a***f
发帖数: 32 | 1 请教大拿,
用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
I tried two times without success. My boss said it has been done
successfully by others. My gene is not immune-related. It involves in
metabolism. I’m pretty sure that I did not make any technical mistakes.
Anybody can share what may cause it? I do not want to it again if there is
no hope
Thanks you! | z*******6
发帖数: 679 | 2 我的老鼠（不是我做的）是表达人基因的...不知道反过来...路过帮顶... | b******n
发帖数: 4225 | 3 多大的基因啊
这种外源表达，实在不好说
?
is
【在 a***f 的大作中提到】
: 请教大拿,
: 用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
: I tried two times without success. My boss said it has been done
: successfully by others. My gene is not immune-related. It involves in
: metabolism. I’m pretty sure that I did not make any technical mistakes.
: Anybody can share what may cause it? I do not want to it again if there is
: no hope
:
: Thanks you!
| s******s
发帖数: 13035 | 4 你要知道，BAC里面就是你要的基因 + 调控序列 - epigenetic markers
虽然很多被epigentically repressed的基因很可能表达，但是你缺少必要
的TFs的话就没有办法了。
?
is
【在 a***f 的大作中提到】
: 请教大拿,
: 用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
: I tried two times without success. My boss said it has been done
: successfully by others. My gene is not immune-related. It involves in
: metabolism. I’m pretty sure that I did not make any technical mistakes.
: Anybody can share what may cause it? I do not want to it again if there is
: no hope
:
: Thanks you!
| w******n
发帖数: 767 | 5 鼠的基因在人细胞表达完全没问题。但是看启动子，不知道你这个基因应该在哪个组织
表达，BAC因为是基因本身的启动子，所以如果不是看家基因，可能需要特定的细胞系
。比如特定在肝脏表达的鼠基因要到对应人的肝脏细胞去表达。
?
is
【在 a***f 的大作中提到】
: 请教大拿,
: 用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
: I tried two times without success. My boss said it has been done
: successfully by others. My gene is not immune-related. It involves in
: metabolism. I’m pretty sure that I did not make any technical mistakes.
: Anybody can share what may cause it? I do not want to it again if there is
: no hope
:
: Thanks you!
| l**********1
发帖数: 5204 | 6 why only try BAC vector method only
alternatively you can try PB transposons vector
>Transfection of naked DNA has been used for large-cargo delivery.
Pronuclear injection of bacterial artificial chromosomes (BACs) has been
successful for transgenesis of up to 300 kb. However, the integrity,
integration site and copy number can not be controlled. BAC vectors have
also been used for targeting large cargos to defined genomic positions in ES
cells via homologous recombination (Valenzuela et al., 2003), but the
efficiency is locus-dependent and can be very low.
Recombinases such as Cre have also been used to deliver BACs to a predefined
genomic location by recombination-mediated cassette exchange (Wallace et al
., 2007; Prosser et al., 2008); however, pre-engineering of target sites in
the genome is necessary. While these methods are useful for certain
applications, all have limitations and most of them are not able to revert
the insertion of large DNA fragments.
full text link:
URL address:
f-t-p ://ftp.sanger.ac.uk/pub4/theses/ml8/chapter7.pdf
?
is
【在 a***f 的大作中提到】
: 请教大拿,
: 用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
: I tried two times without success. My boss said it has been done
: successfully by others. My gene is not immune-related. It involves in
: metabolism. I’m pretty sure that I did not make any technical mistakes.
: Anybody can share what may cause it? I do not want to it again if there is
: no hope
:
: Thanks you!
| a***f
发帖数: 32 | 7 Thanks a lot for the kind response from Zhang.., Bull..., Lot..., Wak....
and Shak...
My gene is about 2500 aa. I used in kidney cells. Have to use this strategy
as boss want to do it in this way.
:-(
I agree that it is important to have the appropriate transcription factors
in the expression system. In other words, I need to have the right host
cells. If the human orthologue is expressed, can I assume that the mouse
version of the gene will be expressed too, in the human cells?
In addition, besides RT-PCR, is there any dry lab method or database to
check if it is expressed in a given tissue type.
Thanks!
?
is
【在 a***f 的大作中提到】
: 请教大拿,
: 用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
: I tried two times without success. My boss said it has been done
: successfully by others. My gene is not immune-related. It involves in
: metabolism. I’m pretty sure that I did not make any technical mistakes.
