The AFF1 Knockout HT29 Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal cell population derived from the HT29 human colorectal adenocarcinoma line, enabling loss-of-function investigations of AFF1. These cells harbor targeted disruptions in the AFF1 gene introduced via CRISPR/Cas9, producing a heterogeneous knockout pool that reduces clonal bias. This polyclonal format is ideal for functional studies of AFF1-dependent transcriptional elongation in an epithelial cancer model.
HT29 is a widely utilized human colorectal adenocarcinoma cell line with epithelial morphology, established from a primary colorectal tumor. It serves as a model for intestinal epithelial cells and is commonly applied in cancer research, differentiation studies, and drug screening. HT29 cells carry mutations in p53 and APC, making them a relevant platform for examining signaling pathways controlling proliferation, apoptosis, and transcriptional regulation in colorectal cancer.
AFF1 encodes a scaffold protein integral to the super elongation complex (SEC), which regulates RNA polymerase II (Pol II) transcription elongation. AFF1 recruits positive transcription elongation factor b (P-TEFb), composed of CDK9 and Cyclin T1, to phosphorylate serine 2 of the Pol II C-terminal domain, facilitating productive elongation. It directly interacts with SEC components ELL, ENL, and AF9, and its activity is modulated by upstream MLL fusion proteins and CDK9. AFF1 also bridges the SEC to MENIN and DOT1L, linking elongation to epigenetic modifications. Downstream, AFF1 promotes expression of target genes including the HOXA cluster, MEIS1, MYC, and BCL2, which are critical in oncogenesis.
Though AFF1 is predominantly associated with mixed-lineage leukemia, where MLL-AFF1 fusions drive constitutive HOX and MEIS1 activation, the general transcriptional role of SEC suggests AFF1 may influence gene expression in solid tumors. In HT29 colorectal adenocarcinoma cells, AFF1 knockout allows dissection of SEC-dependent pathways in an epithelial context, potentially affecting MYC and BCL2 levels and thereby modulating proliferation and apoptosis. This model expands the utility of AFF1 research into non-hematopoietic cancers.
Key experimental applications include Western blot validation of AFF1 knockout, RT-qPCR for HOX and MEIS1, and RNA-seq for transcriptome analysis. Functional assays such as cell proliferation, apoptosis, and drug sensitivity tests with CDK9 or DOT1L inhibitors can be performed, along with flow cytometry for cell cycle analysis. Co-immunoprecipitation experiments can assess SEC complex integrity in the knockout cells. These applications support studies of transcriptional elongation, MLL-rearranged leukemia drug target validation, and broader AFF1 roles in cancer. For additional information, contact Ascent Research.
