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Yes, and rescue experiments are uniquely valuable for isoform-specific studies: • Construct design: use codon-modified PKM1 or PKM2 isoform-specific cDNAs (differ in exon 9 versus exon 10) with small C-terminal tags (FLAG, HA). Each isoform has the same catalytic architecture but different regulatory properties. • Isoform-specific rescue: separate rescue with PKM1 (constitutively active tetramer) or PKM2 (allosterically regulated) enables comprehensive isoform-function studies. • Allosteric mutant rescue: PKM2 K433E mutation disrupts FBP allosteric activation, useful for studying PKM2 regulation. • Functional readout: rescue should restore pyruvate kinase activity and glycolytic flux; isoform-specific rescue reveals distinct metabolic and proliferation phenotypes. A-549 transduces efficiently with lentivirus and supports systematic isoform-specific rescue experiments.

Primary applications: • PKM1 vs PKM2 isoform-specific rescue: separate rescue with PKM1 or PKM2 cDNAs enables comprehensive isoform-function studies in cancer metabolism context. • Pyruvate kinase activity: cellular pyruvate kinase activity (lactate generation, ¹³C-glucose tracing) characterization. • Warburg effect studies: glycolytic flux analysis (Seahorse ECAR) and metabolite levels under glucose-replete and -depleted conditions. • PKM2 activator pharmacology: critical genetic control for TEPP-46, DASA-58, and other PKM2 tetramerization activators in cancer drug development. EDITGENE recommends this model for researchers investigating cancer metabolism, Warburg effect mechanisms, and PKM2-targeted therapeutic development.

The choice depends on whether you are studying PKM (pyruvate kinase muscle isoform)'s role in glycolysis or its functions as the principal Warburg-effect-associated PKM2 isoform in cancer. The Knockout line is the standard tool for asking whether PKM is required for cellular pyruvate generation — PKM produces two splice isoforms, PKM1 (constitutively active tetramer) and PKM2 (allosterically regulated, dimer-tetramer dynamic), with PKM2 predominating in cancer cells and supporting metabolic flexibility through reduced enzymatic activity. Overexpression is useful for studying isoform-specific PKM functions or for testing PKM2 activators. Important consideration: PKM knockout eliminates both PKM1 and PKM2 isoforms — PKLR (liver-erythrocyte PK) provides limited compensation in non-hepatocyte contexts. For cancer metabolism research, the EDITGENE PKM Knockout in A-549 is highly relevant — A-549 is an NSCLC model expressing predominantly PKM2 isoform, and PKM2 is a validated cancer metabolic target. Rescue with isoform-specific cDNAs (PKM1 versus PKM2) enables comprehensive isoform-function studies. The knockout is a critical specificity control for PKM2 activators (TEPP-46, DASA-58) and PKM2-selective inhibitors in cancer drug development.

Yes. NLRP3 rescue experiments are well-established for microglial neuroinflammation research: • Construct design: use a codon-modified Nlrp3 sequence with a small C-terminal tag (FLAG, HA). Mouse Nlrp3 has N-terminal PYD, NACHT, and C-terminal LRR — preserve all elements. • CAPS mutation rescue: patient-derived activating mutations (R262W, D305N, Y570C, V200M corresponding human numbering) introduced for genotype-function studies of cryopyrin-associated periodic syndromes. • Activation-deficient rescue: Walker A motif mutations in NACHT abolish NTP binding and inflammasome activation. • Functional readout: rescue should restore LPS-priming + nigericin-induced ASC speck formation, caspase-1 cleavage, IL-1β/IL-18 release, and gasdermin-D-mediated pyroptosis. BV-2-specific considerations: • BV-2 is an immortalized murine microglial cell line (v-raf/v-myc transformed C57BL/6 microglia) — the most widely used continuous microglial cell line for in vitro neuroimmunology research. • Lentiviral transduction is supported with moderate efficiency; characterize basal microglial activation state (M1/M2 markers) before phenotypic assays. • BV-2 retains key microglial markers (CD11b, Iba1, CD68) and TLR/inflammasome responses, but immortalization may alter some primary microglial features — confirm relevant phenotypes in independent assays.

