pLenti-UQCRH-sgRNA

Cat. No.: P751663

사양

Product Name

pLenti-UQCRH-sgRNA

Product Line

생물 제품

보관 및 배송

보관 조건

Store at -80°C

배송

Dry ice packs + Cold packs

안정성 및 스토리지

Stored at -20 ℃, valid for 3 months;Stored at -80 ℃, valid for at least one year.

Application Protocols

No item-specific, tested application protocols are provided in the Product Data. The following outlines general workflows; adjust to your cell model and consult the vector map/CoA.

A. Lentiviral transduction (general):

1. Thaw on ice. Optional spin to collect condensate; keep cold.
2. Seed target cells 12–24 h prior to achieve 40–70% confluence at transduction.
3. Add virus to cells at desired MOI in serum-free medium for 4–8 h; optionally include polybrene if compatible.
4. Replace with complete medium; incubate 48–72 h.
5. Apply selection if the backbone includes a selectable marker (not specified here). Validate editing at 3–7 days.

B. Plasmid transfection (general):

1. Use nuclease-free technique; prepare complexes per transfection reagent instructions.
2. Co-deliver Cas9 (expression plasmid or RNP) if cells do not express it.
3. Change medium after 6–24 h; analyze editing after 48–96 h.

Controls and QC:

• Include non-targeting and positive-control sgRNAs; confirm on-target edits via TIDE/ICE or NGS.
• For mitochondrial phenotypes, assess OCR/ECAR or ETC complex activities.

Note: Volumes, reagent ratios, selection antibiotics, and titer are not specified for this SKU; refer to the CoA/spec sheet for any product-specific recommendations.

Biological Roles

Target gene: UQCRH (ubiquinol-cytochrome c reductase hinge protein), a component associated with the mitochondrial cytochrome bc1 complex (Complex III) of the electron transport chain.

Biological context (literature):

• Function: UQCRH contributes to electron transfer from ubiquinol to cytochrome c within Complex III, influencing proton motive force and ATP production via oxidative phosphorylation.
• Localization: Mitochondrial inner membrane as part of the respiratory supercomplex landscape.
• Pathway integration: Impacts mitochondrial membrane potential, ROS generation, and metabolic flux through TCA–ETC coupling. Perturbation can alter cellular redox balance and respiratory capacity.
• Genetic studies: CRISPR-mediated disruption of UQCRH is commonly used to interrogate Complex III dependency, metabolic plasticity, and compensatory responses (e.g., glycolytic upregulation) under varying oxygen or nutrient conditions.

Reagent role:

• The pLenti-UQCRH-sgRNA construct provides a programmable guide RNA to direct Cas9/dCas9 to UQCRH genomic loci for knockout or expression modulation experiments.

Note: Specific guide sequence(s), exon targeting, and any validated on-target efficiency metrics are not provided in the Product Data; consult the CoA for sequence-level details to align with your genome build and isoform of interest.

Buffer Applications

This product is not a buffering reagent. It is a genetic tool (sgRNA in a lentiviral/plasmid backbone) and is not used to prepare or maintain pH in biochemical assays.

Practical note:

• If supplied in an aqueous buffer, that buffer is part of the formulation vehicle for stability and sterility only. Composition and pH are not specified for this item; refer to the CoA/Spec Sheet if you need compatibility information with downstream assays.

Green Alternatives

Greenness considerations for nucleic acid/viral reagents focus on biosafety, cold-chain efficiency, and consumable usage rather than solvent replacement.

Opportunities (general):

• Reduce freeze–thaw cycles by aliquoting once, decreasing waste from degraded material.
• Use reusable cold packs and validated insulated shippers to minimize dry ice consumption without compromising stability (follow your institution’s policies).
• Prefer enzymatic clean-up kits and aqueous workflows over phenol–chloroform for downstream nucleic acid processing.
• Consolidate transductions to minimize single-use plastics; employ multi-channel setups to reduce tip usage where sterility permits.

Trade-offs:

• High-titer viral preparations may require ultracentrifugation or precipitation steps that consume energy and materials; consider titer needs carefully to avoid over-concentrating beyond experimental requirements.

Comparison (conceptual):

• Conventional approach: Frequent re-thawing of a master vial; larger plastic waste from repeated aliquoting after thaw.
• Greener approach: Single thaw, immediate sterile aliquoting into low-bind tubes, barcoded inventory management to reduce losses and repeated shipments.

