μM**)% Activity at given concentration.Float%
25Activity at 0.090 uM (0.0900396μM**)% Activity at given concentration.Float%
26Activity at 0.143 uM (0.143235μM**)% Activity at given concentration.Float%
27Activity at 0.355 uM (0.3546μM**)% Activity at given concentration.Float%
28Activity at 0.511 uM (0.510846μM**)% Activity at given concentration.Float%
29Activity at 0.827 uM (0.826762μM**)% Activity at given concentration.Float%
30Activity at 1.997 uM (1.99735μM**)% Activity at given concentration.Float%
31Activity at 2.948 uM (2.94783μM**)% Activity at given concentration.Float%
32Activity at 4.995 uM (4.99486μM**)% Activity at given concentration.Float%
33Activity at 10.87 uM (10.87μM**)% Activity at given concentration.Float%
34Activity at 16.84 uM (16.8371μM**)% Activity at given concentration.Float%
35Activity at 40.67 uM (40.6704μM**)% Activity at given concentration.Float%
36Activity at 60.70 uM (60.7μM**)% Activity at given concentration.Float%
37Activity at 97.30 uM (97.3009μM**)% Activity at given concentration.Float%
38Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
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