发信人: mweilai (Starbucks), 信区: Biology
标 题: Re: protein turnover...
发信站: Unknown Space - 未名空间 (Sat Oct 30 21:29:49 2004) WWW-POST

Check out this paper:
Xu ZL, Mizuguchi H, Mayumi T, Hayakawa T.
Regulated gene expression from adenovirus vectors: a systematic comparison of
various inducible systems.
Gene. 2003 May 8;309(2):145-51.
Actally I wasted half year on Clonetech's tet-on/off system. It is very leaky
so that in some cell types (MEF is worst) you can never control the basal mRNA
expression level at all. After half year's work, I called the campany and also
found out this paper that compared different commercial inducible systems. The
technical surport from clonetech told me, in MEF, which is only cell type I
want work on, I may not be able to get well controled clone even selecting 100
clones. And you know what, I already selected 30 clones, and nothing showed
the downregulated mRNA. So I gave up the project finally. Think about the
system over and good luck.

【 在 Ras (很颓的猪。) 的大作中提到: 】
: does protein degradation always associate with mRNA degradtion?
: I am just curious.
:
: 【 在 horizon (东方之子) 的大作中提到: 】
: : I am doing some protein turnover rate study by using tet-off system. When
I
: : add Dox into the system to turn off my target gene transcription, I found
: : protein degraded after 5 hours of adding Dox. But when I go back to check
the
: : mRNA level ( because tet-off system is supposed to shut off
transcription), I
: : can't see the mRNA degradtion after 5 hour of adding Dox. I used Bio-rad
SYBR
: : GREEN to do the qPCR, cycle number is around 16-17 and almost same for
each
: : time point.
: : How to explain these result and did I make some mistake?
:
:


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