Hello all
Recently I studied Tango assays, and I have some simple questions
My refenrece paper is down below
Article The genetic design of signaling cascades to record receptor activation
In the paper, HTL cells (an HEK293T-derived cell line containing a stably integrated tTA-dependent luciferase reporter) were transiently transfected with expression plasmids encoding GPCR-TEV cleavage site-tTA and b-arrestin-TEV protease fusion proteins.
When agonist binds with the GPCR-TEV-tTA, b-arrestin with TEV protease interacts with the receptor, which yields protease activity of TEV. As a result, tTA will release to bind with the reporter gene in a nucleus.
The author contends that this assay system is independent on endogenous signaling pathway as genes that are introduced are exogenous.
So, here is my question
: What is exogenous gene?
I first thought that exogenous means "from different organism".
However, I'm pretty sure that HTL cells and GPCR, b-arrestin are all from human.
So what does it mean exogenous?? Does it just mean that those genes are transfected?
And, if you please could answer more, I'd like to ask one more question
: Does endogenous b-arrestin (which is not conjugated with TEV protease) interact with GPCR-TEV site-tTA?
If endogenous b-arrestin interacts with GPCR-TEV site-tTA,
how could I say that b-arrestin bindings with GPCR are all reflected in the signal?
In the similar vein, does interactions with native GPCR and agonist happen in the assay system?
Or, doesn't it matter whether or not it happens??
It may be not an important question to conduct an experiment, but I wonder whether there will be someone who could kindly help me.
in answer to just the difference between exogenous and endogenous an exogenous gene is introduced into the organism from outside and an endogenous gene already existed in the organism
Exogenous gene means a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g. by transformation/transfection), and is also referred to as a "transgene".
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