Effect of MnSOD knockout cells on gene profile expression

ABSTRACT: MnSOD is an essential primary antioxidant enzyme that converts superoxide radicals and protons to hydrogen peroxide (H2O2) within the mitochondrial matrix, generated by respiratory chain activity We used microarrays of cells knocked down for MnSOD and a mock transfected cells as their control (siScramble) to reveal changes in gene expression profile WT-AML-12 cells silenced (si) for MnSOD were prepared, briefly: The backbone vector pSUPER.retro.puro was used for silencing. Transcripts of 60-nt long oligos constituting hairpin RNAs were designed to generate a low level of MnSOD silencing: a nine-loop (9 L) hairpin with 20 nt of MnSOD sense (siMnSOD) and a 9 L hairpin with 20 nt of scramble-no gene recognition control sense (Scr). Retroviral particles were produced by triple transfection of HEK 293T cells (ATCC No. CRL-11268T) with the retroviral vector (2 ug), pMD-gag-pol (1 ug) and pVSV-G (1 ug) using Trans-IT (Mirusbio Corporation, Madison, WI). For stable transfection, the various retroviruses were introduced with WT-AML-12 hepatocytes using polybrene (Sigma-Aldrich, St. Louis, MO, USA) in a six-well plate followed by selection with 10 ug puromycin for 2 weeks. To identify sets of genes that are differentially expressed in siMnSOD AML-12 cells compared to Scr-no sense control AML-12 cells, a Gene Chip approach was utilized. Total RNA was extracted (using TRI reagent) and purified using the RNeasy kit (Qiagen, Valencia, CA). Three different batches of frozen cells were taken to microarray analysis. Microarray analysis was performed on an Affymetrix Mouse Gene 1.0 ST chip containing 28,853 genes. The chips were processed and scaled at the Weizmann Institute of Science (Rehovot, Israel) using Affymetrix MAS5.

ORGANISM(S): Mus musculus

SUBMITTER: Michal pardo 

PROVIDER: E-GEOD-14855 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress