pLenti-ECT2L-sgRNA

Cat. No.: P745518

Especificações

Product Name

pLenti-ECT2L-sgRNA

Product Line

Produtos biológicos

Armazenamento e envio

Condições de armazenamento de armazenamento

Armazenar a -80°C

Enviado em

Dry ice packs + Cold packs

Estabilidade e armazenamento

Armazenado a -20 ℃, válido por 3 meses; Armazenado a -80 ℃, válido por pelo menos um ano.

Application Protocols

Only protocols explicitly validated for this SKU should be listed here. For this item, no tested applications or recommended conditions are provided in the Product Data.

Not specified for this item; refer to CoA/Spec Sheet.

General note: For guidance on typical CRISPR/lentiviral workflows (transfection, packaging, transduction, selection, and validation), see the Reaction Conditions tab. Follow your institutional SOPs and adapt parameters to your cell model and vector map.

Biological Roles

Target gene context (general literature):

• ECT2L (ECT2-like) is annotated as a human gene with homology to ECT2, a Rho guanine nucleotide exchange factor (RhoGEF) involved in cytokinesis and actin cytoskeleton regulation. While ECT2 itself is well characterized as a GEF for RhoA/Rac, ECT2L remains less extensively studied; available annotations suggest putative roles related to Rho-family GTPase signaling.
• Paralogy to ECT2 implies potential participation in cell-shape dynamics, migration, or division; however, definitive biochemical specificity (e.g., cognate Rho GTPases) and in vivo functions for ECT2L are not firmly established in the public literature and may be context-dependent.

Use of an ECT2L-targeting sgRNA (research rationale):

• Functional genomics: Assess the contribution of ECT2L to cytoskeletal organization, cell-cycle progression, or signaling pathways by loss-of-function editing in relevant cell models.
• Genetic interaction studies: Combine ECT2L perturbation with knockouts of related RhoGEFs or downstream effectors to map pathway redundancy or compensation.
• Omics readouts: Pair editing with RNA-seq, proteomics, or phospho-signaling assays to delineate ECT2L-dependent networks.

Note: The specific spacer sequence, on-target site, and predicted off-targets for this SKU are not provided here; empirical validation is required for any mechanistic conclusions.

Buffer Applications

Not typically applicable. This product is a nucleic-acid reagent rather than a buffering agent. Any operational buffer (e.g., for transfection, packaging, or storage) should be chosen based on the experimental workflow and vector map.

General guidance (literature/practice):

• Storage: Plasmid DNA is commonly maintained in nuclease-free water or TE buffer; packaged lentiviral preparations are stored in suitable cryoprotectant-containing media at −80°C.
• Cell culture: Use standard culture media for target cells and packaging lines; avoid divalent-cation-containing buffers when they interfere with transfection chemistries.

Item-specific buffer composition for this SKU is not specified; refer to the CoA/Spec Sheet.

Green Alternatives

Perspective: “Green” considerations for genome-editing delivery focus on biosafety, waste minimization, and resource efficiency rather than solvent replacement.

Potential alternatives and trade-offs (general/literature):

• Lentiviral sgRNA delivery (this product class)

  • Pros: Stable integration, broad tropism, suitable for pooled screens and long-term studies.
  • Cons: BSL-2 handling, generation of biological waste, cold-chain requirements, potential integration-related genotoxicity (research context).

• Non-viral plasmid transfection

  • Pros: No viral waste, simpler biosafety profile, scalable and rapid.
  • Cons: Often transient expression; lower efficiency in primary cells and some lines.

• RNP delivery (Cas9 protein + synthetic sgRNA)

  • Pros: DNA-free, transient exposure minimizes off-target window; reduced integration concerns.
  • Cons: Higher reagent cost; delivery into difficult cells can require electroporation.

• AAV delivery

  • Pros: Lower biosafety risk than lenti; efficient in many cell types.
  • Cons: Packaging size limits (~4.7 kb), single-strand kinetics, still requires cold-chain and specialized handling.

Greener practices with lentiviral workflows (general):

• Consolidate freeze–thaw cycles by aliquoting; use validated MOI to avoid excess material.
• Employ closed-system filtration and bleach neutralization to reduce spill and disinfectant volumes.
• Optimize transfection to increase packaging yield, reducing upstream reagent and plastic consumption.

Pharmaceutical Uses

This product is designated for research use only. It is not intended for diagnostic, clinical, or therapeutic applications.

General formulation/bioprocess relevance (non-clinical):

• Tool for target validation: In preclinical discovery, sgRNA vectors are used to validate genetic targets in cell-based assays prior to medicinal chemistry or biologics campaigns.
• Cell-line engineering: Creation of knockout/knockdown/activation cell lines for assay development, mechanism-of-action studies, or bioproduction optimization.
• Process development research: Stable integration systems can establish reporter or selection cassettes in producer lines during upstream development. Any cGMP suitability is not implied or established for this SKU.

No pharmacopeial status, excipient role, or clinical formulation details are provided or implied for this item.

Physical Properties

Item-specific physical data:

• Appearance, concentration, buffer composition, A260/A280: Not specified for this item; refer to CoA/Spec Sheet.
• Density, boiling point, melting point, refractive index, logP: Not applicable to nucleic-acid constructs in aqueous buffer; not specified for this item.

General/literature context for similar materials:

• Typical form: Supplied as aqueous solution or frozen aliquots (e.g., TE, Tris-based, or proprietary storage buffers) for plasmid DNA, or cryopreserved supernatant for packaged lentiviral particles. Exact formulation for this SKU is not provided.
• Solubility: High solubility in water/buffer; incompatible with most neat organic solvents that denature nucleic acids and proteins.
• Stability considerations: DNA constructs are stable at −20 to −80°C; packaged lentiviral particles are typically stored at −80°C to preserve infectivity. Multiple freeze–thaw cycles reduce performance.
• Optical properties: DNA concentration is often quantified by A260 (1 A260 ≈ 50 µg/mL for dsDNA, literature). Fluorescent markers, if present in the backbone, can aid titering or transduction QC; not specified for this item.

Note: For any parameter required for method validation (e.g., exact concentration, titer, endotoxin), use the item’s CoA/Spec Sheet or contact technical support.

Quality and Grades

Item-specific grade/purity: Not specified for this item; refer to CoA/Spec Sheet.

General expectations for quality control of pLenti sgRNA reagents (literature/practice):

• Sequence identity: sgRNA spacer and scaffold typically verified by Sanger sequencing; backbone integrity confirmed by restriction analysis or NGS. Request sequence verification if required for regulated studies.
• Endotoxin: For cell-culture transfection, low endotoxin DNA may improve transfection efficiency and cell viability. If packaged virus is supplied, endotoxin spec is usually not assigned; instead, sterility and mycoplasma testing of packaging lines are emphasized.
• Functional titer (if viral): Infectious units (IU/mL) or transducing units (TU/mL) quantify activity. Verify MOI empirically on the target cell line.
• Purity for plasmid DNA: A260/A280 ~1.8–2.0 (literature) and supercoiled fraction often reported; nicked/linear DNA can reduce transfection efficiency.
• Antibiotic marker and promoter identity: Critical for selection and expression; consult the vector map for this SKU (not provided here).

What “research use only” means:

• Suitable for laboratory research. Not validated for diagnostic, clinical, or therapeutic use. No cGMP status implied unless explicitly stated on documentation.

Reaction and Applications

This section is adapted to molecular biology workflows for a pLenti sgRNA vector rather than synthetic organic reactions.

Primary research applications (general/literature):

• CRISPR-Cas gene perturbation: Express an sgRNA targeting ECT2L to direct Cas9 (or dCas9-based effectors) for knockout, repression (CRISPRi), or activation (CRISPRa), depending on the system used.
• Stable delivery via lentivirus: Enables transduction of dividing and many non-dividing mammalian cells, facilitating pooled genetic screens and long-term selections.
• Library and screening contexts: Single-guide constructs can be combined into pools for dropout or phenotypic screens; maintain low MOI (e.g., <0.3, literature) to favor single integrations.

Practical considerations (general guidance):

• Vector features: Antibiotic selection markers and promoters vary by backbone; consult the vector map for this SKU (not provided). Choice of marker impacts selection kinetics and cytotoxicity.
• Guide performance: On-target activity depends on spacer sequence, chromatin context, and Cas variant. Validate editing via T7E1, amplicon NGS, or ICE analysis.
• Controls: Include non-targeting sgRNA and positive-control sgRNA with known robust phenotype.
• Off-target risk: Minimize with validated spacers and high-specificity Cas9 variants; confirm with orthogonal assays when phenotype is critical.

Note: This SKU’s exact cassette and selectable markers are not specified; tailor conditions to the actual map provided with the shipment.

Reaction Conditions

Adapted to genome-editing workflows (general literature guidance). Item-specific conditions are not provided for this SKU.

• Transfection (plasmid DNA into packaging or target cells): Optimize DNA:reagent ratios per vendor instructions; maintain healthy cells at 60–80% confluence. Serum presence may affect some reagents; Opti-MEM is commonly used during complex formation.
• Lentiviral packaging (if applicable): Use HEK293T or derivative lines with standard helper plasmids (gag/pol, rev, VSV-G). Collect supernatant 24–72 h post-transfection; clarify by low-speed spin and 0.45 µm filtration. Concentration methods include PEG precipitation or ultracentrifugation.
• Transduction: Determine MOI empirically. Polybrene (hexadimethrine bromide) at 4–8 µg/mL (literature) may enhance entry but can be cytotoxic in some cells. Spinoculation can increase efficiency for difficult lines.
• Selection and expansion: Begin antibiotic selection per vector marker once cells have recovered (commonly 24–72 h post-transduction); exact antibiotic and dose depend on backbone and cell sensitivity (marker not specified for this SKU).
• Editing validation: Allow sufficient time for editing and turnover (typically 3–7 days for protein loss in rapidly dividing cells). Quantify editing by amplicon NGS, TIDE/ICE, or functional assays.

All parameters above are general guidance; consult your lab SOPs and the vector map supplied with the product for precise conditions.

Safety and Handling

Item-specific hazard information (from Product Data):

• GHS Classification / Signal Word / H-Statements / Pictograms: Not specified for this item; refer to the product SDS.

General safety guidance for lentiviral sgRNA vectors (literature/good practice):

• Biosafety level: Packaged lentiviral particles are typically handled under BSL-2 containment with institutional approval and appropriate training. Plasmid DNA containing lentiviral elements can be handled on the bench but should be kept segregated from cell-culture areas to avoid cross-contamination.
• PPE: Lab coat, gloves, and eye protection. Use biosafety cabinet for handling viral supernatants, transduced cells before fixation, and waste decontamination steps.
• Incompatibilities: Avoid DNases/RNases contamination; segregate from strong oxidants and from open flames/UV if fluorophores are present.
• Decontamination: 10% bleach (fresh), followed by appropriate detergent/ethanol is commonly used for surfaces. Autoclave solid waste as per institutional biosafety procedures.
• First aid (overview): If exposure to viral supernatant occurs (splash, sharps), follow institutional exposure protocols; wash affected area with water and seek medical evaluation per policy. For eye exposure, rinse with water for ≥15 min.

Always consult the SDS and your institutional biosafety committee for definitive hazard classifications and approved handling/waste procedures for this product.

Solvent Selection

Applicability: This product is an aqueous nucleic-acid reagent; classical organic “solvent selection” does not apply as it would for small-molecule solutes.

Item-specific solvent guidance:

• Supplied matrix: Not specified for this item; refer to CoA/Spec Sheet. Such reagents are typically provided in water, Tris/EDTA, or proprietary cryoprotectant buffers.

General guidance for use (literature/practice):

• Use nuclease-free water or appropriate physiological buffers (e.g., PBS without Ca/Mg, Opti-MEM for transfections) when diluting or formulating.
• Avoid high percentages of organic solvents (e.g., ethanol, DMSO) that can impair transfection, packaging, or viral infectivity.
• For packaging workflows: Maintain isotonic, serum-compatible media for HEK293-derived packaging cells; filter-sterilize buffers with 0.22 µm membranes.

Comparison (general):

• Nuclease-free water: Ideal for thawing/aliquoting DNA stocks.
• TE buffer (10 mM Tris, 1 mM EDTA, pH ~8): Improves plasmid DNA stability but EDTA can inhibit some enzymatic reactions; dilute into EDTA-free buffer before transfection as needed.
• HEPES-buffered saline: Common for calcium phosphate transfection; pH control is critical.