As always, maintain biosafety compliance and product integrity; do not compromise required containment or cold chain in pursuit of greener practices.

Pharmaceutical Uses

This product is designated For research use only and is not intended for diagnostic, therapeutic, or clinical manufacturing purposes.

Context (general, non-clinical):

• sgRNA lentiviral constructs are widely used in discovery research, target validation, and process development studies in preclinical laboratories. They may inform, but are not themselves, GMP-grade materials.
• If translational work is contemplated, separate sourcing of GMP-grade plasmids/viral vectors, comprehensive release testing (sterility, endotoxin, identity), and regulatory documentation are required—outside the scope of this SKU.

No pharmacopeial status, excipient role, or clinical formulation use is specified or implied for this item.

Physical Properties

Item-specific physicochemical parameters are not provided in the Product Data and cannot be inferred.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Concentration/format (plasmid DNA vs. viral particles): Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility: As a nucleic acid/viral reagent, typically supplied in an aqueous buffer (literature). Exact buffer for this item is not specified.
• pH of formulation: Not specified for this item; refer to CoA/Spec Sheet.
• BP/MP, density, refractive index, logP: Not applicable to macromolecular nucleic acid constructs.

Literature/contextual notes (general):

• Plasmid DNA is water-soluble and commonly provided in nuclease-free water or Tris-EDTA; lentiviral stocks are typically in isotonic, serum-free aqueous buffers. Final osmolality and excipients impact particle stability but are not specified here.
• For practical handling, viscosity and turbidity can indicate high-titer lentiviral preparations; however, rely on titer data if provided on the CoA rather than appearance.

Conclusion: Consult the CoA/specification sheet for concentration, buffer composition, nucleic acid length, and (if applicable) viral titer, as none are specified for this SKU.

Quality and Grades

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
• Research Use Only: For research use only (Product Data). Not for diagnostic or therapeutic use.

Interpretation and context (general):

• For plasmid-format sgRNA reagents, quality is typically described by sequence verification (Sanger/NGS), supercoiled fraction, residual endotoxin level, and A260/280 ratios. None of these item-specific metrics are provided here.
• For lentiviral-format sgRNA reagents, quality is often defined by functional titer (TU/mL), absence of replication-competent virus (RCL testing), sterility, and mycoplasma testing. Item-specific values are not provided.
• Stabilizers or cryoprotectants (e.g., serum albumin, sucrose) may be included in some viral formulations to enhance freeze–thaw stability; this is not specified for this SKU.

Practical guidance:

• Request and review the CoA/specification sheet for sequence identity of the UQCRH guide, vector map, backbone features (promoters, selection markers), QC methods, and acceptable ranges.
• If using for screening, consistency of lot-to-lot titer (viral) or supercoiled fraction (plasmid) is important; consider reserving a single lot for multi-batch studies to minimize variability.

Reaction and Applications

Intended use (Manufacturer Applications): Not specified beyond research use; however, by name, pLenti-UQCRH-sgRNA is suited for CRISPR-based gene perturbation of UQCRH.

Typical applications (literature/general):

• Gene knockout (CRISPR-Cas9): Deliver sgRNA to cells stably or transiently expressing Cas9 to induce indels at the UQCRH locus.
• CRISPRi/CRISPRa: When paired with dCas9-KRAB or dCas9-VP64 systems, the same sgRNA scaffold can repress or activate transcription if guides target promoter regions.
• Stable cell line generation: Lentiviral delivery supports stable integration for long-term studies of mitochondrial Complex III function.
• Pooled or arrayed functional genomics: Use in screens to assess dependencies related to oxidative phosphorylation and mitochondrial biology.

Practical tips:

• Determine vector map features (promoter driving sgRNA, selection marker) from the CoA; optimize MOI for single-copy integration (e.g., MOI 0.2–0.5 in screening) or higher for rapid phenotyping, as appropriate.
• Validate on-target cutting by T7E1, ICE/TIDE analysis, or targeted amplicon NGS; functionally confirm by measuring Complex III activity or downstream respiration metrics.
• Off-target mitigation: Use high-specificity guide designs, truncated guides if appropriate, or paired nickase strategies. Incorporate non-targeting and safe-harbor controls.

Note: Exact guide sequence, PAM context, and backbone elements for this SKU are not specified; consult the provided map/CoA before experimental design.

