pLenti-APC-sgRNA

Cat. No.: P743833

Specifications

Product Name

pLenti-APC-sgRNA

Product Line

biological_products

Lagerung und Versand

Storage

Store at -80°C

Verschickt in

Dry ice packs + Cold packs

Stabilität und Lagerung

Stored at -20 ℃, valid for at least 6 months;Stored at -80 ℃, valid for at least one year.

Application Protocols

Tested applications, conditions, and recommended procedures are not specified for this item; refer to CoA/Spec Sheet and SDS.

General workflow references (non‑product‑specific, literature) can include: plasmid preparation, transfection or viral packaging, transduction, selection, and edit validation; however, definitive protocols for SKU P743833 are not provided here.

Biological Roles

• Target gene: APC (adenomatous polyposis coli).
• Cellular functions (literature, general):
  • Negative regulator of Wnt/β‑catenin signaling via destruction complex assembly (with Axin, GSK‑3β, CK1), promoting β‑catenin phosphorylation and turnover.
  • Roles in cytoskeletal organization: microtubule plus‑end tracking via EB1 interaction; actin dynamics modulation; cell migration and polarity.
  • Mitotic functions: chromosome segregation fidelity and spindle dynamics.
• Research impact of APC perturbation:
  • sgRNA‑mediated disruption can elevate β‑catenin activity and Wnt target gene expression, enabling pathway studies and reporter assays.
  • APC loss often alters cell adhesion and migration phenotypes, useful for studying cytoskeleton‑linked processes.
• Genetic context (literature):
  • APC is a large gene with multiple functional domains (armadillo repeats, SAMP repeats, β‑catenin binding/degradation repeats). Guide placement can influence phenotype severity depending on domain targeting; sequence for this item is not specified—review design before experiments.
• Off‑target considerations: Use computational specificity scoring and empirical off‑target assessment when interpreting phenotypes, particularly in pathways with broad transcriptional effects.

Buffer Applications

This product is a plasmid DNA construct rather than a buffering reagent. Classical buffer system design (pKa selection, ionic strength optimization) is not typically applicable. For practical handling, use nuclease‑free water or standard biological buffers (e.g., TE) compatible with downstream transfection/packaging workflows, following your method‑specific guidelines.

Green Alternatives

While plasmid DNA itself is not a solvent or bulk chemical, greener laboratory choices can be considered around its use (literature, general):

• Avoidance of lentiviral steps when possible:
  • Alternative delivery methods such as electroporation or non‑viral transfection reduce biosafety requirements and chemical disinfectant usage. Tradeoff: often lower stability in hard‑to‑transfect cells and lack of stable integration.
• Greener transfection chemistries:
  • Use biodegradable or less toxic polymers/lipids where available. Tradeoff: may have lower efficiency or narrower cell‑type compatibility.
• Process considerations:
  • Minimize single‑use plastics by adopting validated reusable glassware for non‑aseptic steps and using refillable tips where contamination risk is controlled.
  • Replace ethidium bromide with safer nucleic‑acid stains for gel QC.

Comparison (literature, general):

• Lentiviral delivery
  • Pros: broad tropism, stable expression via integration; effective in difficult cell types.
  • Cons: BSL‑2 handling, bleach/quench waste, packaging plasmid footprint, potential insertional mutagenesis risk in research contexts.
• Non‑viral delivery
  • Pros: simpler EHS profile, reduced hazardous waste.
  • Cons: transient expression, lower efficiency in some primary cells.

Pharmaceutical Uses

Not applicable. This product is supplied strictly for research use only (per product data) and is not intended for diagnostic, therapeutic, or manufacturing use in humans or animals. No pharmacopeial status or excipient role is specified for this item.

Physical Properties

• State/appearance: Not specified for this item; refer to CoA/Spec Sheet. (Plasmids are commonly supplied in an aqueous buffer or as a dried film.)
• Concentration: Not specified for this item; refer to CoA/Spec Sheet.
• pH of solution: Not specified for this item; refer to CoA/Spec Sheet.
• UV characteristics (literature, general): DNA shows an A260 peak with A260/A280 ~1.8 for pure samples; extinction coefficient depends on sequence/length.
• Solubility (literature, general): Highly soluble in nuclease‑free water or TE buffer; insoluble in organic solvents.
• Melting/boiling point, density, refractive index, logP: Not applicable to macromolecular DNA constructs; not specified for this item.
• Endotoxin level, residual solvents, salt content: Not specified for this item; refer to CoA/Spec Sheet.
• DNase/RNase status: Not specified for this item; refer to CoA/Spec Sheet.
• Plasmid size (kb) and selection marker: Not specified for this item; refer to CoA/Spec Sheet.

