TOL2 Antibody

FAQs for TOL2 Antibody Research

Advanced Research Questions

How to resolve contradictory data on TOL2-mediated mutagenesis phenotypes?

• Method: Apply revertant analysis.

  • Generate revertant alleles by re-exposing mutant lines to Tol2 transposase .

  • Compare phenotypic rescue (e.g., nup214 revertants showed 100% viability vs. mutant lethality) .

• Critical step: Confirm causality via complementation assays (e.g., morpholino knockdown rescue) .

What strategies optimize TOL2-driven transgene expression in mammalian suspension cultures?

• Protocol:

  • Use dual-promoter vectors (e.g., CMV for transgene + CHX resistance gene) .

  • Co-transfect with Tol2 transposase plasmid (1:2.5 ratio of transposon:transposase) .

  • Apply CHX selection (10–30 μM) post-transfection to isolate high-producer clones .

• Outcome: Clonal lines maintain >90% productivity stability over 12 weeks .

How to address off-target effects in TOL2-based CAR-T cell engineering?

• Methodology:

  • Map integration sites via linear amplification-mediated PCR (LAM-PCR) .

  • Compare profiles to random viral integrations (e.g., lentivirus).

  • Data: Tol2 shows reduced genotoxicity risk vs. Sleeping Beauty (SB) in T-cells .

Data Contradiction Analysis

Discrepancies in TOL2 excision efficiency across studies

Variable glycosylation profiles in TOL2-engineered antibodies

• Root cause: Tol2 integration sites influence promoter accessibility .

• Solution: Use site-specific integration systems (e.g., CRISPR-Tol2 hybrids) to target safe harbors .

Interdisciplinary Applications

Can TOL2 be adapted for in vivo disease modeling?

• Case study: Tol2-mediated nup214 mutagenesis replicated nuclear pore complex defects, validated via revertant analysis .

• Design: Use tissue-specific promoters to limit transposase expression (e.g., pax6b for ocular studies) .

How to integrate TOL2 with CRISPR for high-throughput screens?

• Workflow:

  • Clone sgRNA libraries into Tol2 vectors.

  • Co-deliver with Tol2 transposase and Cas9 mRNA.

  • Apply phenotypic selection (e.g., CHX resistance) .

Key Citations

• Germline excision efficiency

• CHO cell antibody production

• CAR-T cell engineering