IRS1 Knockout HAP1 Cell Line

The choice depends on whether you are studying IRS1 (insulin receptor substrate 1)'s role as the principal insulin/IGF-1 signaling scaffold or modeling its functions in insulin resistance and metabolic disease. The Knockout line is the standard tool for asking whether IRS1 is required for these processes — IRS1 is tyrosine-phosphorylated by the activated insulin receptor (INSR) and IGF-1R kinase, recruiting PI3K p85, GRB2, and other SH2-domain proteins to drive PI3K-AKT and other downstream pathways. Overexpression is useful for studying IRS1 in heterologous expression contexts. Important consideration: IRS2 paralog expression analysis is essential given functional overlap. This product complements the parallel INSR Knockout in HAP1 (also available) for upstream-downstream insulin signaling pathway dissection. Rescue with wild-type or Y608F/Y628F/etc. phospho-resistant IRS1 enables structure-function studies. The knockout is valuable for studying serine kinase-mediated IRS1 inhibition (mTORC1, JNK, S6K1 phosphorylate IRS1 at serine residues to inhibit insulin signaling) in insulin resistance research.

Primary applications: • Insulin/IGF-1 signaling: phospho-IRS1 (Y608, Y628, others), PI3K p85 recruitment, and phospho-AKT analysis following insulin or IGF-1 stimulation in IRS1-null cells. • Insulin resistance modeling: phospho-IRS1 (S307, S612, others) serine phosphorylation analysis given mTORC1/JNK/S6K1-mediated insulin resistance. • IRS2 paralog studies: IRS2 expression analysis to interpret IRS1-specific functions versus paralog compensation. • Combined INSR+IRS1 studies: parallel analysis with INSR Knockout in HAP1 (also available) for upstream-downstream insulin pathway dissection. EDITGENE recommends this model for researchers investigating insulin signaling, insulin resistance mechanisms, and IRS-mediated PI3K-AKT pathway regulation.

Yes. IRS1 rescue experiments require attention to scaffolding architecture: • Construct design: use a codon-modified IRS1 sequence with a small C-terminal tag (FLAG, HA). IRS1 has N-terminal PH and PTB domains (INSR/IGF1R interaction), and large C-terminal region with multiple tyrosine phosphorylation sites (YxxM motifs for PI3K p85 binding) and serine sites (negative regulation) — preserve all elements. • Phospho-tyrosine-resistant rescue: Y608F, Y628F, Y939F mutations in PI3K-binding YxxM motifs abolish PI3K recruitment. • Phospho-serine-resistant rescue: S307A, S612A mutations remove inhibitory serine phosphorylation sites, generating constitutively active IRS1. • Functional readout: rescue should restore insulin/IGF-1-induced phospho-IRS1 (Y608), PI3K recruitment, and phospho-AKT. HAP1-specific considerations: • Diploidization: HAP1 cells gradually diploidize during extended culture — confirm ploidy by flow cytometry at the time of phenotypic assay. • Integration site sensitivity: position effects on transgene expression are more pronounced in near-haploid backgrounds; generating multiple independent rescue clones is strongly recommended. • Transduction efficiency: HAP1 transduces with lentivirus at moderate efficiency — increase MOI compared to standard immortalized lines.