RNASEH1 Knockout Cell Line (HEK293)

The choice depends on whether you are studying RNase H1's role in degrading RNA:DNA hybrids (R-loops) at replication-transcription conflicts or its essential function in mitochondrial DNA replication. The Knockout line is the standard tool for asking whether RNase H1 is required for R-loop resolution and mitochondrial DNA primer removal — RNase H1 is the primary cellular RNase H activity for these processes. Overexpression is useful for studying R-loop biology or for testing RNase H1's role in antisense oligonucleotide mechanism. Important consideration: RNase H1 is essential for mitochondrial DNA replication — complete knockout may impair mitochondrial function and viability. Characterize growth phenotypes before extended use. For R-loop biology research, the EDITGENE RNase H1 Knockout in HEK293 is a standard mechanistic platform. RNASEH1 mutations cause adult-onset autosomal recessive mitochondrial DNA depletion syndrome with Charcot-Marie-Tooth disease (CMT2 phenotype) — disease variant rescue enables genotype-function studies. Rescue with wild-type or catalytically-dead (D210N) RNase H1 is the standard specificity control.

Primary applications: • R-loop quantification: S9.6 antibody-based imaging or DRIP-seq to quantify R-loop accumulation in the absence of RNase H1. • Mitochondrial DNA replication: mtDNA copy number measurement and replication intermediates analysis given RNase H1's essential mitochondrial function. • Antisense oligonucleotide (ASO) mechanism: testing gapmer ASO activity — RNase H1 is required for the cleavage step of ASO-mediated mRNA knockdown. • CMT2 disease modeling: rescue with patient-derived RNASEH1 mutations for genotype-function studies of mitochondrial DNA depletion syndrome with neuropathy. EDITGENE recommends this model for researchers investigating R-loop biology, mitochondrial DNA replication, antisense therapeutic mechanism, and RNASEH1-related neuropathy.

Yes. RNase H1 rescue experiments require attention to dual cellular localization: • Construct design: use a codon-modified RNASEH1 sequence with a small C-terminal tag (FLAG, HA). RNase H1 is expressed from two alternative start codons producing nuclear and mitochondrial isoforms — isoform-specific rescue may be needed. • Catalytically-dead rescue: the D210N mutation in the catalytic site abolishes RNase H activity and is the standard specificity control. • Localization-specific rescue: rescue with nuclear-only or mitochondrial-only RNase H1 enables compartment-specific function dissection. • Functional readout: rescue should restore R-loop resolution (S9.6 immunofluorescence reduction), mitochondrial DNA replication, and ASO-mediated mRNA cleavage activity. HEK293 transduces efficiently with lentivirus and supports stable rescue line generation.