The AFF4 Knockout HEK293T Polyclonal Cells represent a CRISPR/Cas9-mediated gene-disrupted polyclonal population derived from the HEK293T human embryonic kidney cell line. This product provides a loss-of-function model for studying AFF4, a scaffold protein critical for the assembly and function of the super elongation complex (SEC) that regulates transcriptional elongation by RNA polymerase II. The polyclonal format ensures a heterogeneous knockout population, suitable for pooled functional screens and bulk biochemical analyses, without claims of monoclonality or complete biallelic disruption.
The host HEK293T cell line is a widely used derivative of HEK293, stably expressing the SV40 large T antigen, which facilitates episomal replication of plasmids containing the SV40 origin. This cell line is easily transfectable, making it an ideal host for gene-editing experiments, and is routinely cultured in DMEM supplemented with 10% fetal bovine serum. HEK293T cells are extensively used for protein expression, viral production, and signal transduction studies, providing a robust background for investigating transcriptional mechanisms.
AFF4 encodes a scaffold protein essential for assembling the super elongation complex (SEC), which includes P-TEFb (CDK9/Cyclin T1), ELL2, ENL, AF9, and DOT1L, among other factors. This complex is recruited to promoters by BRD4 and other adaptors, where P-TEFb phosphorylates the carboxyl-terminal domain of RNA polymerase II to release paused polymerase and enable productive elongation. Consequently, AFF4 is a key regulator of genes downstream of MLL fusion proteins, including HOXA9, HOXA10, MEIS1, and MYC, which are critical for hematopoiesis and leukemogenesis. Disruption of AFF4 impairs SEC integrity, reducing transcriptional elongation of these targets and providing a versatile tool for dissecting SEC-dependent pathways.
In HEK293T cells, the AFF4 knockout model enables targeted investigation of SEC function in a well-characterized epithelial environment, complementary to hematopoietic models. While HEK293T is not a leukemia line, its robust transfectability and genetic tractability allow for detailed structure-function analyses, including reconstitution experiments with mutant AFF4 constructs. The polyclonal nature of this population supports studies on AFF4-dependent transcriptional programs without clonal artifacts, and it can be used to explore the interplay between the SEC and other chromatin regulators. This model is particularly relevant for understanding how SEC dysregulation contributes to MLL-rearranged acute leukemia, as AFF4 is a critical cofactor for MLL chimeric proteins.
Typical applications include chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) to measure RNA polymerase II occupancy, reverse-transcription quantitative PCR (RT-qPCR) to assess expression of downstream targets like MYC and HOXA9, and RNA sequencing (RNA-seq) for genome-wide transcriptional profiling. Additionally, co-immunoprecipitation and western blotting can evaluate SEC complex assembly and the expression of interacting factors such as CDK9, Cyclin T1, and ELL2. In leukemia functional genomics, these cells serve as controls for drug target validation or proliferation and viability assays with oncogene overexpression. For further information, contact Ascent Research.
