pLenti-CBLB-sgRNA

Cat. No.: P744450

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

Application Protocols

Because vector map, markers, and sgRNA sequence are not provided, the following are general, adaptable protocols for pLenti sgRNA constructs (literature/general). Verify against the CoA/Spec Sheet for this item.

• Quick start (plasmid DNA):
  1. Thaw on ice. 2) Briefly spin down. 3) Keep on ice during setup. 4) For transfection, dilute in serum‑free medium and mix with transfection reagent per manufacturer instructions. Use 0.5–2 µg DNA per well (6‑well) as a starting point; optimize by cell line.
• Lentiviral packaging (BSL‑2/2+):
  • Plate HEK293T cells to ~70% confluence. Co‑transfect transfer, packaging, and envelope plasmids. Replace medium after 6–12 h. Collect supernatant at 48 and 72 h, filter (0.45 µm). Optionally concentrate. Titrate by p24, RT assay, or functional MOI.
• Cell transduction and selection:
  • Infect target cells at desired MOI with polybrene if compatible. After 24 h, replace medium. Begin antibiotic selection or fluorescence‑based sorting per vector features (not specified here).
• Editing verification:
  • Extract gDNA at day 3–7. PCR amplify the CBLB target region. Analyze with TIDE/ICE or NGS. For protein‑level effects, use immunoblotting/flow cytometry with appropriate antibodies (not part of this product).
• Archiving:
  • Freeze validated cell lines in cryoprotectant (e.g., 10% DMSO, 90% FBS) in liquid nitrogen vapor phase.

Always include appropriate biosafety and negative/positive controls. Adjust DNA amounts, reagent ratios, and timing to your cell system.

Biological Roles

CBLB (Cbl proto‑oncogene B, E3 ubiquitin protein ligase) encodes an E3 ubiquitin ligase implicated in receptor‑proximal signaling and protein turnover (literature/general):

• Domain architecture: An N‑terminal tyrosine kinase binding (TKB) domain (comprising SH2 and four‑helix bundle), RING finger catalytic domain, proline‑rich regions, and C‑terminal ubiquitin‑associated motifs enable substrate recognition and ubiquitin transfer (literature/general).
• Signaling contexts: Negatively regulates activation thresholds in immune receptor signaling (e.g., T‑cell receptor and costimulatory pathways) by ubiquitinating signaling intermediates, promoting their internalization or proteasomal degradation (literature/general). It can modulate PI3K, ZAP‑70, and other adaptor interactions depending on cell type.
• Cellular consequences: Fine‑tunes proliferation, activation, and receptor desensitization via ubiquitin‑mediated proteostasis. Redundancy/compensation may occur with related Cbl family members (literature/general).
• Genetic perturbation: CRISPR‑mediated disruption of CBLB allows interrogation of ubiquitin‑signaling circuits, identification of downstream effectors, and mapping of context‑dependent regulatory nodes at the cellular level (literature/general).

This product provides an sgRNA construct designed to target the CBLB locus; exact sgRNA sequence, PAM context, and targeted exon are not specified for this item (refer to CoA/Spec Sheet). Researchers should confirm on‑target editing and assess potential off‑target sites using current design tools and empirical validation.

Buffer Applications

Not typically applicable. This is a DNA construct rather than a buffer reagent. For handling, use nuclease‑free water or TE buffer for dilution and storage (literature/general). See Storage & Reconstitution for practical guidance.

Green Alternatives

This product is a DNA construct rather than a chemical solvent or reagent with conventional environmental substitution options. Nevertheless, greener practices can be applied to its use (literature/general):

• Prefer aqueous buffers over phenol/chloroform for DNA cleanup; use column‑based or magnetic‑bead purification systems that minimize hazardous waste.
• Use endotoxin‑free kits that avoid guanidinium salt carryover; collect and dispose of chaotropic waste according to local regulations.
• Scale packaging/production to experimental need to reduce consumables and cold‑chain burden.
• Consider transient transfection (non‑viral) when appropriate to avoid generating viral waste streams; where lentiviral delivery is essential, follow institutional waste neutralization policies to minimize environmental release.

No item‑specific greener substitute is applicable for an sgRNA plasmid; optimize workflows and waste handling for sustainability.

