Toolbox

All the vectors are self-inactivated.

The 5’U3 region is from the Rous sarcoma virus (RSV).

They all bear the encapsidation signal, the Rev-responsive element (RRE), the central polypurine tract with its termination sequence (cPPT/cTS) and the woodchuck hepatitis virus responsive element (WPRE).

 

https://jahia-prod.epfl.ch/repository/default/content/sites/cpg/files/images/Transfert%20Mat%C3%A9riel/Fichier%201.png

 

Gene over-expression

The promoter can be an ubiquitous one such as hPGK, UbC, EF1alpha, CAG or tissue specific (ie CamKII, mTTR…)

 

https://jahia-prod.epfl.ch/repository/default/content/sites/cpg/files/images/Transfert%20Mat%C3%A9riel/Fichier%201.png

 

Knock down

Depending on the vector there is also either a reporter gene such as the eGFP or a selection marker ie puromycin.

 

Different  marker genes are present in the plasmid database : eGFP, CFP, YFP, dsRed, Tomato, LacZ, muSEAP, Puromycin, Neomycin…

 

https://jahia-prod.epfl.ch/repository/default/content/sites/cpg/files/images/Transfert%20Mat%C3%A9riel/Fichier%201.png

 

Regulation

Tet-on system

This system allows gene expression in the presence of doxycycline an analogue of tetracycline (Barde I.,Mol Ther. 2006). This all in one vector in the uninduced state can be kept at background levels and induction factors of 100-fold are repeatedly obtained over months both in tissue culture and in vivo.

tTRKrab system

The system is composed of an hybrid protein, tTR-KRAB, in which the tetracycline repressor (tTR) from Escherichia coli Tn10 is fused to the KRAB domain of human Kox1 . KRAB is an approximately 75-amino-acid transcriptional repression module found in many zinc finger-containing proteins, which can suppress, in an orientation-independent manner, both polymerase II- and polymerase III-mediated transcription. When linked to the DNA-binding domain of tTR, KRAB can modulate transcription from an integrated promoter juxtaposed with tet operator (tetO) sequences. In the absence of DOX, tTR-KRAB binds specifically to tetO and suppresses the activity of the nearby promoter. Conversely, in the presence of DOX, tTR-KRAB is sequestered away from tetO, thus permitting gene expression. The reverse system is also available.(Wiznerowicz M, J Virol. 2003).