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Scientific Background
Transforming growth factor receptor beta 2 (TGFBR2), or TGFβRII, encodes the TGFβ receptor serine/threonine kinase, which is a transmembrane protein that forms a heterodimeric complex with other receptor proteins and binds TGFβ. The association of TGFβ with TGFβRII leads to the phosphorylation of proteins, such as SMADs, involved in cell proliferation, cell cycle arrest, wound healing, and immunosuppression. Dysfunction of the TFGβ signaling tends to result in cancer development and progression. In the case of solid tumors, TGFβ signaling plays a role in creating a highly immunosuppressive TME (tumor microenvironment), restricting the efficacy of CAR (chimeric receptor antigen)- T cells, which have proved successful in the treatment of hematological cancers. Recently, the use of CAR-T cells armored with a dominant negative form of TGFβRII, missing the intracellular kinase domain, for the treatment of prostate cancer resulted in promising outcomes. 5 out of 13 patients did suffer cytokine release syndrome, which continues to be a concern with CAR-T applications, but on the whole the use of a dominant negative TGFβ receptor to armor CAR-T cells appears to be an approach that deserves further attention in the cancer therapy field.
Product Description
HIV-based · VSV-G-pseudotyped · SIN=YesDominant Negative TGF-β Receptor Type II (TGF-βRII) 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 dominant negative TGF-βRII, missing the intracellular kinase domain (NM_003242.6; amino acid 1-191), driven by an EF1a promoter and a puromycin selection marker.
Technical Details
| Vector Type | HIV-based, VSV-G-pseudotyped lentiviral vector |
|---|---|
| Payload / Construct | Dominant Negative TGF-βRII |
| Target Antigen / Gene | TGF-βRII (Dominant Negative) (TGFBR2 DN) |
| 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 dominant negative TGF-βRII in cells of interest.
- Generate cell pools or stable cell lines expressing human TGF-βRII following puromycin selection.
Biosafety & Safety
To generate a dominant negative TGF-βRII expressing 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 TGF-βRII (Dominant Negative)-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.
- Ikushima H. and Miyazono K., 2010 Nature Reviews Cancer 10:415-424.
- Narayan V., et al., 2022 Nat Med 28:724-34.
