|
| Status |
Public on Jul 18, 2011 |
| Title |
ZF-TF 64, 2nd infection |
| Sample type |
RNA |
| |
|
| Source name |
MCF7
|
| Organism |
Homo sapiens |
| Characteristics |
retroviral transduction: zinc finger transcription factor 64
|
| Treatment protocol |
293T cells were seeded at 5 x 105 cells per 10 cm culture dish, transfected with 3.2 μg of ZT-TF p65 activator library DNA and 2.4μg and 0.8μg of PMD-MLV and PMD-G plasmid DNA using Fugene 6 according to the manufacture’s protocol and retroviral supernatant harvested 48 hrs later. MCF7-R73 cells (6 x105 per 10 cm plate) were infected with 1.33 x 105 ZF-TF retrovirions in media containing polybrene. Two days post-infection the cells were exposed to puromycin (0.4mg/ml), and three days later were subjected to continuous combined puromycin and fulvestrant (100nM) selection for an additional 42 days. Retroviral DNA was recovered by PCR, subcloned into the pDONOR plasmid using Gateway technology (Invitrogen), and subject to DNA sequencing that revealed 46 unique ZF-TF arrays. The 46 ZF-TFs were re-cloned into the retroviral destination plasmid using Gateway technology, 46 ZF-TF retroviral supernatants generated, and MCF-R73 cells infected and subjected to puromycin and fulvestrant selection. Six unique ZF-TFs conferred drug resistance in the presence of continuous fulvestrant exposure. A control MCF7 cell line (MCF7-R238) was generated by infecting MCF7-R73 cells with a retrovirus bearing the p65 activation domain only. T47D cells were infected with the six unique ZF-TF bearing retroviruses and subjected to fulvestrant exposure as described for the MCF7 cells.
|
| Growth protocol |
MCF7-R73 cells, a clonal isolate derived from the MCF7 cell line were kindly provided by Dr. Toshi Shioda (MGH, Charlestown, MA) and maintained in a xenoestrogen controlled environment as described in Coser KR, Wittner BS, Rosenthal NF, Collins SC, Melas A, et al. (2009) Antiestrogen-resistant subclones of MCF-7 human breast cancer cells are derived from a common monoclonal drug-resistant progenitor, Proc Natl Acad Sci U S A 106: 14536-14541
|
| Extracted molecule |
total RNA |
| Extraction protocol |
QIAGEN RNeasy protocol and reagents for extraction of total RNA
|
| Label |
biotin
|
| Label protocol |
Affymetrix One-Cycle Target Labeling and Control Reagents (Part#900493) + Enzo BioArray High Yield RNA Transcript Labelling Kit (Part#42655)
|
| |
|
| Hybridization protocol |
Microarray = Affymetrix GeneChip Human Genome U133 Plus 2.0. Affymetrix Hybridization Oven= 645. Affymetrix Fluidics Station= 450. Fluidics Protocol= EukGE-WS2
|
| Scan protocol |
Affymetrix GeneChip Scanner= 3000-7G. Controller= GCOS
|
| Description |
ZF-TF.64.b4
fulvestrant sensitive MCF7 clone retroviraly induced with zinc finger transcription factor 64, 2nd infection
|
| Data processing |
MAS5 algorithm as implemented by call.exprs function of Bioconductor simpleaffy package version 2.14.05, 2% trimmed mean set to 100. The expression values were corrected for batch effect on a per-probe-set basis as follows. For each batch N other than the first, we added to the expression values of batch N the difference between the average expression value on the first batch and the average expression value on batch N.
|
| |
|
| Submission date |
Feb 22, 2011 |
| Last update date |
Jul 18, 2011 |
| Contact name |
Ben S. Wittner |
| E-mail(s) |
[email protected]
|
| Organization name |
Massachusetts General Hospital
|
| Department |
Center for Cancer Research
|
| Lab |
Lawrence
|
| Street address |
149 13th Street
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE27444 |
Expression data from zinc-finger-transcription-factor-induced Fulvestrant-Resistant MCF7 Cell Lines |
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