Express Gene-to-Lentivirus Service

The purification processes for in vitro grade and in vivo grade lentivirus are different. Both grades undergo sterile filtration before release, but in vivo grade purification involves additional stringent steps such as ultracentrifugation and ultrafiltration. In vitro grade lentivirus is intended for cell culture experiments, while preclinical-grade lentivirus can be used for both cell culture and in vivo animal studies.

You only need to provide 1-4 µg of plasmid. We will handle the plasmid preparation necessary for lentivirus packaging. You don’t need to purchase an additional plasmid prep service unless you wish to receive more plasmid from us. Please note, the timeline in our quote already includes the plasmid preparation.

Lentivirus quality control (QC) encompasses various tests to ensure the integrity and safety of lentiviral vectors for research and therapeutic applications.Our release QC testing primarily focuses on post-transduction titer, which provides a true functional titer(infectious titer) to prevent overestimation.

Post-transduction qPCR involves infecting cells with lentivirus followed by quantifying viral titers using quantitative PCR (qPCR), providing essential information about lentiviral transduction efficiency.

Transduction tests are performed by infecting cells with lentivirus and diluting them to count fluorescent cells. Bright field and fluorescent microscopy images are then analyzed to evaluate transduction efficiency.

Additional QC tests include p24 ELISA to measure the core capsid protein of HIV for accurate lentivirus titration, PCR-based mycoplasma testing to ensure absence of contamination, bioburden testing to quantify live microorganisms, and endotoxin testing using the Limulus amebocyte lysate (LAL) assay to confirm absence of endotoxin contamination.

These comprehensive QC tests ensure that lentiviral vectors are of high quality, free from contaminants, and suitable for downstream research and therapeutic applications.

The 3rd generation lentivirus packaging system offers several benefits. It is inherently safer as it does not replicate and self-inactivate, ensuring enhanced biosafety. This system utilizes four plasmids, each carrying different HIV genes, including the removal of tat, which is advantageous for optimizing safety profiles. Additionally, it features a partially removed 5′ LTR followed by a strong promoter such as CMV, RSV, EF1, or U6 for RNA, enhancing transduction efficiency and reducing the risk of insertional mutagenesis. Furthermore, the removal of U3 renders the lentivirus self-inactivating (SIN), minimizing the risk of unintended integration and enhancing the safety profile of lentiviral vectors for gene delivery applications.

Lentivirus can be stored for up to 6 months at -80°C, although its titer may decrease after this period. To maintain optimal titer, we recommend avoiding freeze-thaw cycles and aliquoting the lentivirus into smaller portions upon the first thaw. We also offer aliquoting services. The first 10 vials aliquoting is complimentary, with a charge of $5 per additional vial.

The lentivirus genome size between LTRs is about 9.3kb. However, to ensure optimal yield during lentivirus production, we recommend keeping the lentivirus genome size between LTRs below 6.5kb. If the genome size exceeds this limit, we cannot guarantee the desired yield. Additionally, if the initial yield does not meet expectations, we will initiate a second batch production to ensure accuracy.

In contrast to the common practice among most vendors, who typically measure lentivirus titer directly using methods such as qPCR or p24 ELISA. The Traditional p24 ELISA kit is the most commonly published method for measuring lentiviral titer. The method is suitable for tittering native or purified recombinant virus. However, in crude (unpurified) lentiviral supernatant, significant concentrations of overexpressed p24 protein may be present that are not assembled into viral particles. This causes an extreme overestimation of lentiviral titer.

We employ a different approach. Our titer measurement focuses on the post-transduction titer determined by qPCR, which helps to eliminate any concerns regarding overestimation, providing infectious titers that are significantly higher (100-1000 times) than the physical titer measured by p24, ensuring greater accuracy and consistency for experimental applications.

Additionally, if the lentivirus carries a fluorescent protein, we can further validate the post-transduction titer by examining bright-field and fluorescent microscopy images obtained from serial dilutions of the virus.

Viral titers are typically reported in two forms: functional titer(infectious titer), expressed as transduction units (TU/mL), and physical titer, measured in viral particles (VP/mL). The physical titer indicates the total amount of virus present, often quantified by assessing levels of viral proteins like p24 or viral nucleic acids. In contrast, the functional titer(infectious titer) reflects the virus’s ability to infect cells and is usually 100 to 1000 times lower than the physical titer. Although direct measurement of functional titer(infectious titer) is more accurate for calculating the multiplicity of infection (MOI), it is often more labor-intensive. At PackGene, lentivirus scale is based on post-transduction titer, providing infectious titers that are significantly higher than the physical titer measured by p24, ensuring greater accuracy for experimental applications.

Yes. We offer piVector Designer, an online-tool to help you design the lentivirus plasmid. The plamid backbones of various application we provided in piVector Designer is experimentally validated by us, and we provide a vast library of gene elements including promoter, reporter/marker, regulatory elements and polyA. Also, our Ph.D. level techincal support team will evaluate