: Anybody can share what may cause it? I do not want to it again if there is
: no hope
:
: Thanks you!
| l**********1
发帖数: 5204 | 8 just try YFP labeling
paper link:
//www.ncbi.nlm.nih.gov/pmc/articles/PMC2889740/pdf/mt201047a.pdf
>besides RT-PCR, is there any dry lab method or database to
strategy
【在 a***f 的大作中提到】
: Thanks a lot for the kind response from Zhang.., Bull..., Lot..., Wak....
: and Shak...
: My gene is about 2500 aa. I used in kidney cells. Have to use this strategy
: as boss want to do it in this way.
: :-(
: I agree that it is important to have the appropriate transcription factors
: in the expression system. In other words, I need to have the right host
: cells. If the human orthologue is expressed, can I assume that the mouse
: version of the gene will be expressed too, in the human cells?
: In addition, besides RT-PCR, is there any dry lab method or database to
| s******s
发帖数: 13035 | 9 我和你说吧，如果有相应的human orthologue表达，那么九成以上你的
老鼠基因能表达，有很小的物种不兼容的可能性你的不表达，很小。
如果相应的Human orthologue不表达，有两种可能，A缺少TF，B分化过
程中被actively epigenetically repress了。如果你得基因是一个重要
的调控基因，那么恭喜，很可能human copy是cis repress了，你得老鼠基
因有比较大的概率仍然可能表达；如果你得是一个下游基因，那么多半不表
达是因为A，那就没辙了
strategy
【在 a***f 的大作中提到】
: Thanks a lot for the kind response from Zhang.., Bull..., Lot..., Wak....
: and Shak...
: My gene is about 2500 aa. I used in kidney cells. Have to use this strategy
: as boss want to do it in this way.
: :-(
: I agree that it is important to have the appropriate transcription factors
: in the expression system. In other words, I need to have the right host
: cells. If the human orthologue is expressed, can I assume that the mouse
: version of the gene will be expressed too, in the human cells?
: In addition, besides RT-PCR, is there any dry lab method or database to
| l**********1
发帖数: 5204 | 10 Still you can try HAC Human artificial chromosome not BAC bacterial artificial chromosome
with that mice gene with labeled YFP knock in HAC
then expressed in Human kidney cells.
please refer:
//www.ncbi.nlm.nih.gov/pubmed/21750534
RE:
>如果你得是一个下游基因，那么多半不表
[回复]
发信人: amysf (kdfa), 信区: Biology
标 题: Re: 请教鼠基因在人细胞的表达
发信站: BBS 未名空间站 (Thu Feb 9 10:03:15 2012, 美东)
...
My gene is about 2500 aa. I used in kidney cells.
----------- | | | w******n
发帖数: 767 | 11 如果RT-PCR阳性，蛋白手段检测不到的话，可能表达量比较低，调整检测手段。最近做
几个蛋白也是这样，提高WB一抗浓度后，有改观，可以试一下。
strategy
【在 a***f 的大作中提到】
: Thanks a lot for the kind response from Zhang.., Bull..., Lot..., Wak....
: and Shak...
: My gene is about 2500 aa. I used in kidney cells. Have to use this strategy
: as boss want to do it in this way.
: :-(
: I agree that it is important to have the appropriate transcription factors
: in the expression system. In other words, I need to have the right host
: cells. If the human orthologue is expressed, can I assume that the mouse
: version of the gene will be expressed too, in the human cells?
: In addition, besides RT-PCR, is there any dry lab method or database to
| a***f
发帖数: 32 | 12 Thanks to Lot..., Wak.... and Shak... again!
Sorry for not responding sooner. I did RT-PCR, as expected, the human
endogenous gene is expressed. But the mouse gene (BAC)is not.
I guess it is correct to say that the mouse transgene should have decent
chance to be expressed if the human orthorlogue is expressed, as a general
rule. But for a given gene, it is very hard to be sure. At least, the
organization of CpG elements and regulatory sequence of gene are very
different from mouse to human, right?
In my case, I used a known gene as a technical control. The mouse version of
this gene is expressed, but not gene-of-interest. Both this gene and my
gene-of-interest are all expressed in human kidney cell.
Could I then safely conclude the promoter of the mouse version of my gene-of
-interest does not work in human cell system?
What do you guys think? Thanks!