Pharmaceutical Uses

Not applicable to this catalog item. This product is supplied strictly for research use only (Product Data). It is a molecular biology reagent and is not an active pharmaceutical ingredient, excipient, or a GMP‑manufactured material. No pharmacopeial status is claimed. Do not use for human or veterinary applications.

Physical Properties

This is a nucleic acid reagent rather than a discrete small molecule; conventional thermophysical properties (boiling point, melting point, density, refractive index, logP, pKa) do not apply in the typical sense.

• Item-specific properties: Not specified for this item; refer to CoA/Spec Sheet.
• Typical presentation (literature/general):
  • Supplied as plasmid DNA in an aqueous buffer (e.g., TE or nuclease‑free water) or as a lentiviral transfer plasmid in glycerol stock for bacterial propagation. Exact formulation is not specified for this item.
  • DNA “melting” temperatures are sequence/length dependent and not meaningful as a single value for entire plasmids.
• Solubility (general for plasmid DNA):
  • Readily soluble in nuclease‑free water or low‑salt buffers; poorly soluble in most organic solvents (literature/general).
• Concentration units and OD260 conversions (literature/general):
  • 1 A260 ≈ 50 µg/mL for dsDNA; purity ratios A260/A280 ≈ 1.8–2.0 indicate low protein contamination.

For precise buffer composition, concentration, and volume of this lot, please consult the CoA/Spec Sheet.

Quality and Grades

• Item-specific quality/grade: Not specified for this item; refer to CoA/Spec Sheet.
• Typical quality attributes for plasmid DNA reagents (literature/general):
  • Endotoxin level: Low endotoxin is preferred for mammalian cell transfection or viral packaging; standard minipreps may suffice for cloning/diagnostics, while transfection-grade or endotoxin‑free preps are recommended for functional assays.
  • Supercoiled fraction: Higher supercoiled content generally improves transfection efficiency and cloning performance.
  • OD purity: A260/A280 ≈ 1.8–2.0; A260/A230 > 2.0 indicate low protein/chaotrope carryover. Agarose gel should show a predominant supercoiled band with minimal genomic DNA/RNA.
• Stabilizers and formulation:
  • Plasmid DNA stability is enhanced in TE buffer (10 mM Tris, 1 mM EDTA, pH ~8.0) to chelate divalent cations that support nuclease activity (literature/general). Specific buffer for this item is not specified; refer to CoA/Spec Sheet.
• What the listing implies:
  • The name “pLenti‑CBLB‑sgRNA” indicates a pLenti transfer backbone containing an sgRNA targeting CBLB. Exact backbone variant, antibiotic marker, promoter(s), and insert sequence should be confirmed on the item’s CoA/vector map before experimental planning.

Reaction and Applications

This construct supports CRISPR workflows where an sgRNA targeting CBLB is delivered via a pLenti transfer vector (literature/general). Typical applications include:

• Gene knockout/knockdown studies: Co‑delivery of an SpCas9 (or other CRISPR nuclease) expression system with this sgRNA to induce targeted double‑strand breaks at the CBLB locus, facilitating NHEJ‑mediated indels (literature/general).
• Stable cell line generation: Packaging into replication‑incompetent lentiviral particles using helper plasmids (e.g., gag/pol, rev, VSV‑G) followed by transduction permits stable integration and sustained sgRNA expression (literature/general). Perform under appropriate biosafety level.
• Pooled screening: If compatible with U6/H1 scaffold and barcoding, can be incorporated into pooled CRISPR libraries for functional genomics screens targeting CBLB‑dependent phenotypes (literature/general).
• Mechanistic studies: Dissecting pathways regulated by CBLB (an E3 ubiquitin ligase) in immune receptor signaling and proteostasis at the cellular level (literature/general; see Biological Roles).

Practical tips (general):

• Validate on‑target activity using amplicon NGS, T7E1/TIDE/ICE, or Sanger trace decomposition. Include non‑targeting and rescue controls.
• Use high-quality, low‑endotoxin DNA for viral packaging or sensitive transfections; filter sterilize and confirm absence of mycoplasma in producer cells.
• Optimize MOI and selection conditions empirically for each cell line. Confirm single‑cell clones by limiting dilution or FACS when clonal knockouts are required.

