Hi,
I'm new in the field of virology and am looking into retrovirus (MLV) production at the moment. I've read vaguely that one can measure the titers by infecting NIH3T3 (gold standard) with diluted virus:
% GFP x absolute no. of cells transduced)/ vol of viral supernatant used
Is this true? Is there anything I need to pay attention to?
Thanks in advance!
Dear Justin
Most of the original work in this area were published in 70s . Please visit
http://www.sciencedirect.com/science/article/pii/0042682270903624
http://www.sciencedirect.com/science/article/pii/0042682271901681
Hi Justin,
yes it is possible. You'll have to take your supernatant from your producer cell line. Say you have 1ml of viral suspension. Dilute this viral suspension (vs) serially 1:10 (100ul vs . 900ul PBS or similar agent); perhaps up to 6times. It depends on producer cell line. Take this supernatant and tranfect 10exp4 NIH3T3 cells with the serially diluted vs. One dilution with one NIH3T3 sample. Wait 2-3 days. GFP can be checked quickly by fluorescense microscopy. This is just to verify positive transduction.
Trypsinize and FACS these cells.
(There is one point you'll have to take into account. Native retroviruses protect themself after infection by downregeulation of the receptor in the host cell to prevent superinfection. But you have recombinant viruses. Therefore, with high vs concentration you inevitably end up with multiple transductions per cell.) Therefore you use this serially diluted vs.
After FACS you'll have to take the 2 successive fraction which will give you the same result to calculate your titer:
for ex.: 1e4 cells, 1:100 dilution, 1000 green cells = 100x1000=100.0000 viral particle/ml
1e4 cells, 1:1000 dilution, 100 green cells = 1000 x 100=100.000 viral particle/ml (As your original volume was 1ml)
I hope this helps, Ingo
What strains of MLV do you use? N-3T3 expresses a restriction factor Fv1n, which blocks B-tropic MLV infection. N-tropic and Moloney MLV should be able to infect N-3T3. By MLV, do you mean replication competant virus, or pseudotyped vector virus for single round infection? In our lab we routinely use FACS to determine the single round infectivity of GFP-expressing MLV vector virus.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology