pLenti-U2AF1-sgRNA

Cat. No.: P751465

Specifications

Product Name

pLenti-U2AF1-sgRNA

Product Line

Produits biologiques

Stockage et expédition

Conditions de stockage de stockage

Store at -80°C

Expédié en

Dry ice packs + Cold packs

Stabilité et stockage

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

Application Protocols

No item-specific, tested application protocols are provided for this product. The following is general guidance for pLenti sgRNA workflows; adjust to your system and consult the CoA/Spec Sheet for construct-specific details.

• Overview (general):

  1. Produce lentivirus by co-transfecting packaging and envelope plasmids with the pLenti-U2AF1-sgRNA vector into HEK293T.
  2. Collect, clarify, and optionally concentrate viral supernatant; determine functional titer.
  3. Transduce target cells at desired MOI with appropriate enhancers if compatible.
  4. Apply selection/enrichment as supported by the backbone (marker Not specified for this item).
  5. Validate editing via PCR and sequencing; assess on-target functional consequences.

• Controls:

  • Non-targeting sgRNA control, additional U2AF1-targeting guides, and rescue constructs to verify specificity.

• Notes:

  • Maintain sterile, nuclease-free technique.
  • Optimize MOI and selection stringency per cell type to balance efficiency and cell viability.

For detailed parameters (volumes, incubation times, antibiotics), refer to institutional protocols and the CoA/Spec Sheet.

Biological Roles

This reagent targets U2AF1 (U2 small nuclear RNA auxiliary factor 1), a splicing factor that recognizes the 3′ splice site AG dinucleotide and contributes to spliceosome assembly.

• U2AF1 function (literature):

  • Forms a heterodimer with U2AF2 (U2AF65) to recruit U2 snRNP to the polypyrimidine tract and 3′ splice acceptor.
  • Contains CCCH-type zinc finger domains involved in RNA binding and splice-site recognition.
  • Influences alternative splicing decisions across numerous transcripts, impacting RNA maturation.

• Research relevance (literature):

  • U2AF1 perturbation is used to study pre-mRNA splicing fidelity and regulatory networks.
  • Editing or knockdown of U2AF1 enables functional genomics screens of splicing-dependent phenotypes.

• Product-specific details:

  • The exact sgRNA spacer sequence, target exon, and PAM context are Not specified for this item; refer to CoA/Spec Sheet.

Use appropriate controls (non-targeting sgRNA, additional guides) and transcript-level readouts (RT-qPCR, RNA-seq) to quantify effects on splicing and gene expression.

Buffer Applications

Dedicated buffering applications (e.g., pH control systems for chromatography or enzymology) are not the focus of this product.

• Practical notes (general):
  • Maintain and dilute plasmid/viral stocks in nuclease-free, isotonic buffers compatible with your cells (e.g., PBS, DMEM-based media). Avoid divalent cations if nuclease contamination is a concern.
  • If concentrating or exchanging buffer (e.g., via spin columns), pre-rinse membranes to remove preservatives and verify recovery.

Item-specific buffer composition is Not specified for this item; consult the CoA/Spec Sheet.

Green Alternatives

Green chemistry considerations are less directly applicable to a plasmid/viral-vector reagent, but process-level choices can reduce environmental impact.

• Alternatives and trade-offs (general/literature):

  • Non-viral delivery (e.g., electroporation of RNPs or plasmids) avoids viral packaging waste and associated biosafety requirements; however, it may reduce delivery efficiency in difficult-to-transfect cells compared to lentivirus.
  • CRISPR RNP delivery minimizes persistent DNA and antibiotic markers, potentially reducing selection antibiotics and associated waste; RNPs may require specialized equipment and have transient activity.
  • Use of serum-free or reduced-serum transduction protocols can lower animal-derived inputs; verify cell compatibility.

• Operational improvements:

  • Minimize single-use plastics where feasible; use recyclable packaging streams.
  • Scale reactions prudently to match experimental need, reducing excess viral production.

Choose the method that balances biosafety, efficiency, and waste profile for your laboratory and cell model.