The choice depends on whether you are studying NLRP3 inflammasome activation in microglia or modeling NLRP3-mediated neuroinflammation in Alzheimer's disease, Parkinson's disease, and other neurodegenerative contexts. The Knockout line is the standard tool for asking whether NLRP3 is required for microglial inflammasome assembly — NLRP3 in microglia responds to amyloid-β fibrils, α-synuclein aggregates, tau, and other DAMPs implicated in neurodegeneration. Overexpression is useful for studying CAPS-associated gain-of-function NLRP3 mutations. For neuroinflammation research, the EDITGENE Nlrp3 Knockout in BV-2 is uniquely valuable — BV-2 is the most widely used immortalized murine microglial cell line, providing a tractable system for studying microglial NLRP3 biology relevant to neurodegenerative disease. Rescue with wild-type or CAPS-associated activating mutant (e.g., R262W, D305N, Y570C) NLRP3 enables comprehensive disease genotype-function studies. The knockout is a critical specificity control for MCC950/CRID3 (and clinical candidates inzomelid, somalix) and dapansutrile (OLT1177, in clinical trials for heart failure and gout) in neurological drug development.

Primary applications: • Microglial inflammasome activation: LPS-priming followed by NLRP3 activators (nigericin, ATP, monosodium urate crystals) to characterize NLRP3-dependent IL-1β/IL-18 release. • Neurodegeneration-relevant activation: amyloid-β fibrils, α-synuclein, tau, and other CNS DAMP-induced NLRP3 activation studies in microglia. • CAPS mutation modeling: rescue with patient-derived activating mutations (R262W, D305N, Y570C, V200M) for genotype-function studies of cryopyrin-associated periodic syndromes. • NLRP3 inhibitor specificity: critical genetic control for MCC950/CRID3 (and clinical candidates inzomelid, somalix), dapansutrile (OLT1177), and emerging NLRP3 inhibitors in neurodegenerative disease drug development. EDITGENE recommends this microglial model for researchers investigating neuroinflammation, NLRP3-mediated neurodegenerative disease mechanisms, and CNS-targeted NLRP3 inhibitor development.

Transfecting suspension cells is generally more challenging. However, due to its superior performance, this product works efficiently not only in adherent cells but also in suspension cells. For example, in Jurkat cells, 48 hours post-transfection, editing efficiency can reach up to 97%, demonstrating the product’s high efficiency in suspension cell transfection and meeting demanding requirements.

The product utilizes advanced biomolecular transfection technology. Compared with the toxicity of traditional chemical transfection methods and the physical stress of electroporation, it shows significant advantages in preserving cell viability.

If gene knockout fails when using this kit, EDITGENE will not charge for the kit. Additionally, the fee you paid for the kit can be directly applied toward EDITGENE’s gene knockout service, ensuring that your gene editing experiments proceed without concerns.

The product has been validated in multiple cell types. The RNP system enters cells and begins functioning within 4 hours post-transfection, and the Cas9 protein is degraded within 24-48 hours. This transient, high-efficiency expression enables gene editing without the need for continuous selection.

While suspension cells are generally more difficult to transfect, this kit performs exceptionally well in both adherent and suspension cell types. For example, in Jurkat cells, editing efficiency can reach up to 97% within 48 hours post-transfection, demonstrating the kit’s outstanding performance and suitability for demanding suspension cell applications.

This kit has been validated across multiple cell lines. The RNP complex enters cells and begins gene editing within 4 hours post-transfection. Cas9 protein is degraded within 24–48 hours, allowing efficient and transient expression-driven editing without the need for antibiotic or fluorescent selection.

The kit employs advanced biomolecular transfection technology, offering significant advantages over traditional methods. Unlike chemical transfection, which may be cytotoxic, or electroporation, which can subject cells to physical stress, this approach ensures minimal damage while maintaining high efficiency.

The kit can detect Mycoplasma in common cell culture reagents, such as media and serum, without the need for sample preparation. Simply proceed directly to PCR reaction and gel loading. However, the kit cannot detect Mycoplasma in organic solvents like DMSO or ethanol.

Use a freshly opened negative control for retesting. Ensure to change pipette tips during sample loading and prioritize loading the negative control first to avoid cross-contamination between samples.