s********9
发帖数: 132 | 1 One wants to knock out gene A and gene B.
One would mate A+/- X B+/-.
what if gene A and gene B are on the same chromosome?
Probably dumb question. Thanks. |
d***s
发帖数: 1062 | 2 This is a very interesting question. |
r******g
发帖数: 600 | 3 我自己从来没试过，我问过我老板完全相同的问题
我记得我老板跟我说，gene A and B 在同一个chromosome上面的话，比较难cross
一般情况下，大家都会放弃，选择cross A 和 C, 比如 C gene是B的receptor，这样可
以解释同样的问题
如果A,B 在chromosome上面距离比较远，有可能 重组 成功，但是 需要一定的工作量
如果A,B的gene位置比较近，举个例子，P38MAPK的alpha, beta （MAPK 11,12,13,14）
。。。。 他们隔得非常近 位于同一个locus，所以 一直没有很好的triple KO, quart
KO model
所以说，crispr很强大呀！ |
s********9
发帖数: 132 | 4 Thanks for the reply.
But in pratice, if one has to do this. What I can think of are:
1) as you said, hoping the homologous recombination during meiosis.
2) get a B+/- allele in A+/- background? 50% chance it would be on the same
chromosome.
Just don't know what people actually do in reality.
BTW, one pertinent questtion, when one makes transgenic mouse, is it routine
to pinpoint the integration site? (People often cross a transgenic mouse
with a KO mouse or transgenic mouse)
Especially for Cre-transgenic mouse, people use them to cross with all kinds
of LoxP-KO. The chance of CRE and LoxP on the same chromosome would be
inevitable for certain genes?
THanks.
quart
【在 r******g 的大作中提到】
: 我自己从来没试过，我问过我老板完全相同的问题
: 我记得我老板跟我说，gene A and B 在同一个chromosome上面的话，比较难cross
: 一般情况下，大家都会放弃，选择cross A 和 C, 比如 C gene是B的receptor，这样可
: 以解释同样的问题
: 如果A,B 在chromosome上面距离比较远，有可能 重组 成功，但是 需要一定的工作量
: 如果A,B的gene位置比较近，举个例子，P38MAPK的alpha, beta （MAPK 11,12,13,14）
: 。。。。 他们隔得非常近 位于同一个locus，所以 一直没有很好的triple KO, quart
: KO model
: 所以说，crispr很强大呀！
|
a*******a
发帖数: 4233 | 5 primary culture mouse A-/- ES
quart
【在 r******g 的大作中提到】
: 我自己从来没试过，我问过我老板完全相同的问题
: 我记得我老板跟我说，gene A and B 在同一个chromosome上面的话，比较难cross
: 一般情况下，大家都会放弃，选择cross A 和 C, 比如 C gene是B的receptor，这样可
: 以解释同样的问题
: 如果A,B 在chromosome上面距离比较远，有可能 重组 成功，但是 需要一定的工作量
: 如果A,B的gene位置比较近，举个例子，P38MAPK的alpha, beta （MAPK 11,12,13,14）
: 。。。。 他们隔得非常近 位于同一个locus，所以 一直没有很好的triple KO, quart
: KO model
: 所以说，crispr很强大呀！
|
h**********r
发帖数: 671 | 6 做真菌mating拿double knockout的时候会看一下摩尔根距离，算一下挑多少个孢子
fail的概率 |
D*a
发帖数: 6830 | 7 那你的project就跪了呗
先cross下试试吧，不行再说。 |
r******g
发帖数: 600 | 8 integration site 不知道的话，文章一样发啊
90年代 做transgenic mice 很多都不知道loci的
你准备怎么cross 老鼠呢？ 讲来听听，我挺好奇的。我自己从来没遇到过这种情况
我想了想，觉得 先cross 出heterozygous A+/-. B+/- 然后 再跟WT cross
直接 同时PCR A和B, 得到double positive 为成功的haplotype。 貌似用这种办法，
screening strategy比较简单
lz准备怎么操作？纯属好奇
same
routine
kinds
【在 s********9 的大作中提到】
: Thanks for the reply.
: But in pratice, if one has to do this. What I can think of are:
: 1) as you said, hoping the homologous recombination during meiosis.
: 2) get a B+/- allele in A+/- background? 50% chance it would be on the same
: chromosome.
: Just don't know what people actually do in reality.
: BTW, one pertinent questtion, when one makes transgenic mouse, is it routine
: to pinpoint the integration site? (People often cross a transgenic mouse
: with a KO mouse or transgenic mouse)
: Especially for Cre-transgenic mouse, people use them to cross with all kinds
|
r******g
发帖数: 600 | 9 还有 lz
如果 你最后成功的 把A/B cross出来了，私信 或者 回个帖，说说 最后的 % 是多少 |
s********n
发帖数: 248 | |
|
|
s********9
发帖数: 132 | 11 I simply brought this up out of curiosity for discussion. I have NO hands-on
experience with mouse genetics whatsoever.
For double KO. the chance gene A and B on the same chromosome would be 1/20.
People have done so many double KO. I figure this could be a practical issue.
For crossing CRE-transgenic mouse with LoxP mouse to produce tissue-specific
KO.
The chance are also 1/20 the CRE-transgene and your LoxP-cassette being on
the same chromosome.
I don't think it's necessary impossible to achieve. One could always get a
ES cell with A KO, then get B KO allele on top of this. Just wonder whether
there are other options.
