pLenti-B2M-sgRNA

Cat. No.: P744047

Specifications

Product Name

pLenti-B2M-sgRNA

Product Line

Produits biologiques

Stockage et expédition

Conditions de stockage de stockage

Conserver à -80°C

Expédié en

Dry ice packs + Cold packs

Stabilité et stockage

Stocké à -20 ℃, valable pendant au moins 6 mois ; Stocké à -80 ℃, valable pendant au moins un an.

Application Protocols

Item-specific validated applications are not provided in the Product Data. The following are generalized, literature-based protocols for pLenti sgRNA plasmids (adapt as needed and confirm backbone features for this SKU).

A. Packaging into lentivirus (general)

• Day −1: Seed HEK293T to reach 70–80% confluence on transfection day.
• Day 0: Transfect transfer plasmid (this SKU) with packaging plasmids (e.g., gag/pol and VSV-G) using PEI or lipid reagent; use serum-free medium for complexing.
• Day 1: Replace medium after 6–16 h.
• Days 2–3: Collect supernatant at 48 and 72 h; filter (0.45 µm); concentrate if required. Store aliquots at −80 °C.

B. Cell transduction and selection (general)

• Add virus to target cells with polybrene (4–8 µg/mL) as tolerated; spinoculation optional.
• After 24 h, replace medium; begin antibiotic selection 48–72 h if backbone encodes a marker (not specified here).
• Expand cells and assess editing at day 3–7 by PCR/Sanger (ICE/TIDE) and by HLA-I flow cytometry to phenotype B2M loss.

C. Direct transfection (if using plasmid without viral packaging)

• Complex with lipid/PEI per manufacturer; change medium after 6–16 h; analyze 48–96 h post-transfection.

Controls and QC

• Include non-targeting sgRNA and a known on-target control.
• Verify plasmid identity by Sanger sequencing of the sgRNA insert prior to use.

Biological Roles

Focus: The encoded sgRNA targets B2M (beta-2-microglobulin). Sequence details are not provided for this item and must be obtained from the CoA/Spec Sheet.

Literature biology of B2M (context for research use)

• Function: B2M is a non-glycosylated component of the MHC class I complex, non-covalently associated with HLA heavy chains, critical for peptide loading and stable surface expression on nearly all nucleated cells.
• Pathways: Involved in antigen processing and presentation via MHC I; impacts CD8+ T cell recognition. Loss of B2M disrupts MHC I transport to the cell surface.
• Cellular consequences of B2M loss (research observations): Reduced HLA-I surface levels; altered susceptibility in co-culture assays with cytotoxic lymphocytes and NK cells; changes in interferon signaling readouts due to impaired antigen presentation.
• Model systems: B2M knockouts are widely used to study immune evasion mechanisms, tumor–immune interactions, and to create HLA class I–deficient lines for MHC engineering studies.

Experimental notes (general)

• Validate on-target edits by sequencing B2M exon(s) targeted by the sgRNA. Complement with functional flow cytometry using pan-HLA class I antibodies.
• Consider off-target analysis for guides designed against conserved exons.

All biological roles described here are general literature context and not claims about any therapeutic or diagnostic application. This product is for research use only.

Buffer Applications

This SKU is a plasmid DNA reagent; classical small-molecule buffering systems are not the focus. However, buffer choice is important for DNA stability and downstream performance.

General guidance (literature)

• Long-term storage: TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5–8.0) helps protect DNA from nucleases; EDTA chelates Mg2+.
• Short-term/enzymatic use: Nuclease-free water or low-ionic-strength Tris (≤10 mM, no EDTA) to maintain compatibility with restriction enzymes, ligases, polymerases, and transfection reagents.
• Cell culture compatibility: Avoid high salt and EDTA in solutions intended for direct complexation with lipids/PEI or for electroporation; exchange into water or electroporation buffer if needed.
• Sterility: For work involving cell exposure, maintain sterility by using sterile-filtered buffers and aseptic technique.

