Project description:KLF3, a member of the Krüppel-like factor (KLF) family, is expressed in a wide range of cell types. It is involved in hematopoiesis of several blood cell lineages including erythrocyte and B lymphocyte. However, the research of regulatory roles on hematopoiesis of KLF3 in K562 cells has been still largely limited. To comprehensively assess the regulatory roles of KLF3 on hematopoiesis in K562 cells, a microarray analysis was performed in KLF3-deficient K562 cells. The differentially expressed genes were applied to IPA analysis to observe the perturbed hematopoiesis-associated functions, networks, and molecular pathways. This study will extensively assess the regulatory roles of KLF3 on hematopoiesis in K562 cells. We used microarrays to study the genome-wide expression profiles of KLF3-deficient K562 cells.
Project description:K562-shX cells are made in an effort to validate TFBS data and ChIP-seq antibodies in Myers lab (GSE32465). K562 cells are transduced with lentiviral vector having Tet-inducible shRNA targeting a transcription factor gene. Cells with stable integration of shRNA constructs are selected using puromycin in growth media. Doxycycline is added to the growth media to induce the expression of shRNA and a red fluorescent protein marker. A successful shRNA cell line shows at least a 70% reduction in expression of the target transcription factor as measured by qPCR. For identification, we designated these cell lines as K562-shX, where X is the transcription factor targeted by shRNA and K562 denotes the parent cell line. For example, K562-shATF3 cells are K562 derived cells selected for stable integration of shRNA targeting the transcription factor ATF3 gene and showed at least a 70% reduction in the expression of ATF3 gene when measured by qPCR. Cells growing without doxycycline (uninduced) are used as a control to measure the change in expression of target transcription factor gene after induction of shRNA using doxycycline. For detailed growth and culturing protocols for these cells please refer to http://hudsonalpha.org/sites/default/files/pdf/shRNA%20K562%20protocol.pdf. To identify the potential downstream targets of the candidate transcription factor, analyze the mRNA expression profile of the uninduced and induced K562-shX using RNA-seq. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Make K562-shX cells as described in the http://hudsonalpha.org/sites/default/files/pdf/shRNA%20K562%20protocol.pdf. Measure the mRNA expression levels in uninduced K562-shX and induced K562-shX cells in two biological replicates using RNA-seq. Identify the potential downstream targets of the candidate transcription factor.