y*********u
发帖数: 183 | 1 如图
我要knock in表达的原基因有4个exon，我想插入一个EGFP reporter
，于是把exon1和exon2 replace掉（留下一小部分flanking sequence）
因为这个基因的3'UTR的调控做得很热，因此我就不能引入外源polyA 比如SV40, BGH
polyA等
粗看过去似乎问题不大，我用的EGFP的编码做过修改不含有splicing context
但是也有人说不加外源polyA的knock in表达很靠运气？ | y*********u
发帖数: 183 | | m******5
发帖数: 1383 | 3 ORZ, 楼主故意设计的结果和我因为设计失误造成的结果一样……
我还不知道有没有表达 | g***y
发帖数: 201 | 4 设计一个vector应该尽量实现多重目的。
像你这个设计，我觉得实现的功能比较少。
你这个原则上就是个 complete knockout 的设计
如果你只是想把原基因的前两个exon替换成egfp，那么是想看在transcription level
这个基因的表达。
那么随便从什么地方买个 gene trap就可以了，为什么还要自己费劲做呢？
另外，你这个设计也有潜在的问题，就是如果egfp本身带了stop codon,我 建议你去搜
non-sense mediated decay.
如果efgp没有带stop codon,你想让它作为fusion protein 跟 exon3,4 接在一起，那
我不确定是不是后面的序列会影响
gfp fluorescence,可以随便在什么cell line 里表达看看。
总之，建议你多看看别人的conditonal knockout vector 的设计。
【在 y*********u 的大作中提到】
: 如图
: 我要knock in表达的原基因有4个exon，我想插入一个EGFP reporter
: ，于是把exon1和exon2 replace掉（留下一小部分flanking sequence）
: 因为这个基因的3'UTR的调控做得很热，因此我就不能引入外源polyA 比如SV40, BGH
: polyA等
: 粗看过去似乎问题不大，我用的EGFP的编码做过修改不含有splicing context
: 但是也有人说不加外源polyA的knock in表达很靠运气？
| m******5
发帖数: 1383 | 5 I have a question on this:
Thus said, the method proposed by LZ would be a very good strategy to
produce hypolymorphic allele? since most mRNA are subjected into non sense
mediated decay, so the protein level would be reasonably low.
And where to search for EGFP/LACZ trapped ES cell line? I searched around
but found only few available trapped locus, could you recommend some company
making good use of it?
level
【在 g***y 的大作中提到】
: 设计一个vector应该尽量实现多重目的。
: 像你这个设计，我觉得实现的功能比较少。
: 你这个原则上就是个 complete knockout 的设计
: 如果你只是想把原基因的前两个exon替换成egfp，那么是想看在transcription level
: 这个基因的表达。
: 那么随便从什么地方买个 gene trap就可以了，为什么还要自己费劲做呢？
: 另外，你这个设计也有潜在的问题，就是如果egfp本身带了stop codon,我 建议你去搜
: non-sense mediated decay.
: 如果efgp没有带stop codon,你想让它作为fusion protein 跟 exon3,4 接在一起，那
: 我不确定是不是后面的序列会影响
| m******5
发帖数: 1383 | 6 楼主呢？ 我还是想问一下这个问题
company
【在 m******5 的大作中提到】
: I have a question on this:
: Thus said, the method proposed by LZ would be a very good strategy to
: produce hypolymorphic allele? since most mRNA are subjected into non sense
: mediated decay, so the protein level would be reasonably low.
: And where to search for EGFP/LACZ trapped ES cell line? I searched around
: but found only few available trapped locus, could you recommend some company
: making good use of it?
:
: level
| n********k
发帖数: 2818 | 7 there is a place called google...I think this board is great for questions,
but sometimes to reply on it may slow down one's own learning...The process
of researching for what one need may benefit one lots more than one expect..
.BTW, if i understand u right, for your particular question, there are several mouse knockout
consortium out there and it shall be within one click away, let me know if
you don't get them...
