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Scientific Background
HLA-E, or MHC (major histocompatibility complex) class I antigen E, is considered a non-classical MHC class I with low expression and fewer polymorphisms than the remaining HLA. HLA-E is composed of a heavy chain and β-2 microglobulin (B2M). It binds to specific peptides derived from the classical MHC class I (HLA-A, B, C and G), after these have been processed in the endoplasmic reticulum and the proteosome. The complex of HLA-E with the peptide is recognized by NK cells via the inhibitory receptor CD94/NKG2A/B. Binding to CD94/NKG2C however results in NK cell activation. Expression of HLA-E combined with knockout of HLA-A, B and C, in pluripotent stem cell (PSC) and their differentiated cell types, resulted in these cells escaping attack by CD8+ T cells and NK cytotoxicity. This strategy brings us closer to an almost universal cell donor reality, reducing the risk of immune rejection during cell transplants and alleviating the enormous investment of creating a PSC bank that has representation of all the haplotypes.
Product Description
HIV-based · VSV-G-pseudotyped · SIN=YesHLA-E Lentivirus are replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles ready to transduce nearly all types of mammalian cells, including primary and non-dividing cells. These viruses result in expression of human HLA-E (NM_005516.6) driven by an EF1a promoter, and a puromycin selection marker.
Technical Details
| Vector Type | HIV-based, VSV-G-pseudotyped lentiviral vector |
|---|---|
| Payload / Construct | HLA-E transgene |
| Target Antigen / Gene | HLA-E (MHC class I antigen E) |
| Signaling / Architecture | N/A (transgene expression) |
| Selection Marker | Puromycin |
| Reporter | None |
| Biosafety Level | BSL-2 |
| SIN Vector | Yes |
| Formulation | The lentivirus particles were produced in HEK293T cells in medium containing 90% DMEM + 10% FBS. |
| Supplied As | Two vials (500 µl x 2) of lentivirus at a titer ≥107 TU/ml. The titer will vary with each lot; the exact value is provided with each shipment. |
| Storage | −80°C; avoid repeated freeze-thaw cycles |
| Hazardous Shipping | UN3373 |
Applications
- Expression of human HLA-E in cells of interest.
- Generate cell pools or stable cell lines expressing HLA-E following puromycin selection.
Biosafety & Safety
To generate an HLA-E stable cell line, remove the growth medium 48 hours after transduction and replace it with fresh growth medium containing the appropriate amount of puromycin (as pre-determined from a killing curve, bpsbioscience.com/cell-line-faq), for antibiotic selection of transduced cells, followed by clonal selection. The lentiviruses are produced with a SIN (self-inactivation) lentivector which ensures self-inactivation of the lentiviral construct after transduction and after integration into the genomic DNA of the target cells. None of the HIV genes (gag, pol, rev) will be expressed in the transduced cells, as they are expressed from packaging plasmids lacking the packing signal and are not present in the lentivirus particle. Although the pseudotyped lentiviruses are replication-incompetent, they require the use of a Biosafety Level 2 facility. BPS Bioscience recommends following all local federal, state, and institutional regulations and using all appropriate safety precautions. Troubleshooting Guide: Visit bpsbioscience.com/lentivirus-faq for detailed troubleshooting instructions. For further questions, please email support@bpsbioscience.com.
To generate a HLA-E-expressing stable cell line, transduce your cells of interest at an appropriate MOI. Remove growth medium 48 hours post-transduction and replace with fresh medium containing puromycin at a concentration pre-determined by a kill curve. Continue selection until untransduced control cells are fully eliminated.
BSL-2 facilities are required. The lentiviruses are produced using a SIN (self-inactivating) lentivector design, ensuring self-inactivation after integration. HIV accessory genes are not expressed in transduced cells. Always follow applicable institutional and regulatory biosafety guidelines.
Each shipment contains 500 µl × 2 of lentivirus at ≥10⁷ TU/ml. The exact titer is lot-specific and is reported on the Certificate of Analysis provided with each shipment.
This lentivirus encodes a puromycin resistance gene. Following transduction, stable integrants can be selected by puromycin treatment. Determine the optimal puromycin concentration for your cell line using a kill-curve assay prior to selection.
This VSV-G-pseudotyped lentiviral vector has broad tropism and can transduce most mammalian cell types, including primary cells, non-dividing cells, and established cell lines. Transduction efficiency may vary by cell type and MOI.
Store at −80°C immediately upon receipt. Avoid repeated freeze-thaw cycles to preserve titer. Aliquot into single-use volumes before freezing for best results.
BioHippo offers customization and add-on services for selected products. Options may include:
- Custom formulations: Virus particles can be packaged in alternative formulations upon request (additional fees may apply).
- Custom titers: Higher-titer preparations may be available; contact us for feasibility and pricing.
- Bulk orders: Volume discounts are available for qualifying order quantities.
- Technical support: Our scientific team can assist with protocol optimization and troubleshooting.
For all customization inquiries, please use the contact form or email support@biohippo.com.
- Gornalusse G., et al., 2017 Nature Biotechnology 35:765-772
