pSM2C retrovirus vector from Openbiosystems, is it good? - (May/18/2006 )

I just placed an order to buy 2 pSM2C vectors with insertion of shRNA (MSCV-retroviral vector) 2 days ago. But today, I met 2 guys in our institution, they told me they used dozens of pSM2C-shRNA vectors, none of them works. Finally, they changed to lentiviral vector and designed shRNA themself, their RNAi vectors works.
Does anyone have experience with this pSM2C retrovirus vectors and shRNA from Openbiosystem?

Many people suggest me lentivirus vector better. Is there any advantage of pSM2C vectors compared with lentivirus vectors?

If there is no advantage, why does that company still clones shRNAs into pSM2C retroviral vectors and sell them? If their predesigned shRNA rarely works, why does they still sell them with such high price?
Please share me your thoughts, if their pSM2C-shRNA vectors really suck, I will cancel the order ASAP.

I am not sure abt pSM2C, but I am working with Lentiviral system (also with shRNA) and they work very nice. They are easy to handle and production needs a little optimisatio but its not a problem.

I would not trust any company's shRNA sequence unless already published by an independent group. Best is to design and order and clone it in yourself.

hi,
I had tried 3 pSM2c clones from openbiosystems, only one had slightly knockdown in a transient transfection experiment. But after selection, no knockdown was observed. Besides, I used these vectors for retroviral transduction, the titers were extremly low. I wonder is there anyone had good experiences with these vectors?

pSM2 retroviral vectors from Open Biosystems contain microRNA adapted shRNA (shRNAmir) which have been shown to produce increased and more specific knockdown compared to conventional shRNA designs. See attached Silva et al 2005 in Nature Genetics. There have also been several other publications using this vector.

Things to consider when you dont see knockdown are:
(1) Is your DNA prep good. Viral vectors like pSM2 are prone to recombination when cultured for long (over 14hrs) periods. Run your DNA on a gel to make sure that the pSM2 band (~7kb) is prominent.

(2) Transfection efficiency should be monitored. If transfection efficiency is not high you can select your cells with puromycin before assaying. This will ensure that cells that have not taken up the shRNA will be killed and wont affect your readout. Open Biosystems has a really good polymer-based transfection reagent called Arrest-In that has been optimized for transfection of shRNA.

(3) For viral production, if high titers are required retroviral packaging lines that rely on co-transfection of packaging plasmids will usually give higher titers (for eg. Phoenix from Orbigen or Clontech's Amphopack retroviral packaging lines).

Technical support at Open Biosystems are also very good at responding to technical troubleshooting queries.

Hope this helps.
Gwen

PLEASE PAY ATTENTION that there are company people at this forum, giving biased opinions!

Our lab have been trying the retroviral or lentiviral system for half year but have not been able to get any good result out of it. The major problem is virus packging.

We started with the retrovirus system, i.e, the pSM2 shRNAmir constructs and LinX cell line. I did DNA prep exactly following the protocol, and of course checked recombination by running the gel. I did extensive trouble shooting including trying GeneJuice, Arrest-In and Amaxa as transfection method, and testing time course for virus collecting. I got an extremely low titer.

We were told to use Phoenix from Orbigen or Clontech's Amphopack retroviral packaging lines instead of LinX cells to package the virus, by Open Biosystem after complaining our problem. We figured out Amphopack is either on back-order or discontinued by Clontech.

We heard from another lab that they get the retrovirus from open biosystems work OK with Phoenix cell lines. We will probably try that next.

Since we have had good experience with the lentiviral system from Invitrogen, we decided to clone the shRNA into pCMV-GIN-Zeo(the lentiviral vector from openbiosystem), and package with ViralPower from Invitrogen. Though we never had any problems with packaging PLKO lentiviral constructs from Invitrogen using ViralPower, pCMV-GIN-Zeo did not work with ViralPower. We know ViralPower is compatible with pCMV-GIN-Zeo!

Later we were told by another company that they tried all the avaiable lentiviral vectors, and found that open biosystems is the hardest to package.

Another thing is I did try simple tranfection with open biosystem shRNA constructs into 293 cells, and I did not really see knock-down. Based on this I would NOT say the sequence did not work, because knock-down cells might lose growth advantage.

These are my bad experience with this system. Probably I am just the stupid that could not get it work. I would be very happy if anyone indeed ever worked on it tell me it worked well in their hands.

Here also no good experience with pSM2 (Shag Magic v2) used in the Hannon shRNA library shipped by OpenBiosystems and Sigma. No magic at all with 4 vectors. We efficiently transfect the plasmids into insulinoma cell lines by electroporation. We saw RNAi for the same genes with other vectors like pGene. So, my first impression is also negative. I tested the Arrest-In transfection reagent that came with the pSM2 clones. Not better than the electroporation we use but quite toxic to the cells.

Bottom line: I will stop using pSM2 and Arrest-In.

Best, j