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lentiviral system: cloning strategy


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#1 makeit

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Posted 19 October 2009 - 12:08 PM

Hi everyone,
Iím currently thinking in some strategies to clone a cDNA in a lentiviral vector system. Usually when I did this in other common expression mammalian systems (pCDNA 3, for example) I added the Kozak sequence just prior my insert observing the correct frame. However, a friend of mine that uses the lentiviral system said to me this is unnecessary and that he usually amplifies a small part of the 5íUTR (around 50 bp) together with his insert. According to him this is sufficient to get a good expression of the transgene. What do I do? Iím afraid get bad results in gene expression by using my friendís strategyÖ
are there someone expert on this matter??

#2 Functional Screens

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Posted 20 October 2009 - 10:06 AM

Depending on which lenti vector you're going to use, some lentiviral vectors give very good expression and you might say "no Kozak" is still working. However Kozak sequence definitely helps your CDS expression.

#3 makeit

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Posted 21 October 2009 - 09:08 AM

Thanks a lot for your words!!

Perhaps it is better to add this sequence instead assume the risk of getting bad expression levels with my vector, which is a non commercial one (obtained from one of our collaborators).



Depending on which lenti vector you're going to use, some lentiviral vectors give very good expression and you might say "no Kozak" is still working. However Kozak sequence definitely helps your CDS expression.






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