Note: Vector map, sgRNA sequence, and selectable markers are not specified for this item; consult CoA/Spec Sheet.

Reaction Conditions

While not a chemical reaction reagent, standard conditions for using a pLenti sgRNA vector in CRISPR workflows are well established (literature/general). Example guidance:

• Transfection (packaging cells, e.g., HEK293T):
  • Co‑transfect transfer vector (this item), packaging (gag/pol, rev), and envelope (VSV‑G) plasmids at common mass ratios (e.g., 4:3:1 or per kit recommendations). Use PEI, lipofection, or calcium phosphate. Maintain cells at 37°C, 5% CO2.
  • Harvest viral supernatant at 48–72 h; clarify by 0.45 µm filtration; concentrate if needed (PEG precipitation or ultracentrifugation). Handle under BSL‑2/2+.
• Transduction (target cells):
  • Add filtered virus with polybrene (e.g., 4–8 µg/mL, cell‑line dependent). Spinoculation may enhance efficiency. Replace medium after 12–24 h.
  • Apply selection if the vector carries a drug marker (antibiotic and timing are vector/cell‑specific; not specified for this item). Expand for 3–7 days before genotyping.
• Editing validation:
  • Extract genomic DNA; PCR amplify target locus; assess editing by TIDE/ICE or amplicon NGS. For clonal lines, isolate by limiting dilution or single‑cell FACS and re‑genotype.
• Controls:
  • Include non‑targeting sgRNA and a positive‑control sgRNA to benchmark activity; evaluate off‑target risk with in silico tools and empirical assays.

Exact promoters, selection markers, and sgRNA sequence for this item are not specified; refer to CoA/Spec Sheet and adapt conditions accordingly.

Safety and Handling

• GHS/SDS information (item-specific):
  • Signal Word: Not specified for this item; refer to SDS.
  • H-Statements: Not specified for this item; refer to SDS.
  • GHS Classification/Pictograms: Not specified for this item; refer to SDS.
• Biosafety considerations (literature/general):
  • pLenti transfer plasmids (DNA only) can be handled at BSL‑1 for routine cloning/propagation. If producing replication‑incompetent lentiviral particles using helper plasmids, perform all work under BSL‑2/2+ with institutional approvals and appropriate engineering controls (certified biosafety cabinet, validated waste deactivation procedures).
• PPE and engineering controls (general best practice):
  • Lab coat, nitrile gloves, eye protection; use nuclease‑free techniques to avoid degradation.
  • For any viral packaging or transduction work: Class II BSC, validated disinfectants effective against enveloped viruses (e.g., 70% ethanol, freshly prepared 10% bleach followed by ethanol), and proper sharps handling.
• Incompatibilities (general):
  • Avoid nucleases (DNase), microbial contamination, and repeated freeze–thaw cycles that can degrade DNA.
• First‑aid overview (general):
  • Skin/contact: Wash with water/soap. Eye: Rinse with water. Ingestion/inhalation: Seek medical attention as per SDS.

Always consult the product SDS and your institution’s biosafety committee guidance for authoritative safety requirements.

Solvent Selection

Not typically applicable. This product is a nucleic acid reagent intended for molecular biology workflows, not a solute for organic reaction media. For handling:

• Use nuclease‑free water or appropriate low‑salt buffers (e.g., TE) for dilution and storage (literature/general).
• Avoid organic solvents and extremes of pH that can denature or hydrolyze DNA.

See Storage & Reconstitution for practical guidance.

Storage and Reconstitution

• Storage (item-specific):
  • Store at -80°C (Product Data).
  • Shipped on dry ice packs + cold packs (Product Data). Inspect upon receipt; if thawed, store at -20°C for short term only and return to -80°C as soon as possible.
• Handling (general for plasmid DNA):
  • Thaw on ice. Mix gently by flicking or low‑speed vortex; briefly spin down. Keep on ice during setup.
  • Avoid repeated freeze–thaw cycles; aliquot upon first thaw to single‑use volumes.
• Reconstitution:
  • If provided lyophilized (not specified for this item), reconstitute with nuclease‑free water or TE buffer to the desired concentration. Allow to dissolve on ice for 10–15 min, gently pipetting to mix. For solutions, no reconstitution is needed.
• Working dilution and buffer:
  • Use nuclease‑free water or TE (10 mM Tris‑HCl, 1 mM EDTA, pH ~8.0) for dilutions (literature/general). Exact buffer of this item is not specified; refer to CoA/Spec Sheet.
• Stability (general):
  • At -80°C, plasmid DNA is stable for years when protected from nucleases. At 4°C, limit storage to short term (days) to minimize degradation.