Pharmaceutical Uses

This product is supplied strictly for research use only. It is not intended for human or veterinary use, diagnostic purposes, or as a therapeutic.

• Formulation/excipient status: Not applicable; no pharmacopeial status is provided for this research reagent.

• Manufacturing context (general):

  • In research manufacturing workflows, lentiviral/plasmid materials may be handled under research-grade conditions without cGMP controls. Transitioning to clinical-grade materials requires distinct GMP processes, documentation, and regulatory oversight, which are beyond the scope of this product.

Any clinical or diagnostic application would require re-development with appropriate quality systems and regulatory clearance; not applicable here.

Physical Properties

As a nucleic acid-based reagent (plasmid/viral vector), conventional small-molecule physical constants (bp, mp, density, refractive index) are not applicable.

• Item-specific properties:

  • Concentration, buffer composition, and exact size: Not specified for this item; refer to CoA/Spec Sheet.
  • Appearance: Not specified for this item; refer to CoA/Spec Sheet.

• Storage (from Product Data):

  • Store at −80°C. Shipped on dry ice packs + cold packs.

• General/literature characteristics for plasmid DNA in aqueous buffer:

  • Solubility: Freely soluble in aqueous buffers; sensitive to nucleases.
  • pH sensitivity: Stable near neutral pH; avoid acidic conditions that can depurinate DNA.
  • Freeze–thaw: Repeated cycles can shear DNA and reduce viral titer if in viral format; aliquoting recommended.

• For lentiviral stocks (general):

  • Typically supplied in a buffered aqueous medium with cryoprotectant (e.g., 5–10% glycerol), but exact composition for this item is Not specified; refer to CoA/Spec Sheet.

Note: Do not infer molecular weight or sequence-dependent properties without the item-specific sequence map. Consult the CoA/Spec Sheet for the backbone size (bp), antibiotic resistance, promoters, and sgRNA spacer sequence if provided.

Quality and Grades

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

• Typical quality parameters for plasmid/viral CRISPR reagents (general/literature):

  • Plasmid quality: Supercoiled fraction, A260/280 ratio, endotoxin level for transfection (e.g., endotoxin-reduced prep for cell culture).
  • Viral stock quality: Functional titer (TU/mL), sterility, absence of replication-competent lentivirus (RCL) tests under applicable regulations.
  • Sequence verification: Sanger or NGS confirmation of sgRNA spacer and vector junctions.

• What quality means in practice:

  • High-purity plasmids facilitate higher transfection efficiency and lower cytotoxicity.
  • Accurate guide sequence ensures on-target editing; mis-matches reduce activity.

• Stabilizers/additives:

  • Any stabilizers, cryoprotectants, or antibiotics present are Not specified for this item; refer to CoA/Spec Sheet. If present, they can influence storage stability and downstream cell compatibility.

• Documentation:

  • For regulated environments, retain sequence maps, lot-specific CoA, and SDS. Confirm antibiotic selection markers and promoters to match your host cell system.

In the absence of item-specific declarations, verify suitability by consulting the CoA/Spec Sheet and performing small-scale pilot transfections/infections.

Reaction and Applications

Focus: CRISPR/Cas9 genome editing applications targeting U2AF1 via a lentiviral sgRNA vector.

• Primary research uses (general/literature):

  • Stable delivery of sgRNA into mammalian cells using lentiviral transduction to support CRISPR-Cas9 knockout/knock-in workflows when Cas9 is supplied by the host cell or co-transduced.
  • Creation of isogenic knockouts or pooled screens focused on RNA splicing factor pathways (U2AF1).
  • Complementation studies when paired with rescue constructs.

• Practical tips:

  • Validate guide activity in your cell line; efficiency can vary with chromatin context.
  • Use appropriate MOI to control multiplicity; include antibiotic/fluorescent selection if the backbone supports it (exact markers for this item Not specified; refer to CoA/Spec Sheet).
  • Confirm editing by T7E1, TIDE, ICE, or amplicon NGS.
  • Evaluate on- versus off-target effects using in silico tools and empirical assays; design orthogonal guides where needed.

