What our experts have to say

Is gene expression permanent in target cells after several divisions?

Integration
Lentiviral vectors insert the sequence of interest right into the host DNA thus allowing for a stable and long-term expression of the transgene. The gene will be copied every time the cell divides.

Viral integration is built to last

Because these viruses insert the vector sequence including the gene of interest into the host, the gene expression is permanent in target cells even after several divisions.

Such integration mediated by lentiviral vectors occurs primarily into active areas of chromatin and the integration sites are random.

As a result, in a given polyclonal and transduced cell population, all cells exhibit a different integration profile between each other. The integration profile cannot have an impact on the whole cell population.

For the expert who would like to know more about viral integration I suggest to read the excellent article on “The Integration Reaction” from the book “Retroviruses”.


A loss of expression is not related to a loss of integration

Some users report isolated restricted cases of gene silencing after several divisions of specific cells. Several possibilities can explain such results:

  • The sequence of interest itself leads to cell toxicity and lethality. The residual non transduced population will gradually replace the initial predominant transduced cells population. To avoid such a concern, a Tet-inducible system is recommended. The use of an inducible promoter allows for the gene expression to be turned on and off as required through the administration of Doxycyclin.

 

  • Stem cell differentiation induces chromatin remodeling changes that can modify the expression profile of cells. If the sequence of interest has been initially inserted into an open genome area that is later silenced by chromatin remodeling, the expression will also be silenced. Gene expression insulators allow to prevent such issues.

 

  • Complex expression cassettes including several promoters, such as IRES in the lentiviral vector’s cassette, can lead to inefficient gene expression. For the expression of several sequences of interest, a vector containing multiple genes of interest can be engineered during the production step, thus avoiding the requirement of multiple promoters, IRES or T2A sequences.


As a result, if you are trying to obtain a stable and long-term expression of your gene of interest, lentiviral vectors are the ideal tool. Also, keep in mind that it requires only 24 hours to detect the expression, thanks to the vector’s DNA integration!

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