Advanced Lentiviral Vector Solutions: From Bench to Breakthroughs with Creative Biogene

Over recent decades, gene expression systems—mainly used for expressing cDNA in mammalian cells—have evolved into two main types: transient plasmid transfection and viral vector-based delivery. Among viral vectors, those derived from adenoviruses, adeno-associated viruses (AAVs), lentiviruses, and retroviruses are most common. Adenoviruses and AAVs enable high expression without genomic integration, while retroviruses and lentiviruses integrate into the host genome, allowing stable gene expression. However, retroviruses show species specificity and mainly infect dividing cells.

Lentiviral vectors are advanced retroviral tools derived from HIV-1. Like retroviruses, they carry LTRs that mediate integration and expression, and their genomes encode gag, pol, and env—responsible for structure, enzymatic activity, and host tropism. Lentiviruses also encode regulatory genes (tat, rev) and accessory proteins (vif, vpr, vpu, nef), adding complexity but enabling superior control.

Compared to murine retroviral vectors, lentiviral vectors integrate into both dividing and non-dividing cells and have lower genotoxicity. They excel at transducing hard-to-infect cells like neurons, stem cells, and primary cells.

Modern lentiviral vectors support:

Tissue-specific expression using specialized promoters or miRNA targets
Multi-gene expression via 2A peptides, or bidirectional promoters
shRNA/miRNA expression and library construction
Drug-inducible gene regulation

To improve safety and performance, most vectors are modified to:

Replace the 5' LTR with stronger promoters
Delete the 3' LTR U3 region, forming SIN (Self-Inactivating) vectors
Include WPRE and RRE elements to enhance transcription and packaging

Over the years, to enhance safety, the components needed to produce lentiviral particles have been split into multiple plasmids. This gave rise to successive "generations" of packaging systems, each iteration stripping out unnecessary or risky genetic elements:

First-Generation Packaging System

Transfer plasmid: Carries the gene of interest flanked by LTRs
Envelope plasmid: Expresses the envelope glycoprotein, commonly VSV-G, replacing native Env. VSV-G pseudotyping allows for broad tropism (due to phospholipid-binding instead of specific receptors), high particle stability, and tolerance to ultracentrifugation
Packaging plasmid: Driven by a CMV promoter and expresses Gag, Pol, and regulatory proteins

The envelope and packaging plasmids are stripped of LTRs and packaging signals to prevent self-integration and reduce the chance of generating replication-competent lentivirus (RCL).

Second-Generation Packaging System

Builds on the first generation with further deletions: removes vif, vpu, vpr, and nef, cutting down HIV virulence
Retains only four HIV genes: gag, pol, tat, and rev
Transfer plasmid still requires tat to activate expression via the weak 5' LTR promoter—hence must be used with second-gen packaging

Third-Generation Packaging System

Goes even further: splits packaging plasmids into two—one for rev, the other for gag and pol
The transfer vector contains a hybrid 5' LTR fused with a CMV promoter, eliminating the need for tat
Safer than second-gen systems, but more complex—requires transfection of four separate plasmids to successfully produce lentiviral particles

In summary, to produce functional lentiviral particles, you'll typically need:

Transfer plasmid – the central piece carrying your gene of interest flanked by LTRs
One or two packaging plasmids – providing Gag, Pol, and Rev
Envelope plasmid – expressing the viral envelope protein (usually VSV-G)

All plasmids are co-transfected into 293T cells, which are the gold standard due to their high transfection efficiency and stable expression of SV40 large T antigen (enhancing the SV40 ori on the transfer plasmid). After co-transfection, viral particles are secreted into the culture supernatant. The supernatant can then be collected—either directly or after concentration—and used to transduce your target cells.

Efficient and Customizable Lentivirus Production at Creative Biogene

Creative Biogene employs a high-efficiency third-generation lentiviral packaging system, offering both off-the-shelf viral products and tailored solutions. With a well-established platform, in-house reagents, and strict quality control, we deliver end-to-end support from vector construction to virus production—streamlining your gene delivery workflow.

See how our platform supports real-world applications below.

Case Study 1

EphA10 is a receptor tyrosine kinase virtually absent in normal tissues except the male testis, yet significantly upregulated in triple-negative breast cancer (TNBC) where it correlates with tumor progression and poor prognosis. The researchers developed highly specific anti-EphA10 monoclonal antibodies that selectively recognize this receptor on cancer cells, with their lead candidate (clone #4) demonstrating enhanced tumor regression in TNBC mouse models.

The team utilized Creative Biogene's third-generation lentiviral system featuring the pCDH-EF1α-MCS plasmid and comprehensive LentiArt packaging service (pHelp1, pHelp2, and pHelp3). Creative Biogene's optimized transfection protocols and Lentivirus services provided support for the efficient delivery of the anti-EphA10 CAR construct into T cells.

Figure 1 illustrates the design, characterization, and functional evaluation of the EphA10-CAR construct, including flow cytometry analysis, MTT assay, and immunohistochemistry staining of xenograft sections from mouse models. Figure 1. The researchers assessed the specificity and efficacy of the CAR construct through flow cytometry and MTT assays, demonstrating significant inhibition of TNBC growth both in vitro and in vivo. (Cha JH, et al., 2022)

Case Study 2

Breast cancer stem cells (bCSCs) drive tumor progression and therapeutic resistance through mechanisms that remain poorly understood. The researchers identified miR-600 as a critical regulator of bCSC behavior, showing that silencing this microRNA expands the stem cell population while its overexpression reduces self-renewal and tumor growth.

Creative Biogene helped the researchers to construct specialized miR-600-sponge lentiviral vectors, enabling precise manipulation of miR-600 expression in patient-derived breast cancer cells. The high-quality custom vectors produced through Creative Biogene's services allowed for effective gene function studies that revealed miR-600's regulation of the WNT signaling pathway in bCSCs. This case demonstrates how Creative Biogene's expertise in engineering tailored lentiviral constructs accelerates cancer stem cell research and therapeutic development through reliable, customized viral vector design services.

Figure 2 illustrates the experimental workflow and in vivo analysis of miR-600 modulation in patient-derived xenografts (PDXs), including lentiviral infection, tumor implantation, and cancer stem cell frequency assessment. Figure 2. The researchers utilized lentiviral infection to modulate miR-600 expression in patient-derived xenografts (PDXs). By implanting FACS-sorted DsRed-positive cells into NOD/SCID mice, they assessed tumor growth and bCSC frequency. (El Helou R, et al., 2017)

Ready to accelerate your research with powerful lentiviral tools?

Contact Creative Biogene today to discuss your project needs and discover how our customized solutions can streamline your workflow.

* For research use only. Not intended for any clinical use.

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