High Content Cell-Based Imaging Primary HTS to Identify Small Molecules Involved in X-Chromosome Reactivation and Reprogramming Measured in Cell-Based System Using Imaging - 7015-01_Activator_SinglePoint_HTS_Activity_Set2
In the mammalian female, nearly all genes on one of the two X chromosomes are transcriptionally silenced by X chromosome inactivation (XCI), the mechanism of dosage compensation that equalizes gene expression in XX females and XY males. In the inner cell mass of peri-implantation embryos, however, the inactivated X chromosome can be reactivated. In this assay, we aim to identify small molecule more ..
BioActive Compounds: 199
Keywords: X-Chromosome reactivation, Gene dosage, lncRNA
High content imaging assay (GFP)
In the mammalian female, nearly all genes on one of the two X chromosomes are transcriptionally silenced by X chromosome inactivation (XCI), the mechanism of dosage compensation that equalizes gene expression in XX females and XY males. In the inner cell mass of peri-implantation embryos, however, the inactivated X chromosome can be reactivated. In this assay, we aim to identify small molecule reactivators of a GFP reporter gene on the inactive X chromosome. This primary HTS is a high content screen using fibroblast cells in which GFP is under the control of the promoter on the inactive X (Xi) gene (XiGFP). The cells are immortalized with SV40 large T antigen. The fibroblast XiGFP cells will be exposed to compounds for 3 days before being imaged using a high content microscope (ImageXpress Micro automated microscope (Molecular Devices)). The re-induction of the inactive X gene by small molecules will be determined by calculating the percentage of GFP positive cells.
Identify small molecule increasing the percentage of GFP positive cells. Compounds increasing the percentage of GFP positive by at least 10% in both replicates compared to the neutral and positive controls will be considered active hits. Compounds increasing the percentage by 10% in one of the two replicates will be considered inconclusive hits. Compounds increasing less than 10% in both replicates will be considered inactive hits.
- Plate 500 68-5-11 cells (provided by Dr. Derek Lessing from Jeannie Lee's lab at Massachusetts General Hospital, Boston MA) in 50 ml per well in DMEM media in presence of 5-aza-dC (Cayman Chemicals, 11166) (final conc. 0.5 mM)(5-aza-dc should theoretically sensitize cells to X-chromosome reactivator compounds).
- Pin compounds (100 nl) and positive control Ellipticine (topisomerase inhibitor) (6uM final concentration) (Enzo Life Sciences, BML-GR315).
- Aspirate media.
- Fix cells and stain nucleus by adding 50 ul PBS with 4% formaldehyde and Hoechst 33342 (Life technologies, H3570)(dilution 1:5000 (10 mg/ml))
- Incubate 15min at room temperature.
- Aspirate formaldehyde/Hoechst and wash fixed cells once with PBS (50ul) using plate washer
- Add 50ul PBS with Plate washer.
- Image cells on high content microscope (Molecular Devices, MetaXpress software)
analysis: number of Hoechst-positive total cells
and % of GFP-positive cells (nuclear).
DMEM media recipe:
500 mL DMEM (high glucose, glutamax, pyruvate) - Life Technologies 10569-044
60 mL Fetal Bovine Serum - Gibco, 16410
15 mL HEPES - Life Technologies 15630-130
6 mL MEM Non-essential amino acids - Life Technologies 11140-076
6 mL Pen/Strep/Glutamine: Life technologies: 16140-071
1 mL 2-Mercaptoethanol - Life Technologies 21985-023
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 10.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)