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同主题阅读:Rosa26-loxP-stop-loxP-YFP mice
[版面:生物学][首篇作者:amadeus] , 2013年03月21日16:48:19
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amadeus
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发信人: amadeus (amadeus), 信区: Biology
标  题: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Thu Mar 21 16:48:19 2013, 美东)

Why do some of my Rosa26-loxP-stop-loxP-YFP mice show YFP+ even without Cre
or anything else?? Does anyone have experience with these mice from Jax?
Thanks!
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spt009
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发信人: spt009 (cytokine), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Thu Mar 21 16:51:02 2013, 美东)

Just the mice directly from JAX or breeder from your own colony.

If direct from JAX, I would say it is the compensation issue.

【 在 amadeus (amadeus) 的大作中提到: 】
: Why do some of my Rosa26-loxP-stop-loxP-YFP mice show YFP+ even without
Cre
: or anything else?? Does anyone have experience with these mice from Jax?
: Thanks!



--

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flyfish2010
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发信人: flyfish2010 (flyfish), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Mon Mar 25 13:07:46 2013, 美东)

我以前做的Rosa26细胞也是这样.这个东西有自发的翻转或删除.而且表达率还不低,拿
第一代都那样.所以我对科学真得失去了兴致,某些新的发现,其实都是假阳性导致的,可
笑的是一班"科学巨头"发表后,徒子徒生们就不得不从各个方面进行cover.
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muhe1985
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发信人: muhe1985 (chuomiao), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Mon Mar 25 14:35:59 2013, 美东)

这个东西一直有改进,比如后来加入了insulator 来防止transcription read through
, 没你说的那么夸张。
【 在 flyfish2010 (flyfish) 的大作中提到: 】
: 我以前做的Rosa26细胞也是这样.这个东西有自发的翻转或删除.而且表达率
--

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htscorpion
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发信人: htscorpion (snoopy), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Mon Mar 25 15:44:58 2013, 美东)

this is leak, it happens in almost all LoxP-STOP-LoxP system.  The stronger
of the promoter it has, the more leak they might have.

transcription machinery is not like 1 or 0, it has some 0.2 or 0.8 status.

【 在 amadeus (amadeus) 的大作中提到: 】
: Why do some of my Rosa26-loxP-stop-loxP-YFP mice show YFP+ even without
Cre
: or anything else?? Does anyone have experience with these mice from Jax?
: Thanks!



--

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muhe1985
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发信人: muhe1985 (chuomiao), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Mon Mar 25 21:14:29 2013, 美东)


Promoter本来就是一直开着的,不管是r26还是CAG还是CMV还是whatever being used
in this kind of system

这是transcription read through, 通常是因为loxp stop loxp 中的stop太弱








【 在 htscorpion (snoopy) 的大作中提到: 】
: this is leak, it happens in almost all LoxP-STOP-LoxP system.  The
stronger
: of the promoter it has, the more leak they might have.
: transcription machinery is not like 1 or 0, it has some 0.2 or 0.8 status.
: Cre



--

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spt009
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发信人: spt009 (cytokine), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Mon Mar 25 21:42:37 2013, 美东)

My two cents:

How protein can be expressed with a stop code, which cause a frame shift? 

For my personal experience, I used Rosa-Stop-YFP mice from JAX and cannot
see any leakage in Cre negative mice in any lymphocyte populations.

It's a transgene and thus it is possible that the insert allele can move and
incorporate into other promote region.  In that case, you just need to
refresh your line and get a new breeder from JAX.


【 在 htscorpion (snoopy) 的大作中提到: 】
: this is leak, it happens in almost all LoxP-STOP-LoxP system.  The
stronger
: of the promoter it has, the more leak they might have.
: transcription machinery is not like 1 or 0, it has some 0.2 or 0.8 status.
: Cre




--

※ 修改:·spt009 於 Mar 25 23:11:38 2013 修改本文·[FROM: 128.]
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muhe1985
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发信人: muhe1985 (chuomiao), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Tue Mar 26 00:06:38 2013, 美东)

rosa 是在rosa位点的knock in




【 在 spt009 (cytokine) 的大作中提到: 】
: My two cents:
: How protein can be expressed with a stop code, which cause a frame shift? 
: For my personal experience, I used Rosa-Stop-YFP mice from JAX and cannot
: see any leakage in Cre negative mice in any lymphocyte populations.
: It's a transgene and thus it is possible that the insert allele can move
and
:  incorporate into other promote region.  In that case, you just need to
: refresh your line and get a new breeder from JAX.
: stronger



--

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htscorpion
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发信人: htscorpion (snoopy), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Tue Mar 26 17:30:43 2013, 美东)

再说说我的看法吧.

LoxP-stop-LoxP-geneX system will leak more or less.  You did not find
protein expression only means the leakage is too low for your detection. 
When a LSL-geneX mouse grows to adult and Cre recombinase is introduced by
adeno or lenti, the "newly" expressed geneX will not be recognized as a
foreign protein by mouse immune system.



【 在 spt009 (cytokine) 的大作中提到: 】
: My two cents:
: How protein can be expressed with a stop code, which cause a frame shift? 
: For my personal experience, I used Rosa-Stop-YFP mice from JAX and cannot
: see any leakage in Cre negative mice in any lymphocyte populations.
: It's a transgene and thus it is possible that the insert allele can move
and
:  incorporate into other promote region.  In that case, you just need to
: refresh your line and get a new breeder from JAX.
: stronger



--

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htscorpion
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发信人: htscorpion (snoopy), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Tue Mar 26 17:32:35 2013, 美东)

I guess stronger promoter will tend to have higher chance of transcription
read through. 

But I could be wrong.  Correct me with ref.

【 在 muhe1985 (chuomiao) 的大作中提到: 】
: Promoter本来就是一直开着的,不管是r26还是CAG还是CMV还是whatever being used
: in this kind of system
: 这是transcription read through, 通常是因为loxp stop loxp 中的stop太弱
: stronger



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Dua
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发信人: Dua (Dua), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Tue Mar 26 18:12:49 2013, 美东)

看哪个line吧,leak严重么?有的leak对自己的课题也没什么影响,照实写就是了,也
不会因为这个课题就不能做了。
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lotkaeuler11
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发信人: lotkaeuler11 (Fibonacci), 信区: Biology
标  题: Re: Rosa26-loxP-stop-loxP-YFP mice
发信站: BBS 未名空间站 (Wed Mar 27 13:21:26 2013, 美东)


RE LZ


pls refer
PMID  21063464

Liu Y et al.
Tamoxifen-independent recombination in the RIP-CreER mouse. (2010).
PLoS One. 25: e13533.



http://www.ncbi.nlm.nih.gov/pubmed/21063464



>There are at least two possible explanations for
>these discrepancies.

>One possibility is that after many generations
of backcrossing to the C57BL/6 background, the expression of the
randomly integrated RIP-CreER transgene is enhanced, thus
increasing the probability of its nuclear entry, even in tamoxifenuntreated
cells. Another more likely possibility is that the Z/AP
cassette in the Z/AP reporter mouse strain is less prone to
recombination, i.e. it is inserted in an area of the chromatin less
amenable to recombination. Easy access of the CreER to the
target locus may result in both high recombination efficiency and
leakiness of the system as seen in the 3 lines described here. On the
other hand, recombination of a less accessible, heterochromatic
locus could be less efficient, but also less leaky, as in the case of the
Z/AP mouse.


>One way to mitigate such problems is the use of the Pdx1-CreER
>mouse, which was also generated in the Melton lab [14].





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