pLenti-CBLB-sgRNA

Cat. No.: P744449

Especificaciones

Product Name

pLenti-CBLB-sgRNA

Product Line

Productos biológicos

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at -80°C

Enviado en

Dry ice packs + Cold packs

Estabilidad y almacenamiento

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

Application Protocols

Tested applications, recommended dilutions/titers, and positive controls are not specified for this item; refer to CoA/Spec Sheet if available.

General workflow suggestions (literature, for planning only): • If supplied as plasmid: transform into high-efficiency E. coli, isolate plasmid (endotoxin-reduced prep for mammalian use), confirm by restriction digest and Sanger sequencing across the sgRNA insert. • If supplied as viral stock: determine functional titer (e.g., qPCR or reporter-based). Transduce Cas9-expressing cells at low MOI, select if marker present, then validate editing by amplicon sequencing. • Controls: include non-targeting sgRNA and a known positive-control sgRNA.

These are not item-specific instructions; always follow your institutional SOPs and consult any documentation shipped with the product.

Biological Roles

Target gene context (general literature): • CBLB (casitas B-lineage lymphoma-b) encodes an E3 ubiquitin ligase of the CBL family, featuring a tyrosine kinase-binding (TKB) domain, RING finger, and proline-rich regions. It modulates receptor tyrosine kinase turnover and T-cell receptor (TCR) signaling. • In immune cells, CBL-B functions as a negative regulator of activation thresholds by ubiquitinating signaling intermediates and modulating co-stimulatory pathways. Loss-of-function can enhance signaling downstream of TCR and other receptors. • Broader roles: regulation of PI3K/AKT and NF-κB pathway components, and cross-talk with metabolic checkpoints (literature). • Genetic perturbation with sgRNA constructs like pLenti-CBLB-sgRNA enables mechanistic studies of immune signaling, receptor trafficking, and ubiquitin-mediated proteostasis.

Notes: • Species, isoform targeting, and exact spacer sequence are not specified for this item; users should verify genome build and target exon choice relative to their model system. • For comprehensive pathway placement, consult curated databases (e.g., UniProt, Reactome) corresponding to your organism of interest.

Buffer Applications

Item-specific buffer composition is not provided. The following are general, literature-based guidelines for handling nucleic-acid vectors: • Reconstitution (if supplied lyophilized DNA): use nuclease-free water or TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). For cell transfection, dilute into cell-compatible buffers/media immediately prior to use. • Working stocks: for plasmid DNA, maintain at ≥ 10–50 ng/µL to minimize adsorption losses; store aliquots to avoid freeze–thaw. For viral preps, follow supplier buffer—avoid unnecessary dilution and repeated freeze–thaw. • pH range: neutral to slightly alkaline (pH 7.4–8.0) supports DNA stability; avoid acidic conditions that promote depurination. • Additives: EDTA (0.1–1 mM) can chelate Mg2+ and inhibit DNases in storage buffers; avoid EDTA in reactions requiring Mg2+ (e.g., PCR, transfection mixes with cationic lipids may tolerate low EDTA). • Filtration: if sterile filtration is required, use low-protein-binding 0.22 µm filters; avoid shear by excessive pressure.

Because this is a gene-editing reagent, classical “buffering capacity” use in analytical chemistry does not apply; focus on nuclease-free, isotonic, cell-compatible conditions.

Green Alternatives

While “green chemistry” is less directly applicable to biological vectors than to organic solvents, delivery modality choices can impact safety, waste, and resource use.