• Workflow outline (general):

  1. Produce lentiviral particles in HEK293T using standard packaging system; 2) Titer and transduce target cells with polybrene if compatible; 3) Select/enrich edited cells; 4) Genotype and phenotype.

Note: The exact sgRNA spacer sequence, promoters, and selection markers for this product are Not specified; consult the CoA/Spec Sheet for construct-specific workflows.

Reaction Conditions

For genome editing with a pLenti sgRNA construct, the “reaction conditions” translate into transfection/transduction and editing workflows. The following are general literature guidelines; they are not item-specific specifications.

• Lentiviral production (general):

  • Cells: HEK293T/17; Transfection: calcium phosphate or PEI/Lipofectamine in serum-containing medium; Harvest at 48–72 h.
  • Concentration: Optional PEG/NaCl precipitation or ultracentrifugation.
  • Titering: p24 ELISA or functional TU/mL via transduction and marker readout.

• Transduction (general):

  • MOI: 0.1–5 for clonal edits; higher MOI for bulk edits, balancing multiple integrations.
  • Enhancers: Polybrene 4–8 µg/mL if compatible; spinoculation optional.
  • Selection: As per backbone marker (antibiotic/fluorescent), which for this item is Not specified; refer to CoA/Spec Sheet.

• Editing assessment:

  • Timing: 48–96 h post-transduction for initial indels; 7–14 days for stable selection and phenotype readouts.
  • Readouts: TIDE/ICE, amplicon NGS, Western blot, RNA-seq for splicing changes.

Exact buffer compositions, cell densities, and incubation times must be optimized per cell type. Refer to your institutional SOPs and the CoA/Spec Sheet for any construct-specific recommendations.

Safety and Handling

• Hazard 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: Not specified for this item; refer to SDS.
  • Pictograms: Not specified for this item; refer to SDS.

• General biosafety considerations for lentiviral plasmids/sgRNA reagents (literature/best practice):

  • Handle nucleic acid reagents using gloves, lab coat, and eye protection to prevent contamination and exposure.
  • If producing lentiviral particles, follow institutional BSL-2 or higher containment and training requirements; use biological safety cabinets, appropriate disinfectants (e.g., freshly prepared bleach, 70% ethanol), and proper waste inactivation.
  • Avoid aerosol generation when handling viral stocks; use sealed rotors for centrifugation.
  • Dispose of materials according to institutional biosafety protocols.

• Storage incompatibilities (general):

  • Avoid nucleases and nuclease-contaminated consumables; use nuclease-free water/tips/tubes.
  • Avoid repeated freeze–thaw; aliquot upon first thaw.

• First aid (general):

  • Skin/eye contact with solutions: Rinse with water. If irritation persists, seek medical attention. For accidental exposure to viral particles, follow institutional exposure protocol and notify biosafety officer.

Always consult the product-specific SDS and your institutional biosafety committee guidance for authoritative safety requirements.

Solvent Selection

This is a nucleic acid/viral-vector reagent typically supplied in an aqueous buffer. Conventional organic solvent selection is not applicable.

• Practical guidance:
  • Maintain in nuclease-free aqueous buffers (e.g., TE or PBS). Do not dilute in organic solvents.
  • For cell delivery, dilute directly into culture medium or transduction medium as per your protocol. Filter sterilize buffers if needed.

For work that requires small-molecule solvents (e.g., disinfectants, extraction), refer instead to applicable chemical reagent sections; they do not pertain to this product.

Storage and Reconstitution

• Storage (from Product Data): Store at −80°C. Shipped on dry ice packs + cold packs.

• Format-specific notes (item-specific details Not specified; general/literature guidance):

  • If supplied as plasmid DNA in buffer: Store at −20 to −80°C in aliquots; avoid repeated freeze–thaw. Thaw on ice and gently mix; do not vortex vigorously. Use nuclease-free water or buffer for dilution.
  • If supplied as lentiviral stock: Keep at −80°C; thaw on ice just before use; keep on ice during handling. Do not refreeze multiple times—prepare single-use aliquots on receipt.

