CMV-Cre-Puro Lentivirus

Name: CMV-Cre-Puro Lentivirus
SKU: LV00960Z

Cat.No. : LV00960Z

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.	LV00960Z
Description	This lentivirus contains Cre recombinase under the control of CMV promoter and puromycin selection marker under the control of PGK promoter.
Target Gene	Cre
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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How does viral entry targeting work? It has three main parts: First, a targeting ligand with high affinity for a receptor on the surface of the target cell must be displayed on the surface of the lentiviral vector particle. If done correctly, this alone will mediate binding of the vector particle to the target cell type, but not entry and transduction. Second, viral glycoproteins that mediate cell entry must be present. If the native receptor recognition of these glycoproteins is unmodified, then cells expressing the native entry receptor will also be transduced. This can result in cells that are negative for the target receptor but positive for the native receptor being transduced. For example, co-display of an unmodified glycoprotein such as VSV G with a targeting ligand will result in preferential transduction of cells that are positive for the target receptor, an effect that may depend on the relative binding affinities of the target receptor and the native entry receptor.

However, these vectors require expression of the VSV G receptor LDLR for cell entry. As a result, they are not only able to transduce irrelevant LDLR+ cells, but are also incompatible with resting LDLR- lymphocytes. Therefore, while this approach can help increase transduction efficiency of rare cell types, it does not provide true cell type-specific gene delivery to these cells. The latter avoids gene delivery to irrelevant cells and tissues and requires a third engineering step, namely disruption of native receptor binding. This task has not yet been achieved for any viral glycoprotein that combines receptor binding and membrane fusion in one polypeptide, as is the case for VSV G or retroviral envelope proteins. In this regard, an approach incorporating a truncated VSV G into LV particles together with a membrane-anchored targeting ligand yielded promising initial results, but was later shown to mediate transduction of non-target cells.

The developmental pathways that orchestrate the fleeting transition of endothelial cells into hematopoietic stem cells remain undefined. Here, researchers demonstrate a feasible method to completely reprogram adult mouse endothelial cells into hematopoietic stem cells (rEC-HSCs) through transient expression of transcription factor encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase of conversion (days 0-8) is initiated by the expression of FGRS in mature endothelial cells, resulting in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1+FGRS-transduced endothelial cells commit to a hematopoietic fate, giving rise to rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20–28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult hematopoietic stem cells, allowing for clonal engraftment and serial primary and secondary multilineage reconstitution, including antigen-dependent adaptive immune functions. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signaling enhances the generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable hematopoietic stem cells could aid the treatment of hematological disorders.

CXCL12 signals through two receptors, CXCR4 and CXCR7. Since CXCR4 is expressed on both mECs and hematopoietic cells, researchers selectively knocked out Cxcr4 in mECs (Figure 1a). Adult lung mECs were isolated from mice carrying a floxed Cxcr4 allele (Cxcr4^fl/fl). These mECs were transduced with FGRS (dox-on) and VN-EC induction. Cxcr4^−/− endothelial cells were generated by transduction with LV-CMV-Cre-Puro lentivirus followed by 7 days of puromycin selection. Treatment with Cre-recombinase deleted Cxcr4 and impaired Runx1 induction in mECs (Figure 1a). Furthermore, activation of CXCR7 protected vascular fate and blocked the EHT process. Overexpression of CXCL12 by VN-ECs increased the number of VEcad⁻RUNX1⁺CD45⁺ cells. Thus, CXCR4, but not CXCR7, contributes to the conversion to rEC-HSPCs. Furthermore, TGFβ inhibition and activation of BMP and CXCL12 signaling enhanced Runx1 expression. The emergence and expansion of rEC-HSPCs were dependent on CXCL12 signaling (Figure 1b), suggesting that the serial conversion of endothelial cells into rEC-HSPCs provides a feasible approach to screen and identify pathways driving transient EHT.

Figure 1. Deconvolution of vascular niche angiocrine signals in rEC-HSPC generation. (Lis R, et al., 2017)

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