h********n
发帖数: 4079 | 1 4种老鼠, 分别带某个基因(KO/KI/tg), 要把四个基因弄到一个老鼠上, 好像逐个杂交
的工作量太大, 而且到最后比例太低. 不知道可行不可行.
各位有没有做过的, 给点指导?
谢谢 |
g****e
发帖数: 137 | 2 I did so, with 3 different flox/flox and one cre mice |
h********n
发帖数: 4079 | 3 那你到了最后的选老鼠岂不是比例很低? 是不是要很多老鼠来选?
我的有些类似, 一个oncogene, 两个Flox/Flox, 一个Cre老鼠. 其中两个flox/flox我
都要homozygous.
谢谢.
【在 g****e 的大作中提到】
: I did so, with 3 different flox/flox and one cre mice
|
g****e
发帖数: 137 | 4 I need homozygous for 2 flox/flox, heterzygous for one flox/flox and cre, it
took me one whole year to get it. The ratio at beginning is very low, but
you can increase efficiency after a few rounds of mating. |
O******e
发帖数: 4845 | 5 You have to make sure they are not on same chromosomes
【在 h********n 的大作中提到】
: 4种老鼠, 分别带某个基因(KO/KI/tg), 要把四个基因弄到一个老鼠上, 好像逐个杂交
: 的工作量太大, 而且到最后比例太低. 不知道可行不可行.
: 各位有没有做过的, 给点指导?
: 谢谢
|
h********n
发帖数: 4079 | 6 在同一个染色体上会咋样?
【在 O******e 的大作中提到】
: You have to make sure they are not on same chromosomes
|
O******e
发帖数: 4845 | 7 正常分离的话，你拿不到你要的组合啊。重组几率太低。
【在 h********n 的大作中提到】
: 在同一个染色体上会咋样?
|
h****g
发帖数: 439 | 8 同问！
【在 h********n 的大作中提到】
: 在同一个染色体上会咋样?
|
c****l
发帖数: 1086 | 9 即使在同一条染色体上还可以发生homologous recombination而组合在一起啊。
只是不能太近，这个几率可以用cM来测量。如果是在同一染色体的两头，重组的几率还
是不低的。
The centimorgan is equal to a 1% chance that a marker at one genetic locus
on a chromosome will be separated from a marker at a second locus due to
crossing over in a single generation
One cM is about 1 Mb apart.
【在 O******e 的大作中提到】
: 正常分离的话，你拿不到你要的组合啊。重组几率太低。
|
O******e
发帖数: 4845 | 10 I wouldn't count on that. lz doesn't really have to get the homozygotes.
【在 c****l 的大作中提到】
: 即使在同一条染色体上还可以发生homologous recombination而组合在一起啊。
: 只是不能太近，这个几率可以用cM来测量。如果是在同一染色体的两头，重组的几率还
: 是不低的。
: The centimorgan is equal to a 1% chance that a marker at one genetic locus
: on a chromosome will be separated from a marker at a second locus due to
: crossing over in a single generation
: One cM is about 1 Mb apart.
|
|
|
c****l
发帖数: 1086 | 11 If you do not count on science, anything else you will count on?
【在 O******e 的大作中提到】
: I wouldn't count on that. lz doesn't really have to get the homozygotes.
|
O******e
发帖数: 4845 | 12 You missed my point. I wouldn't count on HR for that end. Just too much work
to get what lz wanted.
【在 c****l 的大作中提到】
: If you do not count on science, anything else you will count on?
|
g*******0
发帖数: 2152 | 13 a. make sure the four genes are on different chromosome
b. plan the breeding carefully so that for each round of breeding the
mendelian ratio of getting your target genotype is no less than 1:4 ( below
1:16 is almost mission impossible). This way, in general, it takes one round
of breeding to introduce one mutated allele to the genome. Say you are
planning to introduce 4 mutated genes, 2 homozygous 2 heterozygous, you will
need 4 + 2 = 6 round of breeding. Each round will take at least 9 weeks ( |
h********n
发帖数: 4079 | 14 thank you so much for the advice.