• Reconstitution:

  • Any lyophilized/reconstitution instructions are Not specified for this item; refer to CoA/Spec Sheet. If lyophilized, reconstitute with sterile, nuclease-free buffer to the recommended concentration, mix gently, and allow complete dissolution on ice.

• Stability:

  • Working aliquots kept at 4°C are generally suitable only for short periods (hours to a day). For longer storage, return to −80°C.

Always verify lot-specific buffer composition, concentration, and recommended storage in the CoA/Spec Sheet before first use.

Structure and Identity

This product is described as a lentiviral CRISPR/Cas9 sgRNA construct targeting the human U2AF1 gene (splicing factor). As a genetic reagent, it is a plasmid/viral-vector-based nucleic acid rather than a small molecule.

• Item-specific identifiers:

  • CAS: Not specified for this item; refer to CoA/Spec Sheet.
  • CID: Not specified for this item; refer to CoA/Spec Sheet.
  • InChIKey: Not applicable to plasmid DNA; Not specified for this item; refer to CoA/Spec Sheet.
  • SMILES: Not applicable to plasmid DNA; 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.

• Construct type (general/literature):

  • Lentiviral plasmid backbone (pLenti) carrying a single-guide RNA (sgRNA) expression cassette targeting U2AF1.
  • Typical features of pLenti-style CRISPR sgRNA vectors include: U6 or H1 promoter for sgRNA, lentiviral LTRs/psi packaging signal, and antibiotic selection markers (exact elements Not specified for this item; refer to CoA/Spec Sheet).

• Structural description (general):

  • Circular double-stranded DNA plasmid when provided as plasmid; packaged as viral particles if produced via helper plasmids in cells (format for this item Not specified; refer to CoA/Spec Sheet).
  • The “U2AF1-sgRNA” denotes a 20-nt spacer sequence complementary to U2AF1 genomic DNA adjacent to a PAM (exact spacer and PAM Not specified for this item; refer to CoA/Spec Sheet).

• Stereochemistry/functional groups: Not applicable for nucleic acids; the functional entity is the nucleotide sequence and expression cassettes.

Synthetic Utility

Conventional small-molecule synthetic utility does not apply. For molecular biology, the construct provides utility in genome engineering workflows.

• Vector-level utility (general/literature):

  • Enables stable integration or long-term expression of sgRNA via lentiviral delivery to a broad range of dividing and non-dividing cells, contingent on the backbone design (exact features Not specified; refer to CoA/Spec Sheet).
  • Facilitates pooled CRISPR screening against U2AF1-dependent pathways when combined with Cas9-expressing cells.

• Cloning and customization:

  • Many pLenti sgRNA vectors allow swapping sgRNA spacers by oligo annealing and Golden Gate or Gibson cloning; confirm for this item via the sequence map (Not specified).
  • Optional donor templates (HDR) can be co-delivered separately for precise edits.

• Downstream analytics:

  • Compatible with standard genotyping (PCR, Sanger) and edit quantification (NGS amplicons) to support iterative design–build–test cycles.

This construct’s specific backbone map, multiple cloning sites, and selection markers are Not specified for this item; refer to the CoA/Spec Sheet for exact capabilities.

Target Specificity

• Intended gene target: U2AF1 (general/literature), a component of the spliceosome recognizing 3′ splice sites.
• sgRNA spacer sequence, genomic locus, and PAM: Not specified for this item; refer to CoA/Spec Sheet.
• Species reactivity: Not specified for this item; refer to CoA/Spec Sheet.
• Off-target considerations (general): Use validated design tools and empirical assays to assess potential off-target editing; include non-targeting and alternative-guide controls.

No antibody clone/isotype applies; this is a CRISPR sgRNA lentiviral construct.

Foire aux questions

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-U2AF1-sgRNA); cross-reactivity in untested species may exist but is not guaranteed. Contact technical support for specific species inquiries.