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STUDYING GENE FUNCTION IN EUKARYOTES BY CONDITIONALGENE INACTIVATION
http://www.annualreviews.org/doi/full/10.1146/annurev.genet.36.041002.120114?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed&
Conditional Gene Expression Systems
Conditional gene expression can be achieved through utilization of several established conditional expression systems, including the Tet, Cre, FRT, and ER (estrogen receptor) conditional gene expression systems.
Tetracycline-controlled transcriptional activation (The “Tet” System)
The method of “Tetracycline-Controlled Transcriptional Activation” allows for inducible gene expression. Transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline).
In vivo, the Ptet promoter expresses the tetracylin repressor (TetR), and TetA, the protein that pumps the tetracycline antibiotic out of the cell. The Tet-Off and Tet-On systems are the two most common tetracycline controlled expression systems. They consist of a fusion of the Tet repressor (TetR) and a VP16 activation domain to create a transcriptional activator protein (transactivator) rather than a repressor. This protein is referred to as the tetracycline-controlled transactivator protein (tTA).
Gene expression is activated as a result of binding of the Tet-Off or Tet-On protein (TetR vs tTA) to tetracycline response elements (TREs) located within an inducible promoter. The difference between the Tet-Off and Tet-On systems relates to their respective response to doxycycline (Dox, a more stable tetracycline analogue); Tet-Off activates expression in the absence of Dox, whereas Tet-On activates in the presence of Dox.
Tet-Off
The expressed tetracycline-controlled transactivator protein (tTA) binds elements within a gene promoter referred to as tetracycline-responsive promoter elements (TRE). Binding of tTA to TRE within a promoter transactivates the gene adjacent to the promoter. Absence of tetracyclin derivatives allows for expression of TRE-containing target genes. Introduced tetracycline derivatives bind tTA and render the transactivtor incapable of binding to TRE sequences, thereby preventing transactivation of target genes. In essence, introduction of the tetracyclin antibiotic turns gene expression “off” in the Tet-Off system.
Tet-On
The Tet-On system works in the opposite fashion. In this system, the rtTA protein is capable of binding the operator only when bound by doxycycline. Thus the introduction of doxycyline to the system initiates the transcription of the genetic product. The Tet-On system is sometimes preferred for the faster responsiveness.
Conditional Gene Expression in Mouse Liver
LAP-tTA Expression System
Engineered by the Jackson laboratory (http://jaxmice.jax.org/strain/003563.html), these hemizygous mice (A chromosome in a diploid organism is hemizygous when only one copy is present) are viable and fertile.
These transgenic mice carry the gene encoding the tetracycline-controlled transactivator protein (tTA). The tTA protein is under control of a promoter which ensures expression specifically in the liver. The promoter used is that of the liver-enriched activator protein (PLAP, C/EBP, Cebpb).
Expressing tTA specifically in the liver allows for conditional gene expression upon tetracyclin-induced expression of tTA in the liver. Expressed tTA binds elements within a gene promoter referred to as tetracycline-responsive promoter elements (TRE). Binding of tTA to TRE within a promoter transactivates the gene adjacent to the promoter. Tetracycline derivatives bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of target genes. Thus treatment of the mouse with a tetracyclin derivative (ex. doxycycline or dox administered through drinking water) prevents transcription of the TRE-controlled gene (i.e. this is a Tet-Off expression system).
Example:
LAP-tTA TRE-MYC mice allow for expression of a human MYC cDNA induced specifically in the liver.
(Sage 2011 paper)
When these transgenic mice are mated to a strain carrying the luciferase gene coupled to a tetracycline-responsive promoter element (TRE; tetO), the luciferase reporter is expressed in a liver-specific fashion; treatment with doxycycline (dox) prevented transcription of the luciferase reporter in the liver. C57BL/6J-Tg(tTALap)5Uh mice may be mated to transgenic strains containing a gene of interest coupled to a TRE to study the effects of liver specific expression of the target gene in a dox-inducible fashion. Dox concentration may be administered in the animals' water supply. It should be noted that the chromosomal integration site of a TRE-couple transgene may affect the tissue-specific expression of the target gene, such that target gene expression is not limited to liver.