Comparison (general considerations): • Lentiviral sgRNA delivery

• Pros: stable integration/expression; effective in dividing and many non-dividing cells; scalable for pooled screens.
• Trade-offs: BSL-2 handling, integration risk, specialized packaging and disposal, cold-chain dependence. • Non-viral plasmid transfection (lipid/electroporation)
• Pros: no viral packaging, simplified waste streams, fewer biosafety constraints; rapid turnaround.
• Trade-offs: transient expression; variable efficiency in primary cells. • RNP delivery (Cas9 protein + sgRNA)
• Pros: DNA-free, transient nuclease exposure (reduced off-target window), minimal integration risk; smaller biosafety footprint.
• Trade-offs: cost, handling of RNA, often lower throughput for pooled applications. • AAV delivery
• Pros: broad tropism, lower integration frequency.
• Trade-offs: packaging size constraints; still requires biosafety controls and cold chain.

Selection guidance: Choose RNP or plasmid delivery when transient perturbation suffices and greener, lower-burden workflows are desired. Retain lentiviral delivery for stable, long-term perturbations and screen scalability.

Pharmaceutical Uses

Not applicable for pharmaceutical formulation. This product is supplied strictly for research use only (per Product Data) and is not intended for diagnostic, therapeutic, or GMP manufacturing purposes.

General context (non-clinical): • Similar vector backbones may be used in preclinical research to explore gene function or to develop tools later adapted under separate GMP processes. However, this specific item has no stated pharmacopeial status, release tests, or compliance claims. • If translation toward regulated use is contemplated, separate materials with documented GMP, viral safety, and comprehensive quality attributes (sterility, adventitious agent testing, residuals, full sequence, validated titer) are required.

Physical Properties

Item-specific physicochemical specifications (concentration, buffer, A260/280, endotoxin level, viscosity) are not provided in the Product Data.

General/literature characteristics for nucleic-acid-based lentiviral/plasmid reagents (for context only): • Physical state: typically provided as an aqueous solution or frozen aliquots; may also be supplied as DNA plasmid for subsequent packaging (not specified for this item). • UV absorbance (literature): nucleic acids exhibit a maximum at ~260 nm; A260 of 1.0 ≈ 50 µg/mL for dsDNA and ≈ 40 µg/mL for RNA (approximate). • Density/RI: dominated by the aqueous buffer; water-like at room temperature (literature). • pH/Buffer: often stored in nuclease-free water or low-salt Tris/EDTA to protect DNA; specific composition not specified for this item. • Solubility: nucleic acids are water-soluble; insoluble in most organic solvents (literature). • Stability (literature): improved at ≤ −80°C for long-term storage; repeated freeze–thaw can reduce viral titer and DNA integrity.

Do not treat any literature values above as specifications. For lot-specific data (concentration, buffer composition, titer, and purity), refer to CoA/Spec Sheet.

Quality and Grades

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet. • Typical quality attributes for lentiviral CRISPR sgRNA reagents (general, for context):

• Identity: sequence verification of sgRNA spacer and scaffold; backbone validation by restriction digest and/or NGS.
• Purity: low host genomic DNA/RNA carryover and minimal protein contaminants (for viral preps); for plasmids, high supercoiled content and low endotoxin if intended for transfection.
• Functional titer (if virus supplied): infectious units or transducing units per mL by qPCR or FACS-based assays. Not specified for this item.
• Sterility: negative for microbial contamination and mycoplasma (where applicable). Not specified for this item.
• Residuals: benzonase, packaging plasmid carryover, and endotoxin levels often monitored; not specified for this item. • Implications: if your application is sensitive (primary cells, in vivo models), verify endotoxin, titer, and sequence on receipt. For chromatographic or clinical manufacturing claims—none are made; this product is for research use only.

Reaction and Applications

This reagent enables CRISPR-based genome engineering targeting CBLB via an sgRNA expressed from a lentiviral backbone.

Typical research applications (general, literature): • Stable knockout/knockdown: lentiviral delivery integrates the sgRNA cassette for durable expression across cell divisions; pair with Cas9-expressing cells or co-deliver Cas9. • Functional genomics: perturb CBLB to study signaling, ubiquitination pathways, and immune checkpoint regulation in relevant cell types. • Arrayed or pooled screens: use in focused screens interrogating the CBLB axis or interacting pathways; include multiple independent sgRNAs and non-targeting controls to mitigate off-target/guide-specific effects. • Complementation studies: follow knockout with rescue by sgRNA-resistant cDNA to confirm on-target phenotype.