Reaction Conditions

Experimental conditions depend on format and cell type. The Product Data do not specify format or titer; the following are general literature guidelines.

If plasmid format:

• Transfection: Use a DNA:transfection reagent ratio per vendor guidance; start with 1–2 µg DNA per well (6-well format) and optimize. Maintain cells at 60–80% confluence.
• Cas9 supply: Co-transfect Cas9 or use Cas9-stable cells. Assess editing 48–96 h post-transfection.

If lentiviral format:

• Packaging: Co-transfect transfer vector with packaging plasmids in HEK293T cells; harvest supernatant at 48–72 h. Filter through 0.45 µm. (Packaging system details are user-supplied; not part of this product.)
• Transduction: Aim for MOI appropriate to application (e.g., 0.2–0.5 for single-copy screens; 1–5 for rapid bulk edits). Optional polybrene 4–8 µg/mL if cell-type compatible.
• Selection: Apply antibiotic selection only if the backbone supports it; specifics are not provided for this SKU.

Readouts:

• Genotyping: TIDE/ICE or amplicon NGS for indel spectra.
• Functional assays: Measure oxygen consumption rate (OCR), Complex III activity, or cytochrome c reduction kinetics to confirm phenotypic impact of UQCRH perturbation.

Always consult the CoA/vector map for format, features, and any validated conditions for this SKU.

Safety and Handling

Safety information specific to this item is not provided in the Product Data.

• GHS classification, signal word, pictograms, H-statements: Not specified for this item; refer to SDS.
• Storage conditions: Store at -80°C (Product Data). Minimize freeze–thaw cycles.
• Shipping: Dry ice packs + cold packs (Product Data).

General laboratory safety guidance for lentiviral/sgRNA reagents (literature/best practice):

• Biosafety: Work under an appropriate BSL (often BSL-2 for third-generation, replication-defective lentiviral systems) according to your institution’s IBC approvals. Use a certified Class II biosafety cabinet when handling viral particles.
• PPE: Lab coat, gloves, and eye protection at minimum. Use double-gloving and sleeve covers for viral work. Change gloves frequently to avoid cross-contamination.
• Engineering controls: Use aerosol-resistant tips; decontaminate work surfaces with freshly prepared 0.5–1% sodium hypochlorite or appropriate virucidal agents; follow with 70% ethanol.
• Incompatibilities: Avoid nucleases (RNase/DNase) contamination; maintain nuclease-free technique for sgRNA constructs. For viral stocks, avoid foaming/vortexing which can shear envelopes.
• First aid (overview; defer to SDS): Skin/eye contact—flush with water. Inhalation—move to fresh air. Ingestion—seek medical attention. Report potential exposures according to institutional policy.

Always defer to the SDS, institutional biosafety guidelines, and the CoA for authoritative hazard and handling information.

Solvent Selection

This product is a nucleic acid/viral reagent and is typically supplied in an aqueous, nuclease-free buffer. Organic solvent selection is not applicable.

• Working medium (general): Use sterile, nuclease-free water or suitable cell culture medium/DPBS for dilutions, depending on format. Avoid organic solvents that can denature proteins or disrupt viral envelopes.
• Compatibility notes (literature): For lentiviral transduction, dilute in serum-free medium during adsorption to cells; add polycations (e.g., polybrene) only if compatible with your cell type. These additives are user-optional and not part of the product.

For extraction, chromatography, or non-aqueous chemistry, this section does not apply; see Reaction & Applications and Application Protocols for biologically relevant handling.

Storage and Reconstitution

• Storage: Store at -80°C (Product Data). Maintain a continuous cold chain. Avoid repeated freeze–thaw cycles.
• Shipping: Dry ice packs + Cold packs (Product Data).

Format-specific guidance (general, since format is not specified for this SKU):

• If lentiviral stock: Thaw on ice; gently mix by inversion—do not vortex. Keep on ice during setup. Aliquot immediately into single-use, low-bind, sterile tubes. Store working aliquots at -80°C. Avoid more than one freeze–thaw cycle.
• If plasmid DNA: Thaw at room temperature or on ice; briefly spin down. Store aliquots at -20°C to -80°C in nuclease-free buffer. Avoid repeated freeze–thaw; consider storing a master at -80°C and a working aliquot at -20°C.

Reconstitution:

• If received lyophilized (not specified): Reconstitute with nuclease-free water or TE to desired concentration; allow complete dissolution on ice. Actual diluent and target concentration should follow the CoA.