Example preparations (general; not item-specific)

• Reconstitution: Add nuclease-free water to achieve desired stock (e.g., 0.5–1.0 µg/µL), mix gently, briefly spin, and incubate on ice until fully dissolved.
• Working aliquots: Prepare 10–50 µL aliquots at experimental concentration to avoid repeated freeze–thaw.

Item-specific buffer composition and concentration are not provided in the Product Data; consult the CoA/Spec Sheet for exact formulation.

Green Alternatives

Context: This SKU is a plasmid DNA reagent; “green solvent” frameworks are not directly applicable. Nonetheless, greener and safer workflow choices exist for genome-editing delivery.

Greener/safer workflow considerations (general)

• Delivery modality comparison (literature):
  • Lentiviral sgRNA delivery: High efficiency and stable integration; requires BSL-2 facilities and generates biological waste.
  • Non-viral transfection (lipid/PEI): No viral packaging; reduced biosafety overhead; typically transient unless selection/integration systems are used.
  • RNP delivery (Cas9 protein + synthetic sgRNA): DNA-free, transient; minimizes off-target integration risk; reduces need for antibiotic selection and viral packaging.
• Reagent selection: Use endotoxin-low plasmid prep kits to reduce cellular stress and waste associated with repeated optimizations.
• Process efficiency: Plan multiplexing and barcoding to minimize culture volumes and consumables.

Illustrative comparison (general; choose per project constraints)

• Lentivirus vs. RNP
  • Throughput: Lentivirus supports large screens; RNP better for small-scale edits.
  • Biosafety: Lentivirus requires BSL-2 and additional decontamination; RNP typically BSL-2 without viral components.
  • Waste: Viral work generates more regulated biological waste.

Adopt institution-approved decontamination (10% bleach) and energy-efficient cold storage practices (use aliquots to limit freezer door-open time).

Pharmaceutical Uses

Not applicable for clinical or manufacturing use. This product is labeled for research use only.

General context (informational; not a claim for this item)

• Plasmid DNA constructs such as lentiviral transfer vectors are widely used in discovery research, assay development, and preclinical mechanistic studies. They are not suitable for therapeutic administration without substantial process controls and regulatory compliance.
• Pharmacopeial status: No pharmacopeial monograph is applicable to research-grade plasmid DNA expressing sgRNA.
• Formulation/excipient role: Not applicable. This is a genetic construct intended for cell-based experiments, not an excipient.

If your application requires GMP-grade or clinically compliant materials, source vectors manufactured under appropriate quality systems. For this SKU, consult the CoA/Spec Sheet for research-grade QC only.

Physical Properties

Item-specific facts (from Product Data)

• Appearance, concentration, solvent/buffer, OD260, A260/A280: Not specified for this item; refer to CoA/Spec Sheet.

Literature/general properties of plasmid DNA solutions (for context; not product specifications)

• Physical state: Aqueous solution or lyophilized solid, depending on formulation.
• UV absorbance: Peak at ~260 nm; A260/A280 ~1.8–2.0 indicates relatively pure DNA (literature).
• Extinction coefficient: 1 A260 unit ≈ 50 µg/mL for dsDNA (literature).
• pH stability: Stable in mildly basic TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5–8.0); EDTA chelates divalent cations to limit nuclease activity (general guidance).
• Temperature stability: Long-term storage recommended at ≤ −20 °C; minimal freeze–thaw cycles preserve integrity (general practice).
• Viscosity/conductivity: Dependent on buffer and concentration; highly concentrated DNA can be viscous (general observation).

Handling-related notes (general)

• Avoid repeated freeze–thaw; aliquot on first receipt.
• Use nuclease-free plasticware; avoid metal ions that catalyze nuclease activity.
• If lyophilized, rehydrate in nuclease-free water or TE buffer to the desired concentration; mix gently to avoid shearing.

Any specific numeric specifications (concentration, volume, buffer composition, endotoxin level, residual solvents, contaminants) are not provided in the Product Data and should be confirmed on the CoA/Spec Sheet.