As to the time, I am thinking of breeding A+B and C+D at the same time and
then do ABCD, so as to save some time. Do you think there is any potential
issues there?
below
round
will
3
may
very
【在 g*******0 的大作中提到】
: a. make sure the four genes are on different chromosome
: b. plan the breeding carefully so that for each round of breeding the
: mendelian ratio of getting your target genotype is no less than 1:4 ( below
: 1:16 is almost mission impossible). This way, in general, it takes one round
: of breeding to introduce one mutated allele to the genome. Say you are
: planning to introduce 4 mutated genes, 2 homozygous 2 heterozygous, you will
: need 4 + 2 = 6 round of breeding. Each round will take at least 9 weeks (
|
n********k
发帖数: 2818 | 15 I was gonna reply to you but it is not an easy task, and got a bit busy
today...I would seriously suggest you
take a day or two to sit down:
1: Know your goals(what genotypes do you ultimate need, what's proper
control...do you want Cre
control or not, all will dramatically change your breeding stategies)
2: What mice do you have to start with
3: Money versa time (man power as well---sometimes one more generation
seems longer and wasteful
but indeed could save you tons if you don't h
【在 h********n 的大作中提到】
: thank you so much for the advice.
: As to the time, I am thinking of breeding A+B and C+D at the same time and
: then do ABCD, so as to save some time. Do you think there is any potential
: issues there?
:
: below
: round
: will
: 3
: may
|
n********k
发帖数: 2818 | 16 Just saw u have transgene, in that case, likely it is a lot LOT easier....
【在 n********k 的大作中提到】
: I was gonna reply to you but it is not an easy task, and got a bit busy
: today...I would seriously suggest you
: take a day or two to sit down:
: 1: Know your goals(what genotypes do you ultimate need, what's proper
: control...do you want Cre
: control or not, all will dramatically change your breeding stategies)
: 2: What mice do you have to start with
: 3: Money versa time (man power as well---sometimes one more generation
: seems longer and wasteful
: but indeed could save you tons if you don't h
|
h********n
发帖数: 4079 | 17 太感谢了.
我的座位后面有个白板, 我两个月前把breeding strategy写在上面, 然后每天都看看
想想. 主要的问题是, 周围的人都不太有经验, 我有一写经验, 还差很远.
【在 n********k 的大作中提到】
: I was gonna reply to you but it is not an easy task, and got a bit busy
: today...I would seriously suggest you
: take a day or two to sit down:
: 1: Know your goals(what genotypes do you ultimate need, what's proper
: control...do you want Cre
: control or not, all will dramatically change your breeding stategies)
: 2: What mice do you have to start with
: 3: Money versa time (man power as well---sometimes one more generation
: seems longer and wasteful
: but indeed could save you tons if you don't h
|
h********n
发帖数: 4079 | 18 Cre control 是指老鼠跟非Cre mice交配作为control吗? 如果是这个, 我不需要.
我要做的最终的比较组是 gene +/+, +/-, -/-即wt, hetero KO, homo KO. 其中KO是
cre干的.
【在 n********k 的大作中提到】
: I was gonna reply to you but it is not an easy task, and got a bit busy
: today...I would seriously suggest you
: take a day or two to sit down:
: 1: Know your goals(what genotypes do you ultimate need, what's proper
: control...do you want Cre
: control or not, all will dramatically change your breeding stategies)
: 2: What mice do you have to start with
: 3: Money versa time (man power as well---sometimes one more generation
: seems longer and wasteful
: but indeed could save you tons if you don't h
|
n********k
发帖数: 2818 | 19 In general, it would be faster if you don't control for Cre...but I
personally don't recommend as Cre could be a
complicating factor...therefore for my final breeding, I always use one with
double Cre while the other one
with no Cre, and then every pups have cre but different genotype, rather
than the other way: every pups is the
same genotype but either with or without cre...in your case, u might want to
keep only one allele different
between experimental groups while keep the rest the same,
eg
【在 h********n 的大作中提到】
: Cre control 是指老鼠跟非Cre mice交配作为control吗? 如果是这个, 我不需要.
: 我要做的最终的比较组是 gene +/+, +/-, -/-即wt, hetero KO, homo KO. 其中KO是
: cre干的.
|
h********n
发帖数: 4079 | 20 I got your points, which are very helpful to me.
