I transfected HEK293T cells with the 3 plasmid method using PEI and I purified the AAV present in the cells with iodoxanol gradient after 3 cycles of freeze/thaw. However, I need a protocol to precipitate the AAV present in the supernatant so I can perform the iodoxanol gradient to purify the virus.
I would appreciate if anyone has any suggestion or advice.
Thanks
This paper decries the protocol to precipitate AAV from supernatant
Gene Therapy (2010) 17, 503–510; doi:10.1038/gt.2009.157; published online 3 December 2009
High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency
E Ayuso1,2, F Mingozzi3, J Montane1, X Leon1,2, X M Anguela1,2, V Haurigot3,4, S A Edmonson3,4, L Africa3, S Zhou3, K A High3,4, F Bosch1,2 and J F Wright3,5
1Department of Biochemistry and Molecular Biology, Center of Animal Biotechnology and Gene Therapy, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain
2CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain
3Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA
4Howard Hughes Medical Institute, Philadelphia, PA, USA
5Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
Correspondence: Dr JF Wright, Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, ARC 1216C, 3615 Civic Center Blvd., Philadelphia, PA 19104, USA. E-mail: [email protected]
Received 31 July 2009; Revised 15 October 2009; Accepted 26 October 2009; Published online 3 December 2009.
Top of pageAbstract
The purity of adeno-associated virus (AAV) vector preparations has important implications for both safety and efficacy of clinical gene transfer. Early-stage screening of candidates for AAV-based therapeutics ideally requires a purification method that is flexible and also provides vectors comparable in purity and potency to the prospective investigational product manufactured for clinical studies. The use of cesium chloride (CsCl) gradient-based protocols provides the flexibility for purification of different serotypes; however, a commonly used first-generation CsCl-based protocol was found to result in AAV vectors containing large amounts of protein and DNA impurities and low transduction efficiency in vitro and in vivo. Here, we describe and characterize an optimized, second-generation CsCl protocol that incorporates differential precipitation of AAV particles by polyethylene glycol, resulting in higher yield and markedly higher vector purity that correlated with better transduction efficiency observed with several AAV serotypes in multiple tissues and species. Vectors purified by the optimized CsCl protocol were found to be comparable in purity and functional activity to those prepared by more scalable, but less flexible serotype-specific purification processes developed for manufacture of clinical vectors, and are therefore ideally suited for pre-clinical studies supporting translational research.
Use PEG8000.
Prepare a PEG 40% solution in NaCl 2.5M. First prepare the NaCl solution (500ml). Then use 350ml of NaCl 2.5M solution to dissolve your PEG8000. Filtrate (0.22um). Note: dissolve the PEG takes around 2-3 hours. Do not use too much heat, be patient!. Keep your solution at 4C.
To precipitate:
Collect your supernatant and add your PEG solution (8% final concentration). Keep the supernatant+PEG at 4C during 48-72hrs. Centrifuge 3000RPM - 15min, resuspend your pellet in your lysis buffer and proceed with the next steps.
Good luck!
sorry, but I have a doubt... why you use only 350ml of NaCl? which is the final volume you prepare of the PEG 40% stock solution and how much PEG8000 do you add?
Thanks again?
It´s better to use first 300-350 ml because you will use 200gr of PEG. Final volume is 500ml.
Thanks José! after I measured the PEG and I saw the volume I realized why you use just 350ml.
Have a great day
Hi José! I am sorry to bother you again, but when I tried to filter my solution with a 0.22uM filter I was not able because the solution is very thick and viscous. Do you think it would be ok if I just go ahead and use the solution without filter it?
Thanks a lot
Hi Maria, absolutely normal. You have to wait, it take like 30-40minutes (even more) to filtrate everything. Just be patient :) It take like a few minutes before the first drops. This is my email in case you need to contact me directly: [email protected]
Cheers!
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