Quality and Grades

Item-specific facts (from Product Data)

• Grade/purity, concentration, sequence verification, endotoxin levels: Not specified for this item; refer to CoA/Spec Sheet.

General guidance on plasmid DNA quality (for context; not product specifications)

• Sequence identity: For sgRNA constructs, confirm the 20-nt spacer and scaffold integrity by Sanger sequencing. Verify absence of indels or mixed bases.
• Purity tiers (literature/industry):
  • Research-grade: Suitable for transfection/packaging; typical QC includes OD260/280, agarose gel integrity, and restriction digest. Endotoxin may be moderate unless low-endotoxin preps are specified.
  • Transfection-grade/Endotoxin-low: Endotoxin minimized (e.g., <10 EU/mg DNA; literature examples) to reduce cytotoxicity in sensitive cells.
  • GMP-like/clinical grade: Not applicable here; this product is for research use only.
• UV transparency: Not a solvent; “HPLC grade/UV cutoff” concepts do not apply.

Stabilizers and formulation (general)

• Common storage buffers: TE (10 mM Tris, 1 mM EDTA, pH 7.5–8.0) or nuclease-free water. EDTA can inhibit downstream enzymatic reactions at high concentrations; dilute or exchange buffer if needed.

Recommendation

• Consult the CoA/Spec Sheet for this SKU for any item-specific QC metrics (sequence map, antibiotic resistance marker, backbone length, concentration, endotoxin, and recommended selection antibiotics) before experimental use.

Reaction and Applications

Item-specific facts (from Product Data)

• Manufacturer applications: Not specified in Product Data; the name implies CRISPR sgRNA expression targeting B2M.

General applications for pLenti sgRNA plasmids (literature)

• CRISPR knockout/knockdown studies: Express a guide RNA targeting the B2M gene to induce Cas9-mediated indels via non-homologous end joining (NHEJ). Useful for modeling loss of MHC class I surface expression in cell-based assays.
• Stable cell line generation: Lentiviral delivery enables integration and stable sgRNA expression in dividing and non-dividing cells when paired with Cas9 (constitutive or inducible).
• Functional genomics: Dissect antigen presentation pathways, immunopeptidome regulation, and downstream signaling after B2M loss.
• Pooled screens: If configured in a pooled library context, B2M-directed guides serve as positive controls for immune-evasion phenotypes (general concept).

Practical tips (general)

• Verify guide sequence and on-target locus by Sanger before packaging.
• Use high-quality, endotoxin-low DNA for transfection into HEK293T producer cells to improve viral titers.
• Validate editing: Perform genomic PCR and Sanger/ICE analysis, TIDE, or NGS. Confirm phenotypic loss of surface HLA class I by flow cytometry (e.g., anti-HLA-ABC antibodies), recognizing this is a research assay.
• Include non-targeting and safe-harbor controls; evaluate potential off-targets in silico (e.g., CRISPOR, Benchling) and empirically as needed.
• If the plasmid does not encode Cas9, ensure cells express Cas9 via a compatible system prior to sgRNA delivery.

Reaction Conditions

There are no chemical reaction conditions for a DNA plasmid; however, practical conditions for transfection and lentiviral production are provided below as general literature guidance (not item-specific specifications).

Lentiviral packaging (general)

• Producer cells: HEK293T at ~70–90% confluence.
• Plasmid ratios: Typical mass ratio transfer:packaging (gag/pol):envelope (VSV-G) ≈ 4:3:1 (literature). Optimize per reagent.
• Transfection: PEI or lipid-based reagents in serum-free medium; replace with complete medium 6–16 h post-transfection.
• Harvest: Collect supernatant 48–72 h; clarify by 0.45 µm filtration; concentrate if needed (PEG or ultracentrifugation).
• Biosafety: Perform all steps at BSL-2 with appropriate containment.

Cell transduction/editing (general)

• MOI: Titrate to desired editing rate; polybrene (4–8 µg/mL) can enhance entry for some lines.
• Selection: If backbone encodes an antibiotic marker (not specified here), begin selection 48–72 h post-transduction; verify expression.
• Validation: Genomic PCR followed by Sanger/ICE or NGS; phenotype by flow cytometry for HLA-I reduction upon B2M disruption.

