4 Things to Consider When Making Lentiviral Constructs

Home
Blog
4 Things to Consider When Making Lentiviral Constructs

4 Things to Consider When Making Lentiviral Constructs

Posted by Applied Biological Materials (abm) on December 3, 2018

Lentivirus is one of the most commonly used gene delivery systems, yet there are many things to consider before using it for your experiments.

Here are 4 things you should consider when making your lentiviral constructs:

Is lentivirus the right gene delivery system for your experiment?
Is your sequence within the size limit?
Have you optimized your vector backbone design?
Are you choosing the right packaging system?

Find out the answers to these questions below, plus some additional tips for your next experiment.

1. Is lentivirus the right gene delivery system for your experiment?

Here are the advantages of using lentiviruses in your experiments:

Can infect a wide host range with high efficiency and minimal immune response
Able to transduce both dividing and non-dividing cells
Stably integrates into the host genome for long term gene expression

2. Is your sequence within the size limit?

Lentivirus have a defined size limit which is determined by backbone elements like 5’ and 3’ LTRs which flank what is packaged into the viral particle.

Pro Tip: For best packaging results, ensure that the sequence between and including the LTRs is less than 9.4kb in length.

3. Have you optimized your vector backbone design?

Due to the 9.4kb size limit, you have to make wise decisions on what to include into the vector. Common expression cassettes include:

5’ LTR - Promoter – Gene of interest – Promoter – Reporter or Selection Marker – WPRE – 3’LTR

Don’t include a polyA signal between the LTRs, or this will greatly reduce your virus titer.

Pro Tip: For best packaging results, ensure that the sequence between andPro Tip: Include a reporter like GFP or selection marker like Puromycin for ease of monitoring transfection/transduction, as well as stable cell selection.

4. Are you choosing the right packaging system?

Yes, the packaging system matters! Most commonly used are the 2nd Generation and 3rd Generation Packaging Systems, and 293T cells.

2nd Generation: Virus production is dependent on the tat gene supplied by packaging plasmids.
3rd Generation: Tat dependence is eliminated through the use of a chimeric 5’ LTR.

Pro Tip: All of abm’s lentiviral vectors belong to the third generation, but can be packaged using either one of these systems! We recommend 2nd Generation for higher titers, and 3rd Generation for greater safety.

Ready to start? Browse our extensive Lentivirus Library.

Browse Lentivirus Collection

To learn more about the lentivirus system, visit our knowledge base on the topic.

Have questions or need help with your project? Contact our Technical Support team at [email protected]

Molecular Minutes Blog

Educational resources for life scientists and interviews with scientists/science comunicators in the field.

For more in-depth articles, check out our knowledge base, which covers topics such as CRISPR, Next Generation Sequencing, PCR, Cell Culture, and more.

Blog managed by Applied Biological Materials (abm).