I am going back to lab and think of these carefully.
with
to
【在 n********k 的大作中提到】
: In general, it would be faster if you don't control for Cre...but I
: personally don't recommend as Cre could be a
: complicating factor...therefore for my final breeding, I always use one with
: double Cre while the other one
: with no Cre, and then every pups have cre but different genotype, rather
: than the other way: every pups is the
: same genotype but either with or without cre...in your case, u might want to
: keep only one allele different
: between experimental groups while keep the rest the same,
: eg
|
|
|
h********n
发帖数: 4079 | 21 I see your points, which are very helpful to me.
I am going back to lab and think of these carefully.
with
to
【在 n********k 的大作中提到】
: In general, it would be faster if you don't control for Cre...but I
: personally don't recommend as Cre could be a
: complicating factor...therefore for my final breeding, I always use one with
: double Cre while the other one
: with no Cre, and then every pups have cre but different genotype, rather
: than the other way: every pups is the
: same genotype but either with or without cre...in your case, u might want to
: keep only one allele different
: between experimental groups while keep the rest the same,
: eg
|
D*a
发帖数: 6830 | 22 就有一个问题，怎么测哪个是cre homozygote啊？定量PCR？
with
to
【在 n********k 的大作中提到】
: In general, it would be faster if you don't control for Cre...but I
: personally don't recommend as Cre could be a
: complicating factor...therefore for my final breeding, I always use one with
: double Cre while the other one
: with no Cre, and then every pups have cre but different genotype, rather
: than the other way: every pups is the
: same genotype but either with or without cre...in your case, u might want to
: keep only one allele different
: between experimental groups while keep the rest the same,
: eg
|
h********n
发帖数: 4079 | 23 就普通PCR吧. 设计primer跨过endogenous/exogenous sequence, homo/hetero/wt的产
物不同.
【在 D*a 的大作中提到】
: 就有一个问题，怎么测哪个是cre homozygote啊？定量PCR？
:
: with
: to
|
D*a
发帖数: 6830 | 24 你的cre是定点插入的？我都不知道我的cre插在哪儿-_-
另外cre很长的吧？
【在 h********n 的大作中提到】
: 就普通PCR吧. 设计primer跨过endogenous/exogenous sequence, homo/hetero/wt的产
: 物不同.
|
k****o
发帖数: 589 | 25
能加段tag吗？
【在 D*a 的大作中提到】
: 你的cre是定点插入的？我都不知道我的cre插在哪儿-_-
: 另外cre很长的吧？
|
D*a
发帖数: 6830 | 26 cre不是我们自己做的啊
我倒是知道有人鉴定cre homozygote是靠交配来鉴定的。
按这种思路，如果不牵扯background，倒是可以纯合杂和的cre一起用来交配下一代，
至少可以把下一代的cre+ 提高到 > 50%
【在 k****o 的大作中提到】
:
: 能加段tag吗？
|
h********n
发帖数: 4079 | 27 如果觉得不保险, 就设计两对primer, 分别看Wt和Cre.
【在 D*a 的大作中提到】
: 你的cre是定点插入的？我都不知道我的cre插在哪儿-_-
: 另外cre很长的吧？
|
D*a
发帖数: 6830 | 28 不知道cre插在哪儿，怎么看cre+/+ 和cre+/0？？？
【在 h********n 的大作中提到】
: 如果觉得不保险, 就设计两对primer, 分别看Wt和Cre.
|
D*a
发帖数: 6830 | 29 另外刚跟老板讨论这个问题，如果用cre /cre，cre倒是有control了，可是loxp位点没
有control了，也许你加进去的loxp位点somehow影响基因表达呢？所以我们组用的wt
mice实际上是flox/flox, 作图的时候写"control"，不写"wt"
也有人为了这个会多加一个纯粹的wt/wt作为control group, 用来跟flox/flox比较证
明flox/flox是wt的表型。
【在 h********n 的大作中提到】
: 如果觉得不保险, 就设计两对primer, 分别看Wt和Cre.
|
D*a
发帖数: 6830 | 30 你说的两边分别配ab和cd，到最后一起配abcd是完全可以的
另外你永远也不知道cre插在什么地方，只有通过交配才知道，这个是一个风险
还有就是你现阶段不要只想breeding，可以想一下有什么实验是需要set up但是又不需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
节约老鼠且不说了，反正老板花钱^_^，节约时间是很重要的, 我们一个博士的project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
。 |
|
|
h********n
发帖数: 4079 | 31 great point.