Quality and Grades

• Grade/purity: Not specified for this item; refer to CoA/Spec Sheet.
• Research use only: Yes (per product data). Not for human or veterinary use.
• Typical quality attributes for plasmid reagents (literature, general):
  • Supercoiled content (%), A260/A280 and A260/A230 ratios, residual genomic DNA/RNA, and endotoxin level (EU/µg). These parameters influence transfection efficiency and cell compatibility.
  • Sequence identity of sgRNA spacer and scaffold, verified by Sanger/NGS.
• Stabilizers/buffers: Not specified for this item; refer to CoA/Spec Sheet.
• Implications for use:
  • For high‑efficiency transfection or viral packaging, low endotoxin and high supercoiled percentages are preferred (literature).
  • If antibiotic resistance marker, promoter, or reporter details are critical for your workflow, obtain the vector map/sequence from the CoA/Spec Sheet prior to use.
• Lot‑specific documentation: Consult the CoA for exact concentration, buffer, and any sequence verification status associated with SKU P743833.

Reaction and Applications

• Primary application: Expression of an APC‑targeting sgRNA via a lentiviral transfer plasmid for CRISPR‑based genome editing (knockout, repression with dCas9‑KRAB, or activation with dCas9‑VPR; specific effector context determined by co‑expressed Cas9/dCas9 system; not specified for this item).
• Use cases (literature, general):
  • Stable delivery of sgRNA into dividing and non‑dividing mammalian cells via lentiviral transduction, enabling pooled or arrayed loss‑of‑function studies.
  • Generation of APC loss‑of‑function cell models to interrogate Wnt/β‑catenin signaling, cytoskeletal dynamics, and mitotic regulation.
  • Creation of isogenic knockouts for pathway epistasis mapping and synthetic‑lethality screening in cell biology research.
• Practical tips:
  • Verify target site and PAM compatibility with your Cas nuclease (e.g., SpCas9 requires NGG PAM, literature). The actual spacer sequence for this item is not specified—confirm from the CoA/Spec Sheet.
  • For lentiviral packaging, co‑transfect with appropriate packaging and VSV‑G envelope plasmids; use BSL‑2 containment.
  • Validate editing by indel profiling (ICE/TIDE), NGS amplicon sequencing, or functional readouts. Include non‑targeting and safe‑harbor controls.
  • To mitigate off‑target activity, consider truncated guides or high‑fidelity Cas variants; design and verification are user‑dependent.
• Noteworthy: Vector elements (promoter, selection marker, reporter) are not specified for this item; consult documentation before experimental planning.

Reaction Conditions

Item‑specific reaction conditions are not provided. The following are general, literature‑based guidelines for workflows commonly used with lentiviral sgRNA plasmids (not product‑specific):

• Transfection (HEK293‑derived packaging cells):
  • DNA delivery via calcium phosphate, PEI, or lipid reagents in serum‑containing or optimized media; typical DNA amounts 0.5–3 µg per well (6‑well format). Optimize per reagent.
  • Co‑transfect with packaging and envelope plasmids in recommended ratios. Replace media after 6–18 h depending on reagent.
• Viral harvest and transduction:
  • Collect supernatant at 24–72 h, clarify by centrifugation/filtration. Optional concentration by PEG or ultracentrifugation.
  • Transduce target cells with polycation (e.g., polybrene, literature) if compatible; adjust MOI to balance single‑copy integration vs efficiency.
• Selection and validation:
  • Begin antibiotic selection or reporter‑based sorting per vector features (not specified for this item). Validate editing by genotyping and functional assays 3–10 days post‑transduction.
• Temperature: standard mammalian cell culture (37°C, 5% CO2) for production and transduction steps.
• Yield/efficiency: Highly system‑dependent; determine empirically for your cell type and delivery method.

Safety and Handling

• GHS classification, signal word, pictograms, H‑statements: Not specified for this item; refer to SDS.
• Biosafety note (general, literature): Non‑replicating plasmid DNA can be handled at BSL‑1. If used to produce lentiviral particles, follow institutional BSL‑2 or higher practices for viral packaging and transduction.
• PPE: Lab coat, gloves, and eye protection. Use biological safety cabinet (Class II) for any viral production or handling of transduction mixtures.
• Incompatibilities: Avoid nucleases; use nuclease‑free plasticware and solutions. Keep away from strong oxidants and extreme pH that can hydrolyze phosphodiester bonds.
• First‑aid overview (general): In case of skin/eye contact, rinse with water. If inhaled (aerosols during viral work), move to fresh air and seek medical attention per institutional biosafety protocols. If ingested, rinse mouth; obtain medical advice. Always consult SDS.
• Spill response: For plasmid solutions, absorb with paper towels and disinfect area. For lentiviral supernatants, apply appropriate disinfectant (e.g., freshly prepared 10% bleach, literature) with adequate contact time per biosafety guidance.
• Waste: Decontaminate DNA/viral waste according to local regulations (chemical disinfectant or autoclaving, as applicable).