Refer to the CoA/Spec Sheet for lot‑specific concentration, buffer composition, and any additional stabilizers. For research use only (Product Data).

Structure and Identity

This product is a recombinant DNA construct designed for CRISPR applications: a pLenti-based vector encoding a single guide RNA (sgRNA) targeting the human CBLB gene.

• Item-specific identifiers (from Product Data):
  • SKU: P744450
  • Product Name: pLenti-CBLB-sgRNA
  • Storage: Store at -80°C (Product Data)
  • Shipped: Dry ice packs + Cold packs (Product Data)
• Not specified for this item; refer to CoA/Spec Sheet:
  • Molecular formula, Molecular weight, CAS, CID, InChIKey, SMILES
• Construct type (general description):
  • Lentiviral “transfer” plasmid backbone (pLenti) engineered to express an sgRNA under a polymerase III promoter (e.g., U6 or H1; vector-specific, not provided here) (literature/general).
  • Insert: sgRNA sequence targeting CBLB; may be cloned into a gRNA scaffold within the vector (literature/general).
• Structural features (general, vector-class):
  • Circular double‑stranded DNA plasmid (if provided as plasmid DNA) with standard bacterial origin of replication and antibiotic resistance marker for propagation in E. coli (typical for pLenti backbones; literature/general).
  • Lentiviral 5′/3′ LTR-derived elements and packaging signal (Ψ) for packaging into particles when co-transfected with helper plasmids (literature/general).
• 2D structure (verbal): A closed circular dsDNA molecule comprising backbone and regulatory elements flanking an sgRNA expression cassette; no conventional small‑molecule rings/functional groups apply.

Note: Exact map, length (bp), resistance marker, and promoter are not specified for this item; refer to CoA/Spec Sheet.

Synthetic Utility

In the context of molecular biology, “synthetic utility” refers to cloning and genome‑engineering workflows enabled by the construct (literature/general):

• Modularity: pLenti backbones commonly accept sgRNA cassettes via Golden Gate (Type IIS) or Gibson/HiFi assembly; flanking BsmBI/BbsI sites are typical in many CRISPR backbones. Exact sites for this item are not specified; consult the vector map.
• Swapping cassettes: Researchers can replace promoters, introduce alternative scaffolds (e.g., MS2 aptamers), or insert multiplex gRNA arrays using standard cloning strategies.
• Payload compatibility: The transfer vector can be co‑deployed with separate Cas9 vectors (nuclease, nickase, dCas9‑fusions) or in dual‑expression systems for complete CRISPR functionality, depending on experimental design.
• Retrosynthetic value: The CBLB‑targeting insert provides a validated targeting context; the backbone can be repurposed by recloning a different protospacer to retarget other loci while preserving selection and regulatory elements.

Good practice:

• Verify insert by Sanger sequencing using universal primers flanking the gRNA cassette (e.g., U6‑F, gRNA scaffold‑R; primer names are vector‑specific).
• Maintain antibiotic selection in bacterial culture and minimize passages to reduce recombination in repetitive elements. Use recombination‑deficient strains for propagation of LTR‑containing plasmids.

Target Specificity

Only item‑specific target data may be listed here. For this product, no sequence, exon, PAM, species, or off‑target analysis are provided in the Product Data. Therefore:

• Target gene: CBLB (general, inferred from name)
• sgRNA sequence/target site: Not specified for this item; refer to CoA/Spec Sheet.
• Species reactivity: Not specified for this item; refer to CoA/Spec Sheet.
• Off‑target assessment: Not specified for this item; users should perform their own validation.

Note: This section is distinct from antibody “clone/isotype” specificity and does not apply to this DNA construct beyond the general CBLB indication above.