Thanks
需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练
手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的
验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR
genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，
behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还
有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
【在 D*a 的大作中提到】
: 你说的两边分别配ab和cd，到最后一起配abcd是完全可以的
: 另外你永远也不知道cre插在什么地方，只有通过交配才知道，这个是一个风险
: 还有就是你现阶段不要只想breeding，可以想一下有什么实验是需要set up但是又不需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
: 节约老鼠且不说了，反正老板花钱^_^，节约时间是很重要的, 我们一个博士的project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
: 。
|
g*******0
发帖数: 2152 | 32 You always have to do parallel breeding to save time. But
to get A/A B/+ or C/C D/+ you need two round breeding:
(1)A/+ x B/+ --> A/+ B/+ (1:4) and A/+ x A/+ --> A/A (1:4) (start with one
mutant allele)
(2) A/+ B/+ x A/A --> A/A B/+ (1:4)
if you breed AB with CD:
(3)A/A B/+ x C/C D/+ --> A/+ B/+ C/+ D/+ (1:4) and A/A B/+ x A/A B/+ --> A/
A B/B (1:4)
(4) A/+ B/+ C/+ D/+ x A/A B/B --> A/A B/+/C/+ D/+ or A/A B/B C/+ D/+ (1:8)
(5) A/A B/+ C/+ D/+ x A/A B/B --> A/A B/B C/+ D/+ (1:8) (6 mutated alle
【在 h********n 的大作中提到】
: thank you so much for the advice.
: As to the time, I am thinking of breeding A+B and C+D at the same time and
: then do ABCD, so as to save some time. Do you think there is any potential
: issues there?
:
: below
: round
: will
: 3
: may
|
g*******0
发帖数: 2152 | 33 Usually nobody know what locus is the cre located. The safe way to tell cre/
+ or cre/cre is by breeding and look at the Mendelian ratio. But if you pay
enough attention the intensity of PCR genotyping band normally can give you
some clue that two copy PCR band is much brighter than one copy. Better
approach is do cre PCR and a control PCR (say a-actin) in one tube, use the
right cre/+ cre/cre tail DNA control to help you establish the criteria to
judge what is cre/+ or cre/cre (eg. two band wit
【在 h********n 的大作中提到】
: great point.
: Thanks
:
: 需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练
: 手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的
: 验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR
: genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
: project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，
: behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还
: 有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
|
D*a
发帖数: 6830 | 34 多谢，然后还是应该breeding验证的吧～
cre homo的好处就是maintaining起来比较省事，不用每代都去pcr
cre/
pay
you
the
cre,
different
【在 g*******0 的大作中提到】
: Usually nobody know what locus is the cre located. The safe way to tell cre/
: + or cre/cre is by breeding and look at the Mendelian ratio. But if you pay
: enough attention the intensity of PCR genotyping band normally can give you
: some clue that two copy PCR band is much brighter than one copy. Better
: approach is do cre PCR and a control PCR (say a-actin) in one tube, use the
: right cre/+ cre/cre tail DNA control to help you establish the criteria to
: judge what is cre/+ or cre/cre (eg. two band wit
|
h********n
发帖数: 4079 | 35 我昨天在想这个Cre的问题。 我想， 能不能用这个transgene头围的序列设计primer测
序， 测出transgene前后的序列， 然后设计primer根据区分homo hetero and wt.
homo和wt都是一条带， hetero两条带。
cre/
pay
you
the
cre,
different
【在 g*******0 的大作中提到】
: Usually nobody know what locus is the cre located. The safe way to tell cre/
: + or cre/cre is by breeding and look at the Mendelian ratio. But if you pay
: enough attention the intensity of PCR genotyping band normally can give you
: some clue that two copy PCR band is much brighter than one copy. Better
: approach is do cre PCR and a control PCR (say a-actin) in one tube, use the
: right cre/+ cre/cre tail DNA control to help you establish the criteria to
: judge what is cre/+ or cre/cre (eg. two band wit
|
c****l
发帖数: 1086 | 36 How possible?
You basicly have 2 products in that way. You think your sequencing machine
is sensitive enough to detect one molecule of DNA sequence.