Storage and Reconstitution

Item-specific storage and shipping (from Product Data):

• Storage Conditions: Store at −80°C.
• Shipped In: Dry ice packs + Cold packs.

Not specified for this item; refer to CoA/Spec Sheet:

• Exact buffer, concentration/titer, presence of cryoprotectants, and aliquot size.

General handling guidance (literature/practice):

• Minimize freeze–thaw: Aliquot upon first thaw to single-use volumes; keep on wet ice during handling.
• Thawing: Thaw rapidly at 4°C or on ice. Mix gently by inversion; avoid vigorous vortexing, which can shear DNA or reduce viral infectivity.
• Storage buffer: Plasmid DNA is commonly stored in nuclease-free water or TE at pH ~8. Packaged lentiviral stocks remain at −80°C; avoid frost-free freezers.
• Working stocks: Prepare working dilutions in sterile, nuclease-free buffer immediately before use. Discard any thawed viral aliquots after a single use to maintain performance and biosafety.
• Documentation: Retain vector map, sequence file, and CoA with the stored material for traceability and correct experimental setup.

Structure and Identity

Brief description: pLenti-ECT2L-sgRNA is a lentiviral-vector construct designed to express a single guide RNA (sgRNA) targeting the ECT2L locus for CRISPR applications. It is a nucleic-acid-based research reagent, not a small molecule.

Item-specific identifiers (from Product Data):

• CAS: Not specified for this item; refer to CoA/Spec Sheet.
• 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 / InChI / InChIKey: Not applicable to plasmid/viral constructs; not specified for this item.

General structural features (literature/general):

• Backbone: Typical “pLenti” backbones support lentiviral packaging (e.g., 2nd/3rd generation systems) and contain a bacterial origin of replication and antibiotic resistance for propagation/selection in E. coli. Exact map and selection markers for this SKU are not specified.
• Expression cassette: sgRNA is commonly expressed from a U6 (or similar Pol III) promoter and includes a standard gRNA scaffold for Cas9 binding. The specific spacer sequence and promoter for this item are not provided here.
• Functional elements often present in pLenti vectors (schematic description): 5′LTR – Ψ (packaging signal) – promoter(s) – sgRNA cassette – selection marker – 3′LTR. Presence/arrangement for this item is not specified.

2D “structure in words” (general for such vectors): Circular double-stranded DNA plasmid if supplied as a plasmid; when packaged, the functional genome is flanked by LTRs enabling lentiviral transduction. Precise sequence and size are not provided for this SKU.

Synthetic Utility

Classical organic “synthetic utility” does not apply. Instead, utility pertains to genetic engineering workflows.

General molecular biology utility:

• Modular cloning: The sgRNA spacer region in pLenti backbones is often exchangeable via Type IIS restriction sites or Gibson/Golden Gate cloning, enabling rapid re-targeting to alternative loci. Exact cloning sites for this SKU are not specified.
• Assembly and editing systems: Compatible with Cas9, Cas12a (if scaffold/promoter adapted), and dCas9 fusions for CRISPRi/a. Verify scaffold compatibility before use.
• Downstream analytics: Vector can serve as PCR template for amplicon sequencing, junction mapping, or barcode recovery in pooled screens (if barcodes are present; not specified here).

Retrosynthetic/construct design considerations (general):

• Promoter choice for sgRNA (e.g., U6 vs H1) affects expression; poly-T tracts can prematurely terminate Pol III.
• For CRISPRa/i, switch to appropriate scaffolds (e.g., MS2, aptamer-modified) or promoter systems.
• Include orthogonal selection markers if multiplexing with additional constructs.

Target Specificity

Item-derived information:

• Intended target: ECT2L (ECT2-like) gene, as indicated by the product name “pLenti-ECT2L-sgRNA.”

Not specified for this item; refer to CoA/Spec Sheet:

• Species reactivity (e.g., human/mouse ortholog targeting)
• Exact sgRNA spacer sequence and genomic coordinates
• Off-target analysis/validation data
• Cas nuclease compatibility (e.g., SpCas9 vs SaCas9 scaffold)
• Vector selection marker(s), promoter(s), and reporter(s)

Note: Without the exact spacer sequence and genome build, on-target/off-target predictions cannot be provided. Users should verify target specificity experimentally in their system of interest.