Cat.No. : LVIM004Z
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
| Cat. No. | LVIM004Z |
| Description | Lentivirus expressing untagged SV40 Large T Antigen under the control of EF1α promoter, and hygromycin resistance marker for mammalian cell selection. |
| Target Gene | Large T Antigen |
| Species | SV40 |
| Application | SV40 Large T Antigen Lentivirus (EF1α, Hygro) is a recombinant viral vector widely used in molecular and cell biology research. Cell Immortality: The main application is to immortalize primary cells. Many primary cells have a limited lifespan in culture; expression of the SV40 Large T Antigen can extend their ability to proliferate, allowing for extended experimental studies. Generation of Stable Cell Lines: Researchers often use these lentiviruses to create stable cell lines that can continuously express a specific gene of interest. This is particularly useful in protein production, drug screening, and other long-term biological experiments. Cancer Research: Because the SV40 Large T Antigen can promote oncogenic transformation, lentiviruses can be used to study the mechanisms of tumorigenesis. Gene Function Studies: Lentivirus systems can efficiently deliver and stably integrate transgenes. Scientists can study gene function, signaling pathways, and cellular response mechanisms by expressing or knocking out a gene of interest (in conjunction with the SV40 Large T Antigen). High-throughput Screening: In drug discovery, these lentiviral vectors can be used to generate stable and proliferative high-throughput screening platforms, making them suitable for large-scale screening. |
| Titer | Varies lot by lot, for example, ≥1*10^7 TU/mL, ≥1*10^8 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. |
Melanocytes (MCs) are specialized cells that synthesize melanin in melanosomes. Cultured MCs can be used to study their relationship with pigmentation. However, the MC isolation process is cumbersome and the obtained cells can be cultured for a limited time. Here, researchers transformed primary rabbit melanocytes (Pri RMCs) with lentivirus-mediated simian virus 40 Large T (SV40-LT) to establish an immortalized cell line. Morphologically, immortalized RMCs (Im RMCs) were indistinguishable from Pri RMCs, and dendrites were visible after DOPA staining. There was no significant difference in cell proliferation or growth between immortalized RMCs and primary RMCs. Based on melanocyte-specific markers, the expression of MITF, TYR, and TYRP1 was detected by PCR, immunofluorescence staining, and Western blot analysis. Immortalized RMCs did not undergo malignant transformation by karyotyping, soft agar assay, and tumorigenicity assay, indicating that immortalized RMCs can be used as tool cells for future studies of pigmentation mechanisms in fur animals.
Figure 1. Immortalization of rabbit melanocytes using simian virus 40 large T (SV40-LT) lentivirus (A) Isolation and morphological observation of primary melanocytes (100x). (B) Identification of LV-SV40LT lentivirus-expressing cells. (C) Fluorescence analysis of pLVX-IRES-Puro-GFP infection after 48 hours. (D) Monoclonal selection in which 3d/23 d indicates the 3rd/23th day after monoclonal selection. (Chen Y, et al., 2019)
RMC P56 cells were evaluated for SV40-LT, MITF, TYR, TYRP1, and GAPDH mRNA expression by SqPCR (Figure 2A). SV40-LT target bands were observed in immortalized RMCs but absent in Pri RMCs. Western blot and immunofluorescence staining showed SV40-LT expression in RMCs but not in Pri RMCs, indicating successful integration (Figure 2B, C). Expression of melanocyte-specific markers MITF, TYR, and TYRP1 was found in Im RMCs (Figure 2B, C). These findings were consistent with the Pri RMC western blot (Figure 2B) and immunofluorescence analysis.
Figure 2. Tissue-specific gene expression in primary and immortalized melanocytes. (Chen Y, et al., 2019)
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