I would like to express a quite large protein in neurons, around 400KDa, I was told that lentivirus will not do the job.
Do someone has experience with that and what type of virus or other expression system is then recommended?
Thanks in advance for feedback!
400KDa exceeds what most viruses can carry. Rabies virus can carry such large genes if one uses a replication-dead virus and is able to engineer the negative-RNA genome (a few labs can do it: e.g., Klaus Konzelmann in Munich and Marco Tripodi in Cambridge). Gutless Adenovirus 5 can, in principle, reach 30kB of loading capacity and it is simpler to manipulate; vector biolabs offers a full service. Herpesvirus 1 has huge loading capacity (reported up to 50Kb) and it has beuatiful neuronal tropism; you need a minimum BL2 facility because it can easily infect humans. In the past one could get HSV prepared by the core facility of University of South Carolina, for a reasonable price. Alternative options: a trans-splicing AAV may reach 9Kb and a triple-splicing AAV is reported to reach 14Kb (by the Auricchio lab, recent paper); you would need some bioinformatics and optimization of the sequences. Foamy viruses are reported to have a loading capacity in the range of 11Kb, so it may borderline to what you may need. I hope I was useful, somehow.
super helpfull Francesco! I'll look into some of these options and let you know. Thanks a lot again!
I guess you're looking at around 11kb. Why not transient transfection with naked DNA? Lenti can take 11kb but titres aren't great. For lenti, John Counsell's work is good.
Thank you Simon, transfection do not have a nice yield, I would like to have 70-90% of my neuronal population expressing this large protein. I will look at Counsell's work. Thanks!
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus