BioProject

Three 293 cell-derivative cell line were constructed using commercially available Invitrogen Flp-In TREx 293 cells. More...

Three 293 cell-derivative cell line were constructed using commercially available Invitrogen Flp-In TREx 293 cells. The cell lines express wild type HIV-1 Vpr and two mutant Vprs, F72A/R73A and R80A. The mutant Vprs are defective in the Vpr cell cycle arrest phenotype. The cell lines were characterized for their ability to express Vpr at the RNA and protein levels and functionally for the ability of the inducibly expressed Vprs to arrest cell cycle progression at G2/M. To study whether certain host cell genes showed alterations in expression following induced expression of Vpr, we conducted a large scale host cell gene expression profiling study using microarray technologies. To determine whether the differential expression of certain genes was associated with Vpr-induced cell cycle arrest, we compared the gene expression pattern of wild type Vpr expressing cells with the pattern of Vpr mutant F72A/R73A- or R80A-Vpr expressing cells. Doxycycline-induced and uninduced and Flp-In TREx 293/Vpr cells were collected 0, 1, 2, 4, 6, 8, 12, 16 and 24 hours post induction (hpi). Doxycyclineinduced and uninduced Flp-In TREx 293/Blank cells were also collected as a reference for following experiments. Total RNA was isolated using RNAeasy Midi Kit (QIAGEN), according to the manufacturer's protocol. Human long oligonucleotide microarrays were obtained from the National Cancer Institute Microarray Facility, Advanced Technology Center (Gaithersburg, MD). The microarrays (Hs Operon V2) contained 22,434 oligonucleotide (60-70 mer) spots on a glass slide. 25 µg of total RNAs isolated from doxycycline-induced Flp-In TREx 293 stably transfected blank- and FLAG tagged-Vpr cells were reverse transcribed using an oligo (dT) primer and aminoallyl-dNTP (Amersham Pharmacia). Unincorporated dNTPs were removed with QIAquick PCR purification kit (QIAGEN). Purified cDNA was processed for coupling reaction with Cy 5 or Cy 3-ester dye, respectively. Keywords: other Less...