G********e
发帖数: 528 | 1 Something like CreER.
It seems a pretty powerful and established system, but I only found a cell
stem cell article about ATR inducible KO in 2007.
Any big caveats with this system? | G********e
发帖数: 528 | | s******y
发帖数: 28562 | 3 以前实验室里其他人用过，然后我只负责测下游的东西，结论是：太tmd不好使了！！
！！
根本就删不干净！如果你要做的东西有剂量效应的话勉强可以用用，否则的话要三思。
。。
【在 G********e 的大作中提到】
: Something like CreER.
: It seems a pretty powerful and established system, but I only found a cell
: stem cell article about ATR inducible KO in 2007.
: Any big caveats with this system?
| r*******y
发帖数: 48 | 4 probably, it depends on which kind of tissue where you need to ablate that
particular gene, and which kind of system you use. in cardiovascular field,
it is a quite mature technique.
like tissue specific cre activation system, indicible KO usually does not
have a clean KO, again depending on which particular cells where the cre is
induced. Thus, you need to choose carefully before you start.
for whole body cre induction, I believe that b-actin promoter controled or
CMV promoter controled inducible systems should be good, but you need to
check. | G********e
发帖数: 528 | 5 Thank you very much!
It seems that if I want to bypass embryonic lethality, it is a candidate. If
people have been using tissue specific systems, they would be better than
whole body.
I have something in my mind before I want to start it.
If a gene of interest is known critical for embryonic development, is it
really important to bypass the embryonic stage and look at the phenotypes of
loss of function after the mice are fully developed? I think so, but it
seems not many people publish studies using this system, except the ATR one.
In fact, there were not many new findings on ATR using this system.
Everything had been predicted. It doesn't deserve to be published in cell
stem cell.
If this kind of question is still viable, it is also what the inducible
systems are for. Before starting, I have no ideas about the gene and tissue
specificity, since the expression is almost similar in different tissues. Do
you think the whole body inducible system would be in the priority,
compared with tissue specific inducible systems?
No many good projects in the lab, it is very likely I will play with it. | r*******y
发帖数: 48 | 6 if the deletion of the gene of your interest is embryonically lethal, and
there is no significant difference in the expression pattern of this gene in
various tissues/organs, then you need to know:
1. what was the major cause of embrynic death once this gene was deleted
globally? most of genes, although ubiquitously expressed, have more
important roles in the development/function of some organ(s) than others.
depending on the developental stage when the null embryos died, most
possible cause of death should go to the failure of heart (cardiovascular
system), nervous system, or blood system, unless they died at very early
stage like around (pre)implantation.
2. if you have an idea which tissue this gene might target functionally, but
it has not been investigated yet, as an initial step, it will be more
significant to generate tissue specific knockout mode to probe the
underlying mechanisms of functional/developmental regulation of that tissue
by this gene, instead of going straight to the globally inducible knockout
system. | G********e
发帖数: 528 | 7 I'm quite convinced by your logic.
I only know the embryos do not develop beyond E5, which might be considered
quite early. I could not get any hint form embryonic study for tissue
specificity.
Anyway, thank you very much! Really help me think about it. | S******9
发帖数: 2837 | 8 我们用的老鼠是E 12.5天死亡。我们已经完成了 b-actin promoter controlled
systemic inducible cre, deletion 97-99%之间 （ 包括组织和原代细胞培养检测）。
现在是组织特异性的Cre，目前deletion大概是70%左右。 | l**********1
发帖数: 5204 | 9 LZ can refer
Methods in Molecular Biology, vol. 158: Gene Knockout Protocols
Edited by: M. J. Tymms and I. Kola
//www.powells.com/biblio?isbn=9780896035720
full text free download link:
//nhjy.hzau.edu.cn/kech/zwswjs/kc/ziliao/Gene%20Knockout%20Protocols.pdf
PDF file its pp30-32 1.3.1 Cre-loxP Chapter
NB: never used as business benefit
【在 G********e 的大作中提到】
: Something like CreER.
: It seems a pretty powerful and established system, but I only found a cell
: stem cell article about ATR inducible KO in 2007.
: Any big caveats with this system?
|
|
