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Team:LMU-Munich/Jump-or-die/Functional Principle
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=== d) construct 2 and 3 entered the cell === | === d) construct 2 and 3 entered the cell === | ||
| - | PhiC31o will be read off because of CMV promoter. Now the integrase will combine attB and attP site and integrate construct 2 into the genome. Now the gene of interest | + | PhiC31o will be read off because of CMV promoter. Now the integrase will combine attB and attP site and integrate construct 2 into the genome. Now the gene of interest is integrated between the tet-on promoter and bak in the cellular genome. This complex will be translated after tetracycline induction. The doubled stop codon and polyadenylation site directly after eGFP (the gene of interest) will efficiently prevent the tranlation of bak. Thus such cells will express the gene of interest and survive. |
[[Image: Jump14.jpg | 600pxs | Jump recombination]] | [[Image: Jump14.jpg | 600pxs | Jump recombination]] | ||
Revision as of 12:48, 27 August 2010
The Jump-or-Die-system facilitates the integration of a gene of interest into cellular genome and selects the cells successfully integrated with the gene of interest by apoptosis. Therefore three constructs are needed.
Construct 1 contains a tet-on promoter, bacterial attachment site (attB), human bak (bak) and SV40 polyadenylation site (SV40PA). It has to be integrated to the genome of the cellline. The selection of the cells stably transfected with this construct is realized by co-transfection with hygromycine resistance.
Construct 2 consists of the following parts: phage attachment site (attP), eGFP (our gene of interest), doubled stop codon and SV40PA.
This Biobrick contains CMV-promoter, PhiC31o (mouse-codon optimized integrase) and SV40PA. It will be transfected to the cellline and read-off so that PhiC31o can catalyze the integration of Construct 2 into the attB site in genome.
A cellline which has construct 1 integrated into the genome will be co-transfected with construct 2 and 3. Construct 3 will be read-off instantly, in contrast to construct 1 which has to be induced by tetracycline and because of the lack of a promoter, construct 2 will at the beginning not be translated.
The outgoing of this co-transfection has the following possibilities:
a) neither constructs enter the cell
b) only construct 2 enters the cell
c) only construct 3 enters the cell
d) both construct 2 and 3 enter the cell
If neither constructs are taken in by the cell, at the induction of the tet-on promoter, bak will be translated, the protein inserted to mitochandrial membrane and the cell will undergo apoptosis.
Induction of tet-on promoter will cause translation of bak and so induce apoptosis. The gene of interest will not be read off, because there is no promoter in construct 2.
PhiC31o will be translated because of the CMV promoter. But after tet-on induction bak will also be read-off and therefore cause cell death.
PhiC31o will be read off because of CMV promoter. Now the integrase will combine attB and attP site and integrate construct 2 into the genome. Now the gene of interest is integrated between the tet-on promoter and bak in the cellular genome. This complex will be translated after tetracycline induction. The doubled stop codon and polyadenylation site directly after eGFP (the gene of interest) will efficiently prevent the tranlation of bak. Thus such cells will express the gene of interest and survive.
Contents
Jump-or-die-System
Construct 1
Construct 2
Construct 3
Transfection possibilities
a) neither constructs enter the cell
b) only construct 2 enters the cell
c) only construct 3 entered the cell
d) construct 2 and 3 entered the cell