Solvent Selection

This product is a plasmid DNA reagent, not a solvent. Solvent selection per se is not typically applicable.

• Practical notes (literature, general):
  • Use nuclease‑free water or TE buffer for dilution and storage. Avoid organic solvents, which can denature or precipitate DNA.
  • For transfection, follow the solvent/buffer requirements of your transfection reagent (e.g., opti‑formulated media for complex formation) as specified by the reagent manufacturer.
  • For viral packaging, prepare DNA in sterile, endotoxin‑controlled aqueous buffers compatible with your transfection chemistry.

Storage and Reconstitution

• Storage conditions (product data): Store at -80°C.
• Shipping (product data): Shipped on dry ice packs + cold packs to maintain frozen/cold chain.
• Appearance/concentration/buffer: Not specified for this item; refer to CoA/Spec Sheet.
• General handling guidance (literature, not product‑specific):
  • Minimize freeze–thaw cycles; aliquot upon first thaw. Thaw on ice and keep cold during handling to limit nuclease activity.
  • Use nuclease‑free tubes, tips, and solutions. Mix gently; avoid vigorous vortexing that can shear DNA.
  • If provided lyophilized, reconstitute with nuclease‑free water or TE to the desired concentration; allow complete dissolution on ice. Exact reconstitution volume and buffer should follow the CoA/Spec Sheet if specified.
• Stability: Long‑term stability is enhanced at -80°C; short‑term working aliquots may be kept at -20°C as needed (literature). Always verify integrity before critical experiments.

Structure and Identity

• Item type: plasmid DNA construct designed for lentiviral delivery of a single-guide RNA (sgRNA) targeting the APC gene (adenomatous polyposis coli).
• Composition: nucleic acid (double‑stranded circular DNA backbone with an sgRNA expression cassette). Vector map/sequence are not provided here.
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet. (Plasmid mass depends on base‑pair length; typical plasmids are 5–15 kb, literature.)
• SMILES / InChI / InChIKey: Not applicable to plasmid DNA; not specified for this item.
• Structural features (general, literature):
  • Lentiviral transfer backbone carrying an RNA polymerase III promoter (e.g., U6 or H1) upstream of an sgRNA scaffold; exact promoter and scaffold not specified for this item.
  • sgRNA spacer sequence designed to target APC; exact spacer sequence not specified for this item.
  • Usual lentiviral backbones contain LTRs, ψ packaging signal, RRE, and often a selection marker; specific elements not specified for this item.
• Identity confirmation (general practices, literature): agarose gel electrophoresis for size, restriction digest patterning, Sanger/NGS of the sgRNA region, and functional validation by indel analysis (T7E1, ICE/TIDE).

Synthetic Utility

In the context of molecular biology (literature, general), pLenti‑APC‑sgRNA functions as a modular cloning and expression tool rather than a small‑molecule synthetic intermediate.

• Functional elements typically leveraged (exact elements for this item not specified):
  • RNA Pol III promoter → sgRNA spacer → scaffold: enables expression of a defined guide sequence.
  • Lentiviral transfer backbone: allows packaging into pseudotyped particles for stable delivery.
  • Optional markers (antibiotic resistance, fluorescent reporters): facilitate selection and tracking.
• Utility in experimental design:
  • Rapid construction of APC knockout lines when paired with Cas9 expression (stable or transient).
  • Swapping sgRNA spacers (via Golden Gate/Type IIS cloning, literature) to target alternative APC exons or controls.
  • Combining with donor templates for HDR‑mediated edits at APC; donor design and delivery are user‑defined.
• Retrosynthetic perspective (vector engineering):
  • The sgRNA cassette can be excised/replaced, enabling creation of multiplexed guides or conditional expression constructs. Backbone features dictate cloning strategy; consult the map (not provided here).

Target Specificity

• Intended target: APC gene (adenomatous polyposis coli).
• sgRNA spacer sequence, PAM context, species specificity, and any off‑target analysis: Not specified for this item; refer to CoA/Spec Sheet.
• Validation data (on‑target indel rates, functional knockdown/knockout readouts): Not specified for this item; refer to CoA/Spec Sheet.
• Clone ID, backbone details, selectable markers, and promoters: Not specified for this item; refer to CoA/Spec Sheet.