I'm having trouble to transduce NIH3T3 with purified adenovirus serotype 5 (Ad5). I looked for information in the internet and I found some commercials kits available for adenoviral transduction enhancement, like Viraductin (from Cell Biolabs) and Ibiboost (from Ibidi). I would like to know if anyone has experience using any of these kits or knows any other way to enhance adenoviral transduction in NIH3T3 cells.
Any advice will be appreciated.
Dear Douglas,
I can propose two solutions based on physical or biochemical properties to enhance adenoviral infection and transduction in any cell type, permissive or non-permissive (of whom NIH-3T3):
- AdenoMag is based on the Magnetofection technology***. It uses magnetic beads to concentrate adenoviral particles onto the cell surface within minutes. it was tested with Ad5 and worked on several mouse non-permissive cells such as NIH-3T3 or C6.
- AdenoBlast is based on an adenovirus binding peptide that assists transduction by coupling the adenoviral particles to the cell membrane in a CAR independent manner.
These two reagents are very efficient,
would you like to have more information, please do not hesitate to contact me directly at: [email protected]
good luck in your experiment,
best regards,
Cedric
*** The Magnetofection technology uses a magnetic field to attract and concentrate complexes of magnetic nanoparticles and nucleic acid onto the cell surface as demonstrated by Grześkowiak BF et al, Pharm Res. 2014 Jul 18. (http://www.ncbi.nlm.nih.gov/pubmed/25033763).
http://www.ozbiosciences.com/transduction-infection/29-adenomag-magnetofection-reagent-adenovirus-transduction.html
http://www.ozbiosciences.com/transduction-infection/74-adenoblast-transduction-infection-adenovirus-booster.html
An alternative and permanent solution is to generate NIH3T3 cells stably expressing the CAR. These cells lack the coxsackie virus and adenovirus receptor (CXADR or CAR) required for adenovirus binding and infection. We've generated a retrovirus using the MSCV-puro backbone (Clontech) that contains the CAR gene to create cell lines stably-expressing CAR. We've used that retrovirus to make several cell lines permanently infectable with adenovirus (one example transduced with Ad5-GFP) attached. I could send you the construct if you're interested ([email protected]).
3T3-L1 CAR (coxsackie and adenovirus receptor) transgenic cells (Orlicky, D. J., J. DeGregori, and J. Schaack. Construction of stable coxsackievirus and adenovirus receptor-expressing 3T3-L1 cells. J. Lipid Res. 2001. 42: 910–915.) are transduced by adenovirus type 5 vectors at approximately 100-fold-increased efficiency compared to parental 3T3-L1 cells. The transduction efficiency of the modified cells remains considerably lower than that of highly transduceable cells such as HEK 293 and HeLa cells, but the efficiency is sufficient for many types of studies.
We have not tried to enhance transduction of the CAR-expressing cells using Magnetofection technology, but that sounds like a good idea.
I am happy to send a vial of cells.
Jerry Schaack
I have a fair amount of experience in the Ad-transduction of cells that don't express CAR from my postdoctoral time. It is rather difficult, so I would take the advice to seek the 3T3 cells that express CAR. One alternative is that you could also use a type B adenovirus like Ad35 or the Ad5F35 (an Ad5 with the Ad35 fiber protein). This would require a lot of work to make those viruses (and possibly a whole lot of work to construct the chimeric virus, as I am not sure such a vector is commercially available).
If you must get Ad5 to infect the NIH 3T3 cells and the enhancing reagents don't work, you can try to use the Ad5 vector. You will need very high concentration virus, ideally between about 1 and 5 x10^10 pfu/ml. To infect, you would want to add the virus to the cells without diluting it more than about 50%, i.e., 50ul of virus on top of 50ul of cells. You end up needing a lot of virus, so it's quite a pain. (This is why I spend quite a bit of time constructing Ad5F35 versions of my vectors).
Based on the top info provided above, here is some commercial available info from CELL BIOLABS, INC for you. I thought that's what you were looking for and think it's pretty fit to your CAR-less MIH3T3 cells. Good luck!
http://www.cellbiolabs.com/adenovirus-transduction?gclid=CjwKEAiAoo2mBRD20fvvlojj5jsSJABMSc7jmG_ax7o7FRpKc0e_6dMQmw2VIOlzE-8sZ4T28fHGFxoC6ATw_wcB
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