RNP alternative (general)

• Electroporation: Deliver pre-formed Cas9:sgRNA RNP with a DNA donor if HDR is needed; assess editing at 48–72 h.

Exact conditions depend on cell type, transfection reagent, and backbone features; consult the CoA/Spec Sheet for item-specific information.

Safety and Handling

Item-specific facts (from Product Data)

• GHS classification, signal word, H-statements, pictograms: Not specified for this item; defer to the product SDS.
• Storage conditions: Store at −80 °C (per Product Data).
• Shipping: Dry ice packs + cold packs (per Product Data).

General safety guidance for plasmid DNA reagents (information for laboratory practice; consult SDS/IBC policies)

• Hazard profile: Plasmid DNA is generally not classified as hazardous; however, institutional biosafety policies apply, especially if used to generate lentiviral particles.
• Biosafety considerations: The plasmid alone is non-infectious. If packaged into lentivirus using helper plasmids in producer cells (e.g., HEK293T), perform work at BSL-2 with appropriate engineering controls and approvals.
• PPE: Laboratory coat, gloves, and eye protection when handling solutions; change gloves frequently to maintain nuclease-free conditions.
• Incompatibilities: Strong nucleases (DNase), microbial contamination, and repeated freeze–thaw cycles degrade DNA. Maintain nuclease-free workflow.
• First aid (general): In case of accidental exposure to buffers, rinse with water. If ingestion or significant exposure occurs, seek medical attention and provide SDS.
• Waste: Decontaminate surfaces with 10% bleach followed by ethanol/water. Dispose of DNA-containing waste per local biosafety and institutional guidelines.

Special risks (general)

• Lentiviral work: When producing viral particles, follow institutional viral vector SOPs (biological safety cabinets, sealed rotors for centrifugation, surface decontamination, and post-use inactivation).

Solvent Selection

Not a solvent; this is a plasmid DNA reagent. Classical solvent selection does not apply.

Practical medium/buffer considerations (general, literature)

• Reconstitution/Storage buffers: Nuclease-free water or TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5–8.0). Use TE for long-term stability; use water for enzyme compatibility when EDTA is undesirable.
• Working dilutions: Prepare in nuclease-free water or 1× PBS without divalent cations if cells will be exposed directly; avoid Mg2+/Ca2+ in storage to limit nuclease activity.
• Filtration: If sterility is essential (e.g., direct cell exposure), use sterile, low-protein-binding 0.22 µm filters. Avoid shear by using gentle pressure and wide-bore tips.
• Compatibility: Plasmid DNA is compatible with common transfection reagents (PEI, lipids), electroporation buffers, and standard cloning enzymes when salt/EDTA are at appropriate levels.

Selection relative to alternatives

• For maximal stability during storage, choose TE. For immediate enzymatic use or transfection complexation, choose nuclease-free water or low-salt Tris (≤10 mM) without EDTA.

Storage and Reconstitution

Item-specific facts (from Product Data)

• Storage conditions: Store at −80 °C.
• Shipping: Shipped on dry ice packs + cold packs.
• Appearance, buffer, concentration, and volume: Not specified for this item; refer to CoA/Spec Sheet.

General guidance (literature/best practice)

• Upon receipt: Keep frozen; minimize time at ambient temperature. Record lot number and any concentration stated on the CoA.
• Aliquoting: Thaw on ice, mix gently by slow pipetting (avoid vortexing to prevent shearing), briefly spin, and prepare single-use aliquots to avoid repeated freeze–thaw cycles.
• Reconstitution (if lyophilized; not specified): Rehydrate with nuclease-free water or TE buffer to desired stock (e.g., 0.5–1.0 µg/µL). Allow to fully dissolve on ice for 10–15 min; mix gently.
• Short-term storage: −20 °C (days to weeks). Long-term storage: −80 °C (months). Avoid frost-free freezers with defrost cycles.
• Working solutions: Keep on ice during setup; return unused aliquots to −20/−80 °C promptly. For cell work, use sterile, nuclease-free technique.
• Stability considerations: EDTA in TE helps limit nuclease activity but may inhibit some enzymatic reactions; if needed, exchange buffer by spin column or ethanol precipitation before sensitive steps.

