Hi, how can I get an efficient infection of SH-SY5y neuroblastoma cell line with plvx-puro vector?

There are 3 options:

1. That you select the cells with much too high concentration of puromycin. for some cell lines I went as low as 0.5ug/ml- did you test to see what fits your cell line? test control cells with a range of 0.5 to 5 ug/ml. Generally, you need the selection to kill your control cells in about a week, not too fast.

2. *most likely* That you have a very low titer of the virus compared to the control virus, this will affect the efficiency of the infection.

Best way to solve it is to repeat the virus production. before you do that, run your plasmid on agarose to see that it is clean, it could be that your initial concentration is low.

You can improve the efficiency of the infection by up-scaling the volume of viral media that you add, by centrifuging the plates of infected cells and by adding polybrene along with the virus.

3. That while cloning you harmed the selection site. If you did any whole plasmid amplification by PCR in the process, sequence your selection site to be sure that it is not mutated.

Nicolas Grandchamp

Hi Francesca,

It seems you have problems of efficiency given that your control is ok. (Another hypotesis could be interferences of action between puro and your gene of interest. )

To obtain more efficient vetors, three ways:

1. Design

2. Protocol of production

3. Protocol of transduction

1.The plvx vector contains two expression cassettes (trsg of interest+puro) in the same orientation, it is not the best design to induce a double expression because you can have interferences between the two expresssion cassettes. 2A design or bidirectional promoters are often better to induce multiple expression.

2. Try to optimize your protocol (step of centrifugation and/or purification) or order your vector to companies which knows how to produce high efficient vectors

3. You can have a look here to have advices about this point:

https://www.geg-tech.com/knowledge/lentiviral-vectors-multifaceted-tools

Hope this information will help you.

Regards

Nicolas

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