The KRT3 Knockout HEK293T Polyclonal Cells product provides a heterogenous population of HEK293T cells harboring CRISPR/Cas9-mediated disruptions of the KRT3 gene, which encodes the type II intermediate filament protein keratin 3. This polyclonal knockout pool eliminates functional keratin 3 expression, establishing a versatile cellular background for interrogating KRT3 biology. The editing approach targets the genomic locus of KRT3 to create a loss-of-function model, avoiding reliance on transient knockdown methods and enabling stable, long-term studies of keratin 3-deficient phenotypes.
The parental HEK293T cell line is an adherent epithelial line derived from human embryonic kidney tissue and transformed with sheared adenovirus type 5 DNA. These cells constitutively express the SV40 large T antigen, which promotes episomal replication of plasmids containing the SV40 origin, thereby supporting high-efficiency transient transfection and robust recombinant protein production. The well-characterized, fast-growing nature of HEK293T cells makes them a preferred host for CRISPR-based genome editing, viral packaging, and biochemical assays.
Keratin 3 is an essential structural component of corneal epithelial cells, where it forms obligate heterodimers with keratin 12 (KRT12) to assemble intermediate filament networks that maintain mechanical integrity and transparency of the cornea. KRT3 expression is transcriptionally regulated by several factors, including PAX6, KLF4, SP1, and TGF-??1, which orchestrate corneal epithelial differentiation programs. The protein interacts directly with KRT12 and chaperones such as HSP70, as well as 14-3-3 scaffold proteins, and participates in a network that includes desmoplakin, plakoglobin, and transglutaminase-1. Disruption of keratin 3 leads to compromised intermediate filament organization, altered epithelial cell mechanics, and recapitulates key features of Meesmann corneal dystrophy and related fragility syndromes.
Although HEK293T cells do not natively express KRT3 or form mature keratinocyte-like intermediate filament arrays, their use as a knockout host provides a clean null background that circumvents compensatory effects from other keratins present in corneal cell lines. Ectopic expression of wild-type or mutant KRT3 variants in these knockout cells permits precise dissection of filament assembly kinetics, protein-protein interaction requirements, and the pathogenic mechanisms underlying inherited corneal dystrophies. The model is especially useful for interrogating how upstream signaling by TGF-??1, via downstream effectors that include KLF4 and SP1, modulates KRT3 expression and influences the biophysical properties of the intermediate filament cytoskeleton.
Typical research applications include functional assays for KRT3 missense mutations identified in Meesmann corneal dystrophy patients, high-content imaging screens to identify small molecules that correct filament network defects, and co-immunoprecipitation experiments to map interaction interfaces with KRT12 and 14-3-3 proteins. The polyclonal knockout population is compatible with standard readouts such as Western blotting, immunofluorescence, RT-qPCR, and wound healing assays, enabling quantitative analysis of keratin 3-dependent cellular processes. For additional information, including lot-specific validation data and custom engineering options, please contact Ascent Research.