Always consult the product’s CoA/Spec Sheet for item-specific concentration, buffer composition, and any additional handling instructions.

Structure and Identity

Item-specific facts (from Product Data)

• Product: pLenti-B2M-sgRNA (SKU: P744047)
• Type: Plasmid DNA construct encoding an sgRNA that targets the B2M gene (beta-2-microglobulin)
• CAS, CID, InChIKey, SMILES: Not applicable for plasmid DNA; not specified for this item; refer to CoA/Spec Sheet.
• Molecular formula, molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

General description (literature/typical for pLenti sgRNA backbones; may vary by lot/item)

• Backbone: Lentiviral transfer plasmid (“pLenti”) enabling packaging into VSV-G pseudotyped lentiviral particles when co-transfected with helper plasmids. Specific backbone/markers are not specified for this item.
• Typical features (literature): U6 or H1 promoter-driven sgRNA cassette; cloning site for 20-nt protospacer; optional selectable marker (e.g., puromycin) and reporter (e.g., GFP). Do not assume for this product—verify by sequence map/CoA.
• Functional groups/2D description: Circular double-stranded DNA plasmid. Functionally segmented into promoter(s), sgRNA scaffold, bacterial origin of replication, and selection cassettes (exact arrangement not specified).

Identity confirmation (guidance)

• Recommended identity checks (general): Sanger sequencing across the sgRNA insert and junctions; restriction digest to confirm backbone size; whole-plasmid NGS if critical.
• sgRNA target: B2M gene; exact spacer sequence, PAM context, and strand not provided in Product Data—obtain from sequence file/CoA before experimental use.

Synthetic Utility

Classical organic “synthetic utility” does not apply to a plasmid DNA reagent. Instead, consider molecular biology utility.

Molecular biology utility (general)

• Modular cloning: sgRNA spacers are typically introduced via oligo annealing and Golden Gate or Gibson assembly into a pLenti backbone. This enables rapid re-targeting to different B2M exons or alternative genes.
• Vector engineering: The pLenti backbone can be adapted to include different promoters, selection markers, or reporters. Verify the specific features for this SKU via sequence map (not provided in Product Data).
• Combinatorial editing: Co-expression with additional guides (dual-guide architectures) or with donor templates for HDR can be implemented using multi-cassette designs (literature practice).
• Retrosynthetic perspective: For troubleshooting, deconstruct into promoter → sgRNA scaffold → spacer insert → backbone. Validate each module by sequencing to isolate sources of failure.

These are general capabilities of lentiviral sgRNA vectors and not item-specific assertions.

Target Specificity

Item-specific facts (from Product Data)

• Intended target: B2M gene (beta-2-microglobulin) based on product name.
• Exact sgRNA spacer sequence, PAM site, strand orientation, genome build: Not specified for this item; refer to CoA/Spec Sheet.

General considerations (literature)

• On-target site: B2M exons commonly targeted include early coding exons to ensure frameshift and loss of function.
• Off-target risk: Dependent on spacer sequence and cellular context. Use in silico prediction tools and empirical validation (GUIDE-seq, CHANGE-seq, CIRCLE-seq) where critical.
• Species specificity: Human B2M differs from murine and other orthologs; confirm species alignment if using non-human cells. Without the sequence, cross-species activity cannot be assumed.
• PAM requirements: Determined by the Cas nuclease used (e.g., SpCas9 requires NGG PAM). This SKU’s Cas system is not specified; verify in documentation.

Recommendation

• Obtain and review the full sequence map/annotation from the CoA/Spec Sheet to confirm the sgRNA spacer, promoter, scaffold, and any selection markers prior to experimental use.