EF1α-mCherry-Puro Lentivirus

Name: EF1α-mCherry-Puro Lentivirus
SKU: LV00962Z

Cat.No. : LV00962Z

Titer: ≥1*10^7 TU/mL / ≥1*10^8 TU/mL / ≥1*10^9 TU/mL Size: 100 ul/500 ul/1 mL

Storage: -80℃ Shipping: Frozen on dry ice

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Lentivirus Particle Information

Quality Control

Cat. No.	LV00962Z
Description	This lentivirus contains mCherry-IRES-Puromycin under the control of EF1α promoter.
Target Gene	mCherry
Titer	Varies lot by lot, for example, ≥110^7 TU/mL, ≥110^8 TU/mL, ≥1*10^9 TU/mL etc.
Size	Varies lot by lot, for example, 100 ul, 500 ul, 1 mL etc.
Storage	Store at -80℃. Avoid multiple freeze/thaw cycles.
Shipping	Frozen on dry ice

Creative Biogene ensures high-quality lentivirus particles by optimizing and standardizing production protocols and performing stringent quality control (QC). The specific QC experiments performed vary between lentivirus particle lots.
Mycoplasma	Creative Biogene routinely tests for mycoplasma contamination using a mycoplasma detection kit. Cell lines are maintained for approximately 20 passages before being discarded and replaced with a new vial of early passage cells. Approximately 2 weeks after thawing, cell culture supernatants are tested for mycoplasma contamination. Creative Biogene ensures that lentiviral products are free of mycoplasma contamination.
Purity	Creative Biogene evaluates the level of impurities, such as residual host cell DNA or proteins, in prepared lentiviral vectors to ensure they meet quality standards.
Sterility	The lentiviral samples were inoculated into cell culture medium for about 5 days and the growth of bacteria and fungi was tested. Creative Biogene ensures that the lentiviral products are free of microbial contamination.
Transducibility	Upon requirement, Creative Biogene can perform in vitro or in vivo transduction assays to evaluate the ability of lentivirus to deliver genetic material into target cells, and assess gene expression and functional activities.
Proviral Identity Confirmation	All Creative Biogene lentiviral vectors are confirmed to have correctly integrated provirus using PCR. This test involves transducing cells with serial dilutions of the lentiviral vector, harvesting the cells a few days later, and isolating genomic DNA. This DNA is then used as a template to amplify a portion of the expected lentiviral insert.

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The VSV.G pseudotype enables lentiviruses (LVs) to transduce a variety of cell types, including macrophages and dendritic cells (DCs). As a result, recipient antigen-presenting cells (APCs) can present transgene-encoded antigens and efficiently prime antigen-specific CD8+ CTLs, which, once induced, can target transgene-expressing cells for killing. This property has been exploited to trigger anti-tumor and anti-pathogen immunity by delivering tumor-associated antigens or viral determinants via LVs. Following presentation of transgene-derived antigens, B cells can proliferate and differentiate, either into plasma cells (PCs) that secrete high-affinity antibodies against the transgene product or into memory B cells that are prepared for a second encounter with the antigen.

The generation of neutralizing antibodies (nAbs) against lentiviral-encoded proteins may enhance their clearance and/or inhibit their activity through opsonization. These adaptive T and B cell responses occur when naive T and B cells recognize their cognate antigens. Antigen presenting cells (APCs) play a key role in this process as they acquire, process and present antigen epitopes to CD8+ T cells and CD4+ T helper cells in the context of MHC-I and MHC-II molecules, respectively. Following antigen recognition, T cell activation and differentiation are dependent on signal 1, the strength of the interaction between MHC and T cell receptor (TCR), signal 2, co-stimulation, and signal 3, the cytokine milieu at the time of priming. Given that lentiviral particles, like most viruses and viral vectors, have the potential to activate innate immune cells, they may exacerbate the risk of an immune response to transgene products as they induce an inflammatory milieu upon presentation.

Chemokines are key players in the local immune response to tumors. CCL17 (thymus and activation-regulated chemokine, TARC) and CCL22 (macrophage-derived chemokine, MDC) can attract CCR4-bearing cells involved in the cancer immune landscape. However, their direct roles and functional status in tumors remain unclear. Here, researchers show that an active CCL17/CCL22-CCR4 axis is a distinctive feature of the Hodgkin lymphoma microenvironment. CCR4 is widely expressed in immune cells but is highly present on the surface of NK, NKT, and Treg cells. The Balb/C mouse tumor model demonstrated that CCL17 is an anti-tumor chemokine mediated by activated T cell responses. Furthermore, a tumor model in nude mice demonstrated that CCL17 recruited NK cells to inhibit lymphoma growth and enhanced NK-cDC1 interactions to counteract IL4i1-mediated immunosuppression. Interestingly, CCL17-mediated antitumor immune responses are dependent on the lymphoid lineage rather than primarily on the myeloid lineage. Furthermore, it was found from the TCGA database that the CCL17/CCL22-CCR4 axis could not be considered as a biomarker for poor prognosis in most cancer types.

To identify the effects of CCL17 and CCL22 expression on lymphoma cells, researchers designed Lenti-EF1α-Ccl17-p2a-mCherry-IRES-PuroR vector, Lenti-EF1α-Ccl22-p2a-mCherry-IRES-PuroR vector, and Lenti-EF1α-mCherry-IRES-PuroR vector. Then established CCL17-overexpressing A20 cell lines, CCL22-overexpressing A20 cell lines, and control A20 cell lines (Figure 1A). After selection with puromycin, mCherry-positive cell lines were obtained and the mRNA levels of Ccl17 and Ccl22 were evaluated. qPCR results showed that the A20 cell line with overexpression of CCL17 and CCL22 was successfully obtained (Figure 1B). The mRNA and protein expression patterns were inconsistent. Therefore, the researchers further evaluated the protein expression levels of CCL17 and CCL22 (Figure 1C) and excluded cell lines without overexpression of CCR4 ligands (Figure 1D). The transwell cell migration assay showed that the A20-CCL17 and A20-CCL22 cell lines could secrete functional CCR4 ligands and drive migration according to the chemokine concentration gradient. In summary, this study established an effective strategy to evaluate the potential role of CCL17 and CCL22 in the tumor microenvironment.

Figure 1. CCL17 and CCL22 overexpressing lymphoma cell lines were produced using lenti-virus vectors. (Li Y, et al., 2023)

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