Practical tips (general): • Guide validation: confirm editing by amplicon NGS, T7E1/Surveyor, or ICE/TIDE analysis; correlate indel rate with phenotype. • Off-target assessment: use in silico prediction plus empirical validation at high-score sites; consider truncated guides or high-fidelity Cas9 variants to reduce off-targets. • MOI control: aim for MOI ~0.2–0.5 for single-copy integration in population studies; select with appropriate antibiotics if backbone provides a marker (not specified for this item). • Controls: include non-targeting sgRNA, safe-targeting sgRNA, and a positive control sgRNA to benchmark editing efficiency.

Manufacturer Applications: Not specified beyond the product name; the use cases above expand on common workflows for lentiviral sgRNA tools.

Reaction Conditions

Wet-chemistry reaction conditions are not applicable. For genome-editing experiments using a lentiviral sgRNA (general literature guidance): • Packaging (if preparing virus): transfect HEK293T or appropriate producer cells with transfer vector plus packaging/envelope plasmids using a lipid reagent. Maintain at 37°C, 5% CO2; collect supernatant at 48–72 h; filter (0.45 µm). Optional concentration by PEG or ultracentrifugation. • Transduction: expose target cells to virus at controlled MOI (e.g., 0.2–0.5 for single-copy integration). Polybrene (e.g., 4–8 µg/mL) may enhance entry; verify cell compatibility. • Selection: begin antibiotic selection 48–72 h post-transduction if backbone encodes resistance (not specified for this item). Expand for 7–14 days before analysis. • Editing readout: harvest genomic DNA 5–10 days post-transduction; PCR amplify target; assess indels by NGS or mismatch assays. For protein-level validation, analyze by immunoblot or flow cytometry. • Temperature/time: standard mammalian culture conditions; avoid temperature excursions during virus handling; freeze aliquots promptly for long-term storage.

All parameters above are general references; adjust empirically for your cell model and delivery method.

Safety and Handling

• GHS classification, H-statements, pictograms: Not specified for this item; refer to the SDS. • Biosafety considerations (general guidance for lentiviral CRISPR vectors):

• Handle replication-incompetent lentiviral particles and packaging workflows under BSL-2 containment with institutional biosafety approval. Follow local IBC and NIH/WHO guidance.
• Use appropriate PPE: lab coat, gloves, and eye protection. Employ biological safety cabinets (Class II) for manipulations that may generate aerosols.
• Decontamination: treat surfaces and waste with freshly prepared 0.5–1% sodium hypochlorite or appropriate virucidal disinfectant; follow autoclave policies for solid waste.
• Sharps/aerosols: minimize needle use; seal rotors for centrifugation. • Incompatibilities (general): avoid nucleases (RNase/DNase) and extreme pH/temperature that degrade nucleic acids and reduce viral titer. • First aid (general): in case of exposure, follow institutional incident reporting and seek medical evaluation per biosafety plan; rinse skin/eyes with water. Consult SDS for authoritative measures. • Transport: ship on dry ice/cold packs as indicated. Maintain cold chain to preserve integrity and (if applicable) viral titer.

Always defer to the product’s SDS and your institutional biosafety program for definitive safety and handling instructions.

Solvent Selection

This product is a biological nucleic-acid-based construct; classical organic “solvent selection” is not typically applicable.

Practical buffer choices (general literature guidance): • Short-term use (transfection/transduction): nuclease-free water or low-ionic-strength Tris (e.g., 10 mM Tris-HCl, pH 7.5–8.0). Avoid divalent cations that promote nuclease activity. • DNA plasmid storage: TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) helps chelate Mg2+ and protect from DNases; may slightly reduce transfection efficiency relative to pure water—dilute into cell-compatible media just before use. • Viral preps: maintain in supplier buffer; avoid dilution in serum-free media for prolonged periods. For spinoculation or low-MOI settings, add polybrene only as cell-compatible.