【在 w******n 的大作中提到】
: 如果RT-PCR阳性，蛋白手段检测不到的话，可能表达量比较低，调整检测手段。最近做
: 几个蛋白也是这样，提高WB一抗浓度后，有改观，可以试一下。
:
: strategy
| s******s
发帖数: 13035 | 13 normally, there are multiple BACs for the same gene. You can try more
of
【在 a***f 的大作中提到】
: Thanks to Lot..., Wak.... and Shak... again!
: Sorry for not responding sooner. I did RT-PCR, as expected, the human
: endogenous gene is expressed. But the mouse gene (BAC)is not.
: I guess it is correct to say that the mouse transgene should have decent
: chance to be expressed if the human orthorlogue is expressed, as a general
: rule. But for a given gene, it is very hard to be sure. At least, the
: organization of CpG elements and regulatory sequence of gene are very
: different from mouse to human, right?
: In my case, I used a known gene as a technical control. The mouse version of
: this gene is expressed, but not gene-of-interest. Both this gene and my
| w******n
发帖数: 767 | 14 再找小鼠细胞验证一下，确认BAC没问题。
of
【在 a***f 的大作中提到】
: Thanks to Lot..., Wak.... and Shak... again!
: Sorry for not responding sooner. I did RT-PCR, as expected, the human
: endogenous gene is expressed. But the mouse gene (BAC)is not.
: I guess it is correct to say that the mouse transgene should have decent
: chance to be expressed if the human orthorlogue is expressed, as a general
: rule. But for a given gene, it is very hard to be sure. At least, the
: organization of CpG elements and regulatory sequence of gene are very
: different from mouse to human, right?
: In my case, I used a known gene as a technical control. The mouse version of
: this gene is expressed, but not gene-of-interest. Both this gene and my
| l**********1
发帖数: 5204 | 15 Congratulation!
If you try that successful others same BAC and same protocol and same
materials
then you try again and again it is still negative.
And not only you but your college PD does it again and also it is negative=
It seems that paper might had misconduct
likes below blog shown:
//md-anderson-cc.blogspot.com/
or some trigger around it.
That is more interesting than you get positive data from that paper.
回复]
发信人: amysf (kdfa), 信区: Biology
标 题: 请教鼠基因在人细胞的表达
发信站: BBS 未名空间站 (Tue Feb 7 23:13:13 2012, 美东)
请教大拿,
用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
I tried two times without success. My boss said it has been done
successfully by others
of
【在 a***f 的大作中提到】
: Thanks to Lot..., Wak.... and Shak... again!
: Sorry for not responding sooner. I did RT-PCR, as expected, the human
: endogenous gene is expressed. But the mouse gene (BAC)is not.
: I guess it is correct to say that the mouse transgene should have decent
: chance to be expressed if the human orthorlogue is expressed, as a general
: rule. But for a given gene, it is very hard to be sure. At least, the
: organization of CpG elements and regulatory sequence of gene are very
: different from mouse to human, right?
: In my case, I used a known gene as a technical control. The mouse version of
: this gene is expressed, but not gene-of-interest. Both this gene and my
| a***f
发帖数: 32 | 16 Thanks all for input.
I guess I did not make it cleary enough. What I meant is that a BAC
containing my mouse gene was not expressed. But a BAC containing another
known gene was. I did both in parallel and the latter wss used as a control.
【在 l**********1 的大作中提到】
: Congratulation!
: If you try that successful others same BAC and same protocol and same
: materials
: then you try again and again it is still negative.
: And not only you but your college PD does it again and also it is negative=
: It seems that paper might had misconduct
: likes below blog shown:
: //md-anderson-cc.blogspot.com/
: or some trigger around it.
: That is more interesting than you get positive data from that paper.
| l**********1
发帖数: 5204 | 17 Agree with
shakuras said:
[回复]
发信人: shakuras (doskey), 信区: Biology
标 题: Re: 请教鼠基因在人细胞的表达
发信站: BBS 未名空间站 (Sat Feb 11 17:13:35 2012, 美东)
normally, there are multiple BACs for the same gene. You can try more
control.
【在 a***f 的大作中提到】
: Thanks all for input.
: I guess I did not make it cleary enough. What I meant is that a BAC
: containing my mouse gene was not expressed. But a BAC containing another
: known gene was. I did both in parallel and the latter wss used as a control.
|
|
