In chicken DT40 cells, there are six linker histone H1 variants and 12 of coding genes. We have previously reported of 11 out of 12 H1 knock out DT40 cells (Takami et al., Genes to Cell 1997 [PMID:9491804] but complete H1 null DT40 cells could not established, so far. We identified one of the H1 variant, H1R was involved in genomic instabilities(Hashimoto et al., DNA repair (2007) [17613284], so we re-introduced floxed H1R-eGFP and mer-cre-mer into 11 out of 12 H1 knock out DT40 cells. Then we targeted last enedogenous H1, we successfully established conditional H1 KO cells (K11). Then we treated with tamoxifen to loop out floxed H1R-eGFP, and cloning H1 completely null cells (K11-5, and K11-7). We analyzed those gene expression pattern in wild-type, K11, and K11-5 cells.
Treatment protocol
Apoptosis is induced in H1 null cells, we culture DT40 cells to inhibit apoptosis with pan-caspase inhibitor, 100 nM of Z-VAD-FMK.
Growth protocol
Cells were cultured in RPMI1640, 10% FCS, 1% chicken serum, 2 mM L-glutamin, 50 micro-M of 2-Mercaptoethanol, and 100 nM of Z-VAD-FMK at 39.5C
Extracted molecule
total RNA
Extraction protocol
From exponately growing DT40 cells, Total RNAs were extracted with Sepasol I (Nakarai) and RNeasy (QIAGEN) as vendor's mannuals
Label
Used Affymetrix one cycle labeling Kit
Label protocol
Following Affymetrix one cycle labeling Kit mannuals
Hybridization protocol
Following Affymetrix protocols
Scan protocol
Using Affymetrix scanner and mannuals
Description
Study to compare gene expression in wild type, K11 and K11-5 cells
Data processing
Affymetrix CHP files including row data were generated with GCOS