If you require solvent information for HPLC or small-molecule synthesis, this section does not apply; see Reaction & Applications and Buffer Applications for relevant guidance.

Storage and Reconstitution

• Storage conditions (item-specific): Store at −80°C. • Shipping (item-specific): Shipped on dry ice packs + cold packs. Maintain cold chain on receipt; place at −80°C immediately. • Appearance/concentration: Not specified for this item; refer to CoA/Spec Sheet. • Reconstitution (general, if lyophilized DNA is provided): dissolve in nuclease-free water or TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Mix gently; avoid vigorous vortexing for viral samples. • Aliquoting: prepare single-use aliquots to minimize freeze–thaw. Repeated freeze–thaw can reduce viral titer and DNA integrity. • Short-term handling: keep on ice during setup. For viral stocks, thaw rapidly at 37°C, then place on ice and use immediately; do not refreeze thawed remnants. • Light sensitivity: not typically light-sensitive, but protect from prolonged UV exposure (which can damage nucleic acids). • Stability: Item-specific shelf life, titer retention, and buffer composition are not specified; verify performance after storage as needed.

Research Use Note: For research use only.

Structure and Identity

• Product type: lentiviral CRISPR construct containing an sgRNA directed at the CBLB locus (vector-level biological reagent; not a small molecule) • SKU: P744449; Product Name: pLenti-CBLB-sgRNA • Molecular formula: Not specified for this item; refer to CoA/Spec Sheet. • Molecular weight: Not specified for this item; refer to CoA/Spec Sheet. • CAS, CID, InChIKey, SMILES: Not applicable to this biological construct; not assigned. • Structural features (general, literature):

• Backbone: pLenti-type vector used for packaging into lentiviral particles (typically includes a U6/H1 promoter to drive sgRNA, and lentiviral LTRs and packaging elements). Specific backbone elements and map are not specified for this item.
• sgRNA cassette: single guide RNA scaffold plus a gene-specific spacer sequence targeting CBLB; exact spacer sequence and PAM context are not specified for this item. • 2D description (general): circular plasmid map with lentiviral elements and an sgRNA expression cassette; when packaged, yields RNA genome lentiviral particles carrying the sgRNA expression unit. • Stereochemistry: not applicable (nucleic acid polymer).

Synthetic Utility

Classical synthetic chemistry utility is not applicable. For molecular biology, the construct provides “genetic synthetic” utility in cloning and genome engineering workflows.

General/literature use cases: • Modular cloning: swap sgRNA spacers using oligo annealing, Golden Gate (Type IIS) or Gibson assembly into a pLenti backbone; verify by Sanger sequencing. • Multigene constructs: pair the sgRNA cassette with selectable markers or reporters (e.g., puromycin resistance, fluorescent proteins) for selection/screening—specific features for this item are not specified. • Combinatorial perturbations: assemble dual-sgRNA or CRISPRi/a cassettes to interrogate genetic interactions with CBLB. • Packaging utility: co-transfect with packaging plasmids into producer cells to generate lentiviral particles; titer and transduce target cells expressing Cas9.

These utilities are presented as general guidance; consult the CoA/Spec Sheet for backbone-specific sites, selection markers, and cloning strategy if provided for this SKU.

Target Specificity

Item-specific targeting data are limited. Based on the product name, this construct carries an sgRNA intended to target the CBLB gene. • Target: CBLB (gene); species/isoform, exact spacer sequence, PAM, and exon context are not specified for this item; refer to CoA/Spec Sheet if available. • Off-target profile, validated on-target sites, and tested cell lines: Not specified for this item.

Users should confirm genome build compatibility and validate on-target cutting and off-target risk empirically in their system.