【在 m******5 的大作中提到】
: 楼主呢？ 我还是想问一下这个问题
:
: company
| n********k
发帖数: 2818 | 8 BTW, some(actually many) the gene traps may not behave as NMD as you would
hope...check out the principles, you would know why...
company
【在 m******5 的大作中提到】
: I have a question on this:
: Thus said, the method proposed by LZ would be a very good strategy to
: produce hypolymorphic allele? since most mRNA are subjected into non sense
: mediated decay, so the protein level would be reasonably low.
: And where to search for EGFP/LACZ trapped ES cell line? I searched around
: but found only few available trapped locus, could you recommend some company
: making good use of it?
:
: level
| m******5
发帖数: 1383 | 9 in LZ's case, if he made the construct exactly by the strategy he proposed.
It would for sure introduce NMD.
But my questions is that, how extensive would NMD work? it is for sure that
small proportion of accidentally created frame shift should be eliminated
by NMD.
But in LZ's case, what kind of allele would he create exactly?
a null allele producing all mRNA subjected to NMD? or a hypormorphic allele
with the major proportion degraded by still capable of producing an moderate mount of protein?
This is the question I have problem with, because I didn't work in this field, I am overwhelmed by the results produced by Google.
,
process
..
several mouse knockout
【在 n********k 的大作中提到】
: there is a place called google...I think this board is great for questions,
: but sometimes to reply on it may slow down one's own learning...The process
: of researching for what one need may benefit one lots more than one expect..
: .BTW, if i understand u right, for your particular question, there are several mouse knockout
: consortium out there and it shall be within one click away, let me know if
: you don't get them...
| m******5
发帖数: 1383 | 10 it was not the question I wanted to ask, sorry for my language issue. It was rather a separated question, I rephrased it on LS.
【在 n********k 的大作中提到】
: BTW, some(actually many) the gene traps may not behave as NMD as you would
: hope...check out the principles, you would know why...
:
: company
| n********k
发帖数: 2818 | 11 Are u asking about NMD or genetraps? I thought your original questions were
about finding genetraps...as for NMD, as far as my knowledge goes(I have
not updated myself on the research area for 4-5 ys), I don't think we know
enough to answer your question...but I am not sure whether your assertion
about "it would for sure introduce NMD"...hard to say, one would have to
look at the design in details to be more certain...and ultimately it is the
nature rules...I don't think a null allele would allways produce mRNA all
subjected to NMD,it all depends....on the contrary, sometimes, a conditional
allele could produce hypormoph if that's what you look for...
.
that
allele
moderate mount of protein?
field, I am overwhelmed by the results produced by Google.
【在 m******5 的大作中提到】
: in LZ's case, if he made the construct exactly by the strategy he proposed.
: It would for sure introduce NMD.
: But my questions is that, how extensive would NMD work? it is for sure that
: small proportion of accidentally created frame shift should be eliminated
: by NMD.
: But in LZ's case, what kind of allele would he create exactly?
: a null allele producing all mRNA subjected to NMD? or a hypormorphic allele
: with the major proportion degraded by still capable of producing an moderate mount of protein?
: This is the question I have problem with, because I didn't work in this field, I am overwhelmed by the results produced by Google.
:
| m******5
发帖数: 1383 | 12 as the file attached by LZ,assuming the KI sequence contain a stop codon,
there would be at least 3 EJC not removed to recruit NMD.
were
the
conditional
【在 n********k 的大作中提到】
: Are u asking about NMD or genetraps? I thought your original questions were
: about finding genetraps...as for NMD, as far as my knowledge goes(I have
: not updated myself on the research area for 4-5 ys), I don't think we know
: enough to answer your question...but I am not sure whether your assertion
: about "it would for sure introduce NMD"...hard to say, one would have to
: look at the design in details to be more certain...and ultimately it is the
: nature rules...I don't think a null allele would allways produce mRNA all
: subjected to NMD,it all depends....on the contrary, sometimes, a conditional
: allele could produce hypormoph if that's what you look for...
:
|
|