Record-keeping:

• Document aliquot IDs, freeze–thaw counts, and any observed changes in performance. Verify titer/concentration after initial aliquoting when possible.

For exact buffer composition, concentration, and any stabilizers, refer to the CoA/Spec Sheet, as these details are not specified for this item.

Structure and Identity

This product is a lentiviral sgRNA construct targeting the human UQCRH gene (ubiquinol-cytochrome c reductase hinge protein) supplied under the name pLenti-UQCRH-sgRNA.

• Item identity: pLenti-UQCRH-sgRNA (likely a plasmid backbone configured for lentiviral delivery of a single guide RNA)
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES: Not specified for this item; refer to CoA/Spec Sheet.
• InChIKey: Not specified for this item; refer to CoA/Spec Sheet.
• CAS/CID: Not specified for this item; refer to CoA/Spec Sheet.

Structural features (general, literature):

• Typical pLenti sgRNA vectors contain: a U6 or H1 RNA polymerase III promoter driving the sgRNA, a gRNA scaffold (tracrRNA sequence), and lentiviral LTR/packaging elements enabling virus production in trans when co-transfected with packaging plasmids.
• Optional features that may be present in some pLenti backbones include a selection marker (e.g., puromycin, blasticidin) and a reporter (e.g., GFP). Vector-specific features for this item are not specified; consult the vector map/CoA.

2D “structure” description (general):

• As a nucleic acid construct, the functional architecture is modular: 5′ LTR – ψ packaging signal – promoter – sgRNA cassette (target-specific spacer + scaffold) – selection/reporter elements (if any) – 3′ LTR. Exact module composition and spacer sequence for this SKU are not provided in the Product Data.

Synthetic Utility

Traditional organic synthetic utility does not apply. Instead, consider molecular biology “synthetic” workflows where this reagent is central.

Genetic engineering utility (general):

• Modular cloning: sgRNA spacer can be exchanged in many pLenti backbones using type IIS restriction sites or Gibson/Golden Gate assembly (vector-specific details not provided for this SKU).
• Combinatorial perturbations: Multiple guides (if backbone supports) enable dual targeting; otherwise, serial transductions can generate multi-gene knockouts.
• Integration with reporters: Coupling sgRNA delivery with transcriptional reporters facilitates high-content readouts of mitochondrial function upon UQCRH perturbation.

Retrosynthetic design (conceptual):

• To design experiments, “break down” the system into delivery (lentiviral packaging), effector (Cas9/dCas9), and selection/reporting modules. Each module’s specifications should be matched to cell type, division rate, and desired duration of effect.

Note: Cloning sites, antibiotic markers, and promoter identities for this specific product are not specified; refer to the vector map/CoA before planning cloning or selection steps.

Target Specificity

• Intended target: Human UQCRH gene (ubiquinol-cytochrome c reductase hinge protein), mitochondrial Complex III component.
• Guide sequence(s): Not specified for this item; refer to CoA/Spec Sheet.

General considerations (literature):

• Specificity drivers: PAM compatibility (e.g., NGG for SpCas9), GC content (~40–70%), and avoidance of off-target sites with minimal mismatches in seed regions.
• Off-target risk mitigation: Use high-fidelity Cas9 variants, chemically modified guides (if applicable), and orthogonal confirmation of phenotype (rescue with sgRNA-resistant cDNA).
• Isoforms/transcripts: Ensure the guide targets exons common to major UQCRH isoforms if knockout is desired; promoter-targeting guides are preferred for CRISPRi applications.

Validation recommendations:

• In-silico scoring against the relevant genome build; PCR across predicted cut sites; functional rescue to confirm on-target dependence.

Without sequence-level information or validated targets for this SKU, users should confirm guide identity and mapping from the CoA before experimental use.

자주 묻는 질문

How should this product be stored?

Store at ?80 °C. Ultra-low temperature storage is required to maintain the specified shelf life.

How is this product shipped?

This product ships frozen with dry ice and cold packs. Unpack on arrival and transfer it to the storage condition stated above; handle dry ice with insulated gloves in a ventilated area.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.

Which species reactivity has this antibody been validated against?

Validated reactivity is listed on the PDP and on each lot's datasheet. We validate against the most commonly tested species for the target (pLenti-UQCRH-sgRNA); cross-reactivity in untested species may exist but is not guaranteed. Contact technical support for specific species inquiries.