STUDYING GENE FUNCTION IN EUKARYOTES BY CONDITIONALGENE INACTIVATION
http://www.annualreviews.org/doi/full/10.1146/annurev.genet.36.041002.120114?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed&
Conditional Gene Expression Systems
Conditional gene expression can be achieved through utilization of several established conditional expression systems, including the Tet, Cre, FRT, and ER (estrogen receptor) conditional gene expression systems.
Tetracycline-controlled transcriptional activation (The “Tet” System)
The method of “Tetracycline-Controlled Transcriptional Activation” allows for inducible gene expression. Transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline).
In vivo, the Ptet promoter expresses the tetracylin repressor (TetR), and TetA, the protein that pumps the tetracycline antibiotic out of the cell. The Tet-Off and Tet-On systems are the two most common tetracycline controlled expression systems. They consist of a fusion of the Tet repressor (TetR) and a VP16 activation domain to create a transcriptional activator protein (transactivator) rather than a repressor. This protein is referred to as the tetracycline-controlled transactivator protein (tTA).
Gene expression is activated as a result of binding of the Tet-Off or Tet-On protein (TetR vs tTA) to tetracycline response elements (TREs) located within an inducible promoter. The difference between the Tet-Off and Tet-On systems relates to their respective response to doxycycline (Dox, a more stable tetracycline analogue); Tet-Off activates expression in the absence of Dox, whereas Tet-On activates in the presence of Dox.
Tet-Off
The expressed tetracycline-controlled transactivator protein (tTA) binds elements within a gene promoter referred to as tetracycline-responsive promoter elements (TRE). Binding of tTA to TRE within a promoter transactivates the gene adjacent to the promoter. Absence of tetracyclin derivatives allows for expression of TRE-containing target genes. Introduced tetracycline derivatives bind tTA and render the transactivtor incapable of binding to TRE sequences, thereby preventing transactivation of target genes. In essence, introduction of the tetracyclin antibiotic turns gene expression “off” in the Tet-Off system.
Tet-On
The Tet-On system works in the opposite fashion. In this system, the rtTA protein is capable of binding the operator only when bound by doxycycline. Thus the introduction of doxycyline to the system initiates the transcription of the genetic product. The Tet-On system is sometimes preferred for the faster responsiveness.
Conditional Gene Expression in Mouse Liver
LAP-tTA Expression System
Engineered by the Jackson laboratory (http://jaxmice.jax.org/strain/003563.html), these hemizygous mice (A chromosome in a diploid organism is hemizygous when only one copy is present) are viable and fertile.
These transgenic mice carry the gene encoding the tetracycline-controlled transactivator protein (tTA). The tTA protein is under control of a promoter which ensures expression specifically in the liver. The promoter used is that of the liver-enriched activator protein (PLAP, C/EBP, Cebpb).
Expressing tTA specifically in the liver allows for conditional gene expression upon tetracyclin-induced expression of tTA in the liver. Expressed tTA binds elements within a gene promoter referred to as tetracycline-responsive promoter elements (TRE). Binding of tTA to TRE within a promoter transactivates the gene adjacent to the promoter. Tetracycline derivatives bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of target genes. Thus treatment of the mouse with a tetracyclin derivative (ex. doxycycline or dox administered through drinking water) prevents transcription of the TRE-controlled gene (i.e. this is a Tet-Off expression system).
Example:
LAP-tTA TRE-MYC mice allow for expression of a human MYC cDNA induced specifically in the liver.
(Sage 2011 paper)
When these transgenic mice are mated to a strain carrying the luciferase gene coupled to a tetracycline-responsive promoter element (TRE; tetO), the luciferase reporter is expressed in a liver-specific fashion; treatment with doxycycline (dox) prevented transcription of the luciferase reporter in the liver. C57BL/6J-Tg(tTALap)5Uh mice may be mated to transgenic strains containing a gene of interest coupled to a TRE to study the effects of liver specific expression of the target gene in a dox-inducible fashion. Dox concentration may be administered in the animals' water supply. It should be noted that the chromosomal integration site of a TRE-couple transgene may affect the tissue-specific expression of the target gene, such that target gene expression is not limited to liver.
Allele-specific inhibition in cell lines
Example •se allele-specific
inhibitor 3-MB-PP1 on Cdk7as/as
HCT116