Lentiviral infection optimization

Scott Brown via methods%40net.bio.net (by SBrown from ccia.unsw.edu.au)
Mon Aug 30 15:08:25 EST 2010


Hi all,

I have been trying to kd a gene in nalm6 suspension cells. My lentivirus had been well validated in 293s showing good efficiency and corresponding mRNA and protein levels
I seem to have become stuck with the nalm6 infections, basically I plate 7M 293ft cells then the next morning I take the media and replace is with 2% FCS works better than standard stock.
I let the media collect for 24 hrs at which point I spin each tube >13k g for 15 min then filter the supernatent after adding polybrene to give a final 8ug/ml.
So far I have managed kd in the nalms by 50%, following 24hr exposure of the lenti virus and confirmed flow.

My problem is I have flow data telling me the population is 95.% Gfp positive however it doesn't correlate with real time of wb analysis, I'm at a loss, how can gfp get actively transcribed yet the kd level is low.

Does anyone out there have an explanation for this? I would also appreciate any advice.

Lastly I have been cloning with the use of invitrogens virapower packing mix.

Thanks for you're time guys  and i look forward to any insight or suggestion

Kind regards

Scott .

Scott Brown
PhD Candidate
Molecular Carcinogenesis Program
Children's Cancer Institute of Australia for Medical Research
High Street (PO Box 81)
RANDWICK NSW 2031
AUSTRALIA

Phone: +61 2 9382 1829
Fax: +61 2 9382 1850
Email: sbrown from ccia.unsw.edu.au
Web: www.ccia.org.au
Children's Cancer Institute Australia is the only independent medical research institute in Australia solely devoted to research into the causes, prevention and cure of childhood cancer. Our vision is to save the lives of all children with cancer and eliminate their suffering.
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Scott Brown PhD Student Children's Cancer Institute Australia for Medical Research Lowy Cancer Research Centre, UNSW PO Box 81 Randwick NSW 2031 Australia Direct 02 9385 1861 Email SBrown from ccia.unsw.edu.au Web www.ccia.org.au Children's Cancer Institute Australia
 for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and
 to eliminate their suffering.
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