The only possible way is to do a "Pan-PCR" to clone the insertion site which
could actually be very complicated and time-consuming.
Q-PCR is able to distinguish the difference of one and two copies difference
if you have fine pipetting skill.
For most people, just forget about it unless you are having problem breeding
cre/loxp sites together. Then,y
【在 h********n 的大作中提到】
: 我昨天在想这个Cre的问题。 我想， 能不能用这个transgene头围的序列设计primer测
: 序， 测出transgene前后的序列， 然后设计primer根据区分homo hetero and wt.
: homo和wt都是一条带， hetero两条带。
:
: cre/
: pay
: you
: the
: cre,
: different
|
n********k
发帖数: 2818 | 37 As far as I know, Cre has demonstrated effects in lots of line...while in
general loxP effects shall be controlled easily...if it does, u shall have
hypomoph, right? I don't think ur boss's argument for this particular is
that sound...anyway, people use the other breeding strategy (without control
for cre) all the time. If u have a cell death phenotype, then u need to be
extra careful about cre
【在 D*a 的大作中提到】
: 另外刚跟老板讨论这个问题，如果用cre /cre，cre倒是有control了，可是loxp位点没
: 有control了，也许你加进去的loxp位点somehow影响基因表达呢？所以我们组用的wt
: mice实际上是flox/flox, 作图的时候写"control"，不写"wt"
: 也有人为了这个会多加一个纯粹的wt/wt作为control group, 用来跟flox/flox比较证
: 明flox/flox是wt的表型。
|
n********k
发帖数: 2818 | 38 It is indeed pretty easy to go for another generation of breeding, that way
you know double or single copy of cre and you only need to start with very
few and then get tons of them.., with my own experience, it saves time and
money unless u only need a few animals...
As for to determine the locus, I suppose one could go for FISH like you
point it out...maybe one could kind of go a RACE approach like insertational
mutation genesis(not sure just my guess), or one could even use Parrallel
sequen
【在 c****l 的大作中提到】
: How possible?
: You basicly have 2 products in that way. You think your sequencing machine
: is sensitive enough to detect one molecule of DNA sequence.
: The only possible way is to do a "Pan-PCR" to clone the insertion site which
: could actually be very complicated and time-consuming.
: Q-PCR is able to distinguish the difference of one and two copies difference
: if you have fine pipetting skill.
: For most people, just forget about it unless you are having problem breeding
: cre/loxp sites together. Then,y
|
c****l
发帖数: 1086 | 39 Not as easy as you thought, let's say you will got a litter of ten mice, are
you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
homo Cre? Since the mice are good for breeder only 6 months or so, by the
time you figure out which is homo Cre, you are only left with 3 months. I do
not think it is practical to do this routinely.
RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
cDNA, has nothing to do with insertional site analysis.
not sure about par
【在 n********k 的大作中提到】
: It is indeed pretty easy to go for another generation of breeding, that way
: you know double or single copy of cre and you only need to start with very
: few and then get tons of them.., with my own experience, it saves time and
: money unless u only need a few animals...
: As for to determine the locus, I suppose one could go for FISH like you
: point it out...maybe one could kind of go a RACE approach like insertational
: mutation genesis(not sure just my guess), or one could even use Parrallel
: sequen
|
D*a
发帖数: 6830 | 40 其实我觉得，一旦发现breeding死活拿不到想要的老鼠，也就差不多确定cre的位置了
吧。。。project也就算是挂了吧。。。也就没必要测cre到底具体在哪儿了吧。。。
如果想要的老鼠频率比较低，那就凑合着用吧。。。
way
insertational
【在 n********k 的大作中提到】
: It is indeed pretty easy to go for another generation of breeding, that way
: you know double or single copy of cre and you only need to start with very
: few and then get tons of them.., with my own experience, it saves time and
: money unless u only need a few animals...
