CAG-mCherry Lentivirus

Name: CAG-mCherry Lentivirus
SKU: LV00963Z

Cat.No. : LV00963Z

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.	LV00963Z
Description	This lentivirus contains mCherry under the control of CAG (CBA) 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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Lentiviruses (LV) belong to the Retroviridae family, but their genomes are more complex than those of gammaretroviruses. All retroviruses contain essential genes required for viral infection and replication. These genes include the gag gene, which encodes structural proteins, the pol gene, which encodes enzymatic proteins, and the env gene, which encodes envelope glycoproteins. LV also contains the rev and tat genes, which are essential for viral replication, as well as the accessory genes vif, vpr, vpu, and nef. The structural proteins are essential for packaging motifs around the RNA in the core of the viral particle. The viral core contains two identical copies of RNA, as well as the enzymatic proteins integrase, reverse transcriptase, and protease in complex with nucleocapsid proteins. Outside the core, there is a layer of matrix proteins that are able to interact with the outer lipid membrane. The glycoproteins encoded by env are embedded in this outer layer.

Viral infection begins with the binding of glycoproteins encoded by the enveloped virus (env) to receptors on the cell membrane. The most commonly used envelope protein in LV-mediated gene therapy applications is the glycoprotein of vesicular stomatitis virus (VSV), which confers broad tropism to the virus by binding to the low-density lipoprotein (LDL) receptor and its similar family members. This binding allows the virus to fuse with the cell. After binding, the viral contents are translocated to the interior of the cell. After entering the cell, the viral envelope is stripped away and the viral core components are released into the cytoplasm. This includes the single-stranded RNA genome, which is converted into cDNA and transported to the nucleus. During gammaretroviral infection, the cell must undergo cleavage (i.e., nuclear membrane lysis) before the viral DNA can enter the nucleus. On the other hand, LVs have additional features, including a central DNA flap and other undetermined factors, which allow their DNA to pass through an intact nuclear membrane. Both gammaretroviruses and LVs integrate their DNA (now double-stranded) into the host genome and then use the host machinery to transcribe their genes back into RNA. Once the wild-type retroviral RNA has been replicated and returned to the cytoplasm, it is translated and packaged into new virions, which then bud out of the cell, completing the life cycle.

The cerebral cortex underwent rapid expansion and increased complexity during recent hominid evolution. Gene duplications are major evolutionary forces, but their impact on human brain development remains unclear. Using custom RNA sequencing (RNA-seq), researchers analyzed the spatial and temporal expression of great ape-specific duplicated (HS) genes in the human fetal cortex and identified a repertoire of 35 HS genes that exhibit robust and dynamic patterns during cortical neurogenesis. Among them, NOTCH2NL, a human-specific paralog of the NOTCH2 receptor, stood out for its ability to promote the maintenance of cortical progenitors. NOTCH2NL promotes clonal expansion of human cortical progenitors, ultimately leading to higher neuronal output. At the molecular level, NOTCH2NL activates the Notch pathway by inhibiting cis-Delta/Notch interactions. These studies identify a large number of recently evolved genes active during human corticogenesis and reveal how human-specific NOTCH paralogs promote the expansion of the human cortex.

Here, researchers used lentivirus-based clonal analysis to measure the potential effects of NOTCH2NL on the clonal amplification and differentiation of individual cortical progenitors (Figures 1A and 1B). Human ESCs were first differentiated into cortical cells for 30 days and then infected with low-titer lentiviruses (Lenti-CAG-mCherry control and Lenti-CAG-NOTCH2NLB-ires-EGFP). Under control conditions, cortical progenitors gradually expanded, resulting in a doubling of clone size after 20 days of culture (Figures 1C-1I), while the number of SOX2-positive progenitors per clone decreased over the same period (Figures 1J-1R). Notably, NOTCH2NLB-expressing clones increased in size almost threefold over the same period (Figure 1I), with a continued increase in the proportion of SOX2-positive progenitors (Figures 1Q and 1R), reflecting the enhanced ability of NOTCH2NLB-expressing progenitors to expand clones. Furthermore, quantification of the number of neurons per clone showed that NOTCH2NLB clones produced a greater neuronal output than controls (Figure 1P). These data suggest that NOTCH2NLB expression in cortical progenitors results in larger clone size, slower exhaustion of the progenitor pool, and ultimately the generation of more neurons.

Figure 1. NOTCH2NLB Overexpression Leads to Clonal Expansion of Human Cortical Progenitors. (Suzuki I K, et al., 2018)

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