Can the insertion of shRNA into host cell genome influence certain gene expression? How can I know the inserted site of lentiviral shRNA in a stably transduced cell clone?
Lentivirus tends to insert in active genes (see paper), this is why lentiviral transduction usuallty yields more comparable expression levels between different insertions (clones) than transfection - where the transfected DNA can insert everywhere, including heterochromatin. So that is a decided advantage of lentiviral transduction over transfection.
Gene activity can indeed be disrupted by lentiviral insertion, but then the other allele is still active. And indeed, like Wen-Yuan says, work with early transductant pools, then the insertion effect will cancel out.
Do make sure you do not have too many insertions per genome -wise for all kinds of genetic modification. This is achieved by careful titration of the lentiviral transduction. Aim for 2-3 insertions per target cell, max.
Yes, the insertion site is very important and can influence the expression of the inserted element. If you want to find out the sequence of the adjacent DNA you will have to do inverse PCR. It is not very difficult. Good luck!
Yes, the insertion site can influence the host cell genome and the genome can influence the expression of the genes expressed from the lentivirus. A simple example would be an integration site that disrupts an exon of a gene. In some cell types vector expression can be silenced and may influence surrounding genes as well.
You would need to do inverse PCR to find the region of integration. However, you can control for the site of integration by selecting multiple stable cell lines. The integration site should be different especially if chosen from cells transduced in different wells.
Good luck!
You may want to pay more attention on the integration site(s) if you pick colony(ies), generally the sites of integration should not be matter much if selection in a pool of integrations/cell population.
In general,lentiviruses, being part of the family of Retroviruses, insert their genome in multiple sites within the host-cell genome. For retroviral stem cell therapy, this is currently one of the biggest issues - trying to decipher where the genes will be inserted, and in how many copies. Depending on your system,you may have a similar scenario, however, not all inserted sites may be transcribed. Thus, the best way to screen your transfected cells is really by sequencing the whole genome, and finding this out. However, have a look at the stem cell therapy literature - you may find preferred insertion sites in there. Hope this helps.
Lentivirus tends to insert in active genes (see paper), this is why lentiviral transduction usuallty yields more comparable expression levels between different insertions (clones) than transfection - where the transfected DNA can insert everywhere, including heterochromatin. So that is a decided advantage of lentiviral transduction over transfection.
Gene activity can indeed be disrupted by lentiviral insertion, but then the other allele is still active. And indeed, like Wen-Yuan says, work with early transductant pools, then the insertion effect will cancel out.
Do make sure you do not have too many insertions per genome -wise for all kinds of genetic modification. This is achieved by careful titration of the lentiviral transduction. Aim for 2-3 insertions per target cell, max.
P.S. The paper in my previous post also contains a method to determine lentiviral insertions sites in the human genome.
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