: As for to determine the locus, I suppose one could go for FISH like you
: point it out...maybe one could kind of go a RACE approach like insertational
: mutation genesis(not sure just my guess), or one could even use Parrallel
: sequen
|
|
|
D*a
发帖数: 6830 | 41 我觉得不用等3个月，既然小老鼠也不需要，买几只OF1，一窝生十几个的，等三个星期
，生出一窝来把尾巴一剪，一天pcr就出来了
如果是为了breeding用，那就看你需要的老鼠数量了。
假设十只老鼠都是cre+，其中三只是cre+/+, 为了省后续工作，那么等三个星期测cre
也许值得
假如自己就四五只cre，我觉得还是管他纯合杂合，所有cre都放进去breeding，先拿到
后代进行前期实验，之后再看哪只cre纯合来用于批量生产。
是不是可以这样，pcr少P几圈，找个定量的仪器差不多定量下，排除几只肯定是杂合的
就是了，剩下不能确定的去breeding等三个星期。
are
is
do
【在 c****l 的大作中提到】
: Not as easy as you thought, let's say you will got a litter of ten mice, are
: you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
: homo Cre? Since the mice are good for breeder only 6 months or so, by the
: time you figure out which is homo Cre, you are only left with 3 months. I do
: not think it is practical to do this routinely.
: RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
: cDNA, has nothing to do with insertional site analysis.
: not sure about par
|
h********n
发帖数: 4079 | 42 Can I just use genotyping primer to run qPCR on genomic DNA?
are
is
do
【在 c****l 的大作中提到】
: Not as easy as you thought, let's say you will got a litter of ten mice, are
: you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
: homo Cre? Since the mice are good for breeder only 6 months or so, by the
: time you figure out which is homo Cre, you are only left with 3 months. I do
: not think it is practical to do this routinely.
: RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
: cDNA, has nothing to do with insertional site analysis.
: not sure about par
|
c****l
发帖数: 1086 | 43 why not. check out sybr green. make sure no non-specific band or primer
dimer which will interfere with your Q-PCR readout. Usually the Q-PCR
machine will do a dissociation curve.
【在 h********n 的大作中提到】
: Can I just use genotyping primer to run qPCR on genomic DNA?
:
: are
: is
: do
|
n********k
发帖数: 2818 | 44 sure, I know RACE, that's why I said kind of like RACE...
are
is
do
【在 c****l 的大作中提到】
: Not as easy as you thought, let's say you will got a litter of ten mice, are
: you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
: homo Cre? Since the mice are good for breeder only 6 months or so, by the
: time you figure out which is homo Cre, you are only left with 3 months. I do
: not think it is practical to do this routinely.
: RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
: cDNA, has nothing to do with insertional site analysis.
: not sure about par
|
h********n
发帖数: 4079 | 45 为啥测序这么难呢? 如果我只要知道Cre插入的前后800bp的序列, 也这么难吗?
谢谢.
which
difference
breeding
roughtly
【在 c****l 的大作中提到】
: How possible?
: You basicly have 2 products in that way. You think your sequencing machine
: is sensitive enough to detect one molecule of DNA sequence.
: The only possible way is to do a "Pan-PCR" to clone the insertion site which
: could actually be very complicated and time-consuming.
: Q-PCR is able to distinguish the difference of one and two copies difference
: if you have fine pipetting skill.
: For most people, just forget about it unless you are having problem breeding
: cre/loxp sites together. Then,y
|
c****l
发帖数: 1086 | 46 tell me how do you figure out Cre插入的前后800bp的序列 or even 1 bp up or downstream?
real deal project!
【在 h********n 的大作中提到】
: 为啥测序这么难呢? 如果我只要知道Cre插入的前后800bp的序列, 也这么难吗?
: 谢谢.
:
: which
: difference
: breeding
: roughtly
|
h********n
发帖数: 4079 | 47 设计primer跟插入片段头尾分别互补, 然后分别往上和往下走, 产物测序?
downstream?
【在 c****l 的大作中提到】
: tell me how do you figure out Cre插入的前后800bp的序列 or even 1 bp up or downstream?
: real deal project!
|
D*a
发帖数: 6830 | 48 你搞清楚cre在哪里干什么？能发paper么？。。。 |
c****l
发帖数: 1086 | 49 How many copies do you think you will get by that way? Is that enough for
sequencing?
Don't you need a PAIR of primers on each side of your inserted transgene?
Without knowing the upstream or downstream sequence of your integration site
, how do you design the primer outside of your transgene?
I start doubting you really understand what is PCR.
