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lentiviral transduction in BLCL cells


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#1 Kumaran Sundaram

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Posted 18 February 2016 - 08:35 AM

Hi,

I am trying to lentiviral transduction in BLCL (B Lymphoblastoid Cell Lines), which is suspension cells. I am not able to success transduction in these cells. If anyone have the experience in lentiviral transduction in these cells?

Thanks

Kumaran 



#2 miST32

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Posted 18 February 2016 - 11:08 AM

Hi Kumaran,

How are you packaging your virus (in particular what envelope proteins are you using?)  The "standard" VSV-G system should infect human cells.  

Have you titered the virus?  Perhaps it is low MOI and the cells aren't seeing much virus.

I usually perform spinfection on adherent cells.  Perhaps you could try a modified spinfection protocol for your suspension cells?  I'd try the following:

1.  Pellet the cells
2.  Resuspend in small volume of optimem medium + virus 
* can include polybrene or transdux reagent to increase efficiency
3.  Spin the cells for 1hour (500-1000xg) at 37C
4.  Return the cells to culture and let them recover for 24 hours at 37C

Replace the medium the next day and begin antibiotic or flow selection at 72h post-transduction.

If it doesn't work, then maybe consider a different envelope system that provides more selective tropism for your cells?


Edited by miST32, 18 February 2016 - 11:10 AM.


#3 Kumaran Sundaram

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Posted 18 February 2016 - 02:00 PM

Hi, Thank you for your information. 

I am using packaging vector psPAX2 and envelope pMD2.G. I tried spinfection at 32 C for 30 min and 90 minat 800xg.

After 48 h, I did not able to see any GFP cells under fluorescence microscope.



#4 miST32

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Posted 24 February 2016 - 10:40 AM

Hmm, that does sound frustrating.  We also use psPAX2 and pMD2.G and generally get pretty high titers with 4:3:1 (transfer:packaging:envelope) and concentration by ultracentrifuge.

There could be several problems.

1.  Low viral titer/MOI
- During packaging, did you see appreciable fluorescence in the HEK293T cells?  I usually see strong fluorescence during packaging (except as it turns out, when using a transactivated promoter).
- Have you checked the titer on HEK293 cells?

2.  No tropism for your cell type.  

3.  GFP is out of frame or the fusion mRNA isn't being expressed.  Does transfecting the transfer plasmid alone result in expression in your cell type?

I'd look at the viral titer first, and transfect some of the HEKs with the transfer vector at the same time.  If it is infectious for HEK293 but not your target cells, I'd be suspect the tropism to be a problem.  If you don't see fluorescence in either the infected or transfected cells, then something else might be wrong with your construct.

*edit, you could also look at p24 levels via ELISA to determine if you've produced viral particles.
** One other note, sometimes the virus takes >72 hours to integrate and express your GOI.

Good luck!


Edited by miST32, 24 February 2016 - 10:43 AM.





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