"
The Drug
Return to: Project
In nature, Ptet promoters expresses two proteins, one of which, TetR, acts as a repressor and the other, TetA, pumps tetracycline out of the cell.
Based on this, Tet-On and Tet-Off systems are two types of tet-involved activation systems that help induce gene expression, and they are the most commonly used ones in eukaryotic cells. Transcription is up or down regulated when tetracycline or its derivatives are present. Tet-On and Tet-Off systems are different in that Tet-On activates transcription with doxycycline is present while Tet-Off does when dox is absent.
Tet-Off was first developed and published in 1992, featuring a fusion protein, tTA, obtained from TetR and a virus protein. tTA responds to Tet0 sequences and its binding activates transcription, while presence of tetracycline or its derivatives would block its binding.
Fig. 1 Tet-Off system schematic
Tet-On system works similarly to Tet-Off system. The corresponding protein, rtTA, binds to the operator with Tet or its derivatives present.
Fig. 2 Tet-On system schematic
We get Tet-On system from 2013 Tsinghua team. At first, we design a test plasmid to determine the feasibility of applying Tet-On system in our system.
We constructed an mCherry gene after a CYC1 TATA region (TATA region) which is regulated by a tetracycline response element on its upstream. After mCherry gene, a CYC1 terminator is used to terminate the transcription of mRNA.
Fig. 3 Tet-mCherry map
After construction of this plasmid, we transfect it into HEK293T cell to see if the mCherry gene can be expressed under the regulation of the DOX (tetracycline analogue).
Then we do the PCR of the Tet-On system with mCherry to bring in two restrition sites at the terminals. After purification of the PCR products, we cut the vector and the tet on system and ligate them. The plasmid which has AAV vector that we bought from a company has two the same restrition sites(NotI) in its insert rigion. So, to aviod the vector’s self-ligation, we dispose the digestion product with CIAP to remove the phosphate group at the sticky end of the vector.
Fig. 4 pAAV-Tet-mCherry map
After construction of this plasmid, we want to replace this mCherry gene with insulin. Since we want to transfect the AAV vector into somatic cells, we must make sure that the insulin can be processed in these cells. In beta cells, proinsulin was processed by cellular endopeptidases known as prohormone convertases (PC1 and PC2), as well as the exoprotease carboxypeptidase. However, in non-endocrine cells, some of these enzyme are lacking. So, if we want to get mature insulin, we must alter the insulin gene sequence. In non-endocrine cells, an endoprotease called furin which can cleave the concensus processing site, -Arg-4-X-3-Lys/Arg-2-Arg-1. So, we add two 6bp nucleotides into two cleavage sites in insulin CDS to get the furin endoprotease sites.
To get the insulin gene, firstly we want to PCR every exons of its in human genome and then overlap them. However, the sequence of insulin is in a high GC region in the genome. So, our first trial was not successful. Fortunately, the insulin gene is very small. So, we design 12 overlap primers and do one reaction to ligate these oligos. In that step, we insert these two furin sites into our sequence.
Fig. 5 insulin electrophoresis gel image
Fig. 6 insulin map
In this figure we can see the 12 overlap primers extension to the insulin. We cut the gel, do the gel purification and use the other two primers to do the PCR and get more insulin molecules.
Then, we replace mCherry gene in tet system by this insulin gene, and transfect it into HEK293T cells.
Fig. 7 pAAV-Tet-insulin map
Return to: Project