BTW, for those CRE-transgenic mouse, do we know the integration site and
copy number?
Many many thanks.
【在 r******g 的大作中提到】
: integration site 不知道的话，文章一样发啊
: 90年代 做transgenic mice 很多都不知道loci的
: 你准备怎么cross 老鼠呢？ 讲来听听，我挺好奇的。我自己从来没遇到过这种情况
: 我想了想，觉得 先cross 出heterozygous A+/-. B+/- 然后 再跟WT cross
: 直接 同时PCR A和B, 得到double positive 为成功的haplotype。 貌似用这种办法，
: screening strategy比较简单
: lz准备怎么操作？纯属好奇
:
: same
: routine
|
z*******6
发帖数: 679 | 12 我们领域的三个基因直接就是tandem连在一起的，single KO，三种组合的double KO，
还有triple KO都是单独做的，当然这是上世纪末的事情了... |
s********9
发帖数: 132 | 13 Thanks. Could u plz give a PMID?
【在 z*******6 的大作中提到】
: 我们领域的三个基因直接就是tandem连在一起的，single KO，三种组合的double KO，
: 还有triple KO都是单独做的，当然这是上世纪末的事情了...
|
r******g
发帖数: 600 | 14 你这个3个gene连的很近吧 intronic region 不大吧，所以，直接一个 recombination
直接把3个gene都KO掉
如果3个基因隔得有一定距离，相当不好操作的
【在 z*******6 的大作中提到】
: 我们领域的三个基因直接就是tandem连在一起的，single KO，三种组合的double KO，
: 还有triple KO都是单独做的，当然这是上世纪末的事情了...
|
r******g
发帖数: 600 | 15 你这个3个gene连的很近吧 intronic region 不大吧，所以，直接一个 recombination
直接把3个gene都KO掉
如果3个基因隔得有一定距离，相当不好操作的
【在 z*******6 的大作中提到】
: 我们领域的三个基因直接就是tandem连在一起的，single KO，三种组合的double KO，
: 还有triple KO都是单独做的，当然这是上世纪末的事情了...
|
z*******6
发帖数: 679 | 16 http://www.pnas.org/content/96/20/11452.full.pdf
【在 s********9 的大作中提到】
: Thanks. Could u plz give a PMID?
|
z*******6
发帖数: 679 | 17 他们好像是一个一个敲的... 在一个的基础上... 也就是他们那种有钱挥霍的实验室会
干这种事情...
recombination
【在 r******g 的大作中提到】
: 你这个3个gene连的很近吧 intronic region 不大吧，所以，直接一个 recombination
: 直接把3个gene都KO掉
: 如果3个基因隔得有一定距离，相当不好操作的
|
H****N
发帖数: 997 | 18 In most cases, when the two genes are far enough away from each other, it is
not a problem to generate the double null. However, when the two genes are
very close to each other, it will be very hard to create the double null by
just crossing the two single KOs; you may have to knockout the two genes in
the same es cell to generate the mouse. Anybody who has taken a genetics
course should know the underlying mechanism (hint: this has to do with what
happens to the chromosomes during meiosis). |
H****N
发帖数: 997 | 19 You point regarding cre transgene and the floxed gene to be knocked out on
the same chromosome is interesting. This can be a problem in practice.
People don't always map the transgene. However, if you have a hard time
getting the flox/flox;cre mouse, it is likely due to that the transgene and
the gene of interest are closely located on the same chromosome.Of course,
you still need to rule out embryonic lethality first.
same
routine
kinds
【在 s********9 的大作中提到】
: Thanks for the reply.
: But in pratice, if one has to do this. What I can think of are:
: 1) as you said, hoping the homologous recombination during meiosis.
: 2) get a B+/- allele in A+/- background? 50% chance it would be on the same
: chromosome.
: Just don't know what people actually do in reality.
: BTW, one pertinent questtion, when one makes transgenic mouse, is it routine
: to pinpoint the integration site? (People often cross a transgenic mouse
: with a KO mouse or transgenic mouse)
: Especially for Cre-transgenic mouse, people use them to cross with all kinds
|
s********9
发帖数: 132 | 20 Very informative.
I cannot believe people don't know where the cre is when starting the
crossing.
Thanks a lot.
and
【在 H****N 的大作中提到】
: You point regarding cre transgene and the floxed gene to be knocked out on
: the same chromosome is interesting. This can be a problem in practice.
: People don't always map the transgene. However, if you have a hard time
: getting the flox/flox;cre mouse, it is likely due to that the transgene and
: the gene of interest are closely located on the same chromosome.Of course,
: you still need to rule out embryonic lethality first.
:
: same
: routine
: kinds
|
s********9
发帖数: 132 | 21 Plus, just for discussion, if gene A and B are near the two opposite
telomeres on a certain long chromosome (as far as it can be), what kind of
chance would you expect? (This expectation would be critical when you set up
the crossing, right?)
is
are
by
in
what
【在 H****N 的大作中提到】
: In most cases, when the two genes are far enough away from each other, it is
: not a problem to generate the double null. However, when the two genes are
: very close to each other, it will be very hard to create the double null by
: just crossing the two single KOs; you may have to knockout the two genes in
: the same es cell to generate the mouse. Anybody who has taken a genetics
: course should know the underlying mechanism (hint: this has to do with what
: happens to the chromosomes during meiosis).
|
H****N
发帖数: 997 | 22 The efficiency should be the same as you would expect with the two genes on
different chromosomes. |
