Cat.No. : LVIM011Z
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. | LVIM011Z |
| Description | Lentivirus expressing untagged Human CDK4 under the control of CMV promoter, and puromycin resistance marker for mammalian cell selection. |
| Target Gene | CDK4 |
| Species | Human |
| Application | Human CDK4 lentivirus (CMV, Puro) is a research tool used in molecular and cell biology to study gene function, specifically the function of the CDK4 gene encoding cyclin-dependent kinase 4. The following are its main applications: Immortalized cell lines: With the CDK4 lentivirus, researchers can create immortalized cell lines. These cell lines are able to divide and grow continuously, avoiding experimental limitations caused by cellular senescence. This is extremely beneficial for both basic research and drug screening, as these stable cell lines can be used for long-term studies. Cancer research: CDK4 is highly expressed in many types of cancer, and its dysregulation is often associated with tumor formation. By infecting cancer cells with CDK4 lentivirus, researchers can study the role of CDK4 in cancer development and progression in more detail, providing assistance in the search for new therapeutic targets. Gene knock-in/overexpression: Stable overexpression of CDK4 through lentiviral vectors helps to study its role in various cell types. The physiological and pathological functions of CDK4 can be deeply understood by observing changes in cell behavior, activation of signaling pathways, etc. Drug screening: Cell lines that stably express CDK4 can be used for high-throughput drug screening to find potential drugs that can regulate CDK4 activity. This is of great significance for the development of new treatments. Regenerative medicine and stem cell research: Stem cell immortalization: Using CDK4 lentivirus, scientists can generate immortalized stem cells, which provides a valuable tool for stem cell research and regenerative medicine. These stem cells can be used to better understand the mechanisms of cell differentiation and tissue regeneration. |
| 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. |
Biological experiments are often performed in vitro using immortalized cells because they are easier to obtain and propagate than primary cells and live animals. However, immortalized cells may present different proteomic and glycoproteomic characteristics from primary cells due to the introduction of genes that enhance proliferation (e.g., CDK4) or extend telomeres. To demonstrate the changes in phenotype after CDK4 transformation, researchers performed LC-MS/MS glycomic and proteomic characterization of a primary human lung cancer cell line (DTW75) and a CDK4-transformed cell line derived from DTW75 (GL01). The study showed that primary cells expressed higher levels of mixed and complex sialylated N-glycans, while CDK4-transformed cells expressed higher levels of complex fucosylated and sialylofucosylated N-glycans. In addition, CDK4-transformed cells expressed higher levels of RNA binding and adhesion proteins. CDK4-transformed cells showed changes in N-glycosylation after 31 days of cell culture, with a decrease in high mannose and an increase in fucosylated, sialylated, and sialofucosylated N-glycans. Identification of these changes between primary and CDK4-transformed cells will provide useful insights when tuning cell lines to more closely resemble in vivo physiological conditions.
To establish a CDK4-transformed lung cancer cell line, primary cells (DTW75) were obtained from a lung cancer patient with type IIA lung adenocarcinoma, transfected with a CDK4 lentiviral vector, and then single cell populations were isolated and cultured to obtain GL01 cells (Figure 1). Primary and immortalized cells were then characterized and protein glycosylation and protein expression were compared. N-glycome analysis was performed after N-glycan release with PNGase F, followed by cleanup with porous graphitized carbon (PGC), and N-glycome (chipQToF LC MS/MS) analysis. On the other hand, the proteome was obtained by trypsin digestion, C18 cleanup, and proteome (nLC-Orbitrap LC-MS/MS) analysis. The researchers identified more than 300 N-glycan structures and more than 3,000 proteins from both cells.
Figure 1. Study design for determining the effect of CDK4-transformation on glycosylation. Primary cells were obtained from a lung cancer patient and then transformed using a CDK4-lentivirus. Cells were analyzed for N-glycomic and proteomic profiles using Chip-QToF and nLC-Orbitrap, respectively. (Alvarez M R S, et al., 2024)
A: The transduction unit (TU or IFU) of the lentiviral particles was estimated using the formula- 1TU=30 copies of viral genomic RNA. The physical copy numbers of the viral genomic RNA was determined using qRT-PCR. The customer should test the transduction at MOI=0.3, 1, 3, 5, 10 for their specific cell lines in order to get the best transduction efficiency.
A: Lentivirus is sent on dry ice, please put it to -80°C for storage immediately after receipt. Avoid repeated freezing and thawing to affect virus titers.
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