Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

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Effect of expression of wild type KSHV RTA or the activation domain (aa608-651) deleted version of RTA in 293T cells on gene expression profiling.

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Transcription profiling of human 239T cells expressing wild type or mutant forms of gammaherpesvirus transactivator RTA

Effect of expression of wild type KSHV RTA or the activation domain (aa608-651) deleted version of RTA in 293T cells on gene expression profiling.


Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-2

Labeling of samples was carried out according to the manufacturer’s standard protocol

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-123022

Two cell lines were generated, expressing wild type or mutant forms of the KSHV/HHV-8 transcription activator RTA:  1. TRexFLAG-RTAwt expresses wild type FLAG-tagged RTA cDNA.  2. TRexFLAG-RTAD608-651 expresses FLAG-tagged RTA cDNA with an internal deletion of amino acids 608-651 within the activation domain. This region has been shown to be required for RTA to interact with the mediator complex. The mutant is severely defective for transcription activation and unable to reactivate latent KSHV.   The cDNAs were cloned into the pCDNA5/TO vector (Invitrogen) downstream of a tetracycline inducible promoter. The vectors were transfected into 293T-Rex cells which stably express the tetracycline repressor (Invitrogen).  Cell clones were isolated and propagated. The transformed cells are maintained in DMEM with 10% low tetracycline serum (Clontech) and pen/strep with 5ƒÝg/ml Blasticidin (Invitrogen) and 100ƒÝg/ml Hygromycin.    Cells were seeded onto 6 well dishes and induced with 1ƒÝg/ml tetracycline. Extracts were harvested at 0, 4, 8 and 12 hours post induction. All time points were prepared in duplicate.  

P-MEXP-133497

Both cell lines were treated with 1ug/ml tetracycline to induce RTA expression.

P-MEXP-133498

Whole cell RNA was prepared using the standard Qiagen RNeasy kit.  RNA concentrations were measured and 1ƒÝl of each RNA sample was analyzed on a 1% agarose gel.

P-MEXP-133708

Arrays were GCRMA normalized so that the median intensity across genes and arrays is a value of 100.

Transcription profiling of human 239T cells expressing wild type or mutant forms of gammaherpesvirus transactivator RTA

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