【在 h********n 的大作中提到】
: 设计primer跟插入片段头尾分别互补, 然后分别往上和往下走, 产物测序?
:
: downstream?
|
c****l
发帖数: 1086 | 50 Sometimes, like in my case, homo cre cause neurological symptom, do you want
to figure out which gene is disrupted? Could be a potential CNS paper. :-)
【在 D*a 的大作中提到】
: 你搞清楚cre在哪里干什么？能发paper么？。。。
|
|
|
D*a
发帖数: 6830 | 51 这种情况下倒是不错啊 ;D
want
【在 c****l 的大作中提到】
: Sometimes, like in my case, homo cre cause neurological symptom, do you want
: to figure out which gene is disrupted? Could be a potential CNS paper. :-)
|
h********n
发帖数: 4079 | 52 I mean sequencing primer.
when you design primer to sequence sth, you don't need a pair of primers. So
if I design a primer from the head of insertion and go upstream to sequence
the unknown, I only need to know 100 bp sequence. then based on this 100 bp
I can design primer, one primer on transgene and one on upstream, whose
product is cross the insertion starting point, so to distinguish homo vs
hetero.
But I don't know whether the sequencing pcr can be done on genomic DNA.
BTW, when you doubt
【在 c****l 的大作中提到】
: How many copies do you think you will get by that way? Is that enough for
: sequencing?
: Don't you need a PAIR of primers on each side of your inserted transgene?
: Without knowing the upstream or downstream sequence of your integration site
: , how do you design the primer outside of your transgene?
: I start doubting you really understand what is PCR.
|
h********n
发帖数: 4079 | 53 I am thinking of running a microarray, comparing gene expression profile on
beta-actin-Cre homo, hetero and wt mice.
want
【在 c****l 的大作中提到】
: Sometimes, like in my case, homo cre cause neurological symptom, do you want
: to figure out which gene is disrupted? Could be a potential CNS paper. :-)
|
c****l
发帖数: 1086 | 54 The sequencing you mentioned needs the DNA to be certain concentration,
definitely not a few copies. Don't you need do a PCR before sequencing? What
is that step for? To increase your template copies!
So
sequence
bp
【在 h********n 的大作中提到】
: I mean sequencing primer.
: when you design primer to sequence sth, you don't need a pair of primers. So
: if I design a primer from the head of insertion and go upstream to sequence
: the unknown, I only need to know 100 bp sequence. then based on this 100 bp
: I can design primer, one primer on transgene and one on upstream, whose
: product is cross the insertion starting point, so to distinguish homo vs
: hetero.
: But I don't know whether the sequencing pcr can be done on genomic DNA.
: BTW, when you doubt
|
n********k
发帖数: 2818 | 55 that's why I suggest to kind of do a RACE approach, first priming with a Cre
primer, and then do similiar like
RACE...I don't know but I want to believe there is a way to do this since
the insertional mutagenesis has been
done, in a way, it has the same difficulity to determine the location as Cre
transgene...
What
【在 c****l 的大作中提到】
: The sequencing you mentioned needs the DNA to be certain concentration,
: definitely not a few copies. Don't you need do a PCR before sequencing? What
: is that step for? To increase your template copies!
:
: So
: sequence
: bp
|
c****l
发帖数: 1086 | 56 Are you talking about 5' RACE to let the product ligate into a loop? then
you will be able to design anther primer using known sequence. However in
the case of a transgene, it is no as simple as 5'RACE since very often the
transgene construct forms concatemers which greatly increase the complexity
of real products. there are several of these similar techniques such as "Pan
PCR", "selective circularization" or sth etc.
There are also commercial kits available.
Cre
Cre
【在 n********k 的大作中提到】
: that's why I suggest to kind of do a RACE approach, first priming with a Cre
: primer, and then do similiar like
: RACE...I don't know but I want to believe there is a way to do this since
: the insertional mutagenesis has been
: done, in a way, it has the same difficulity to determine the location as Cre
: transgene...
:
: What
|
n********k
发帖数: 2818 | 57 thanks, I c....so I guess that's not the case for insertional mutagenesis,
then? I see your points, however, still can the concatemers problem be
overcame with introducing an extra cloning step with primers designed
containing specific rare enzyme cut sites...
complexity
Pan
【在 c****l 的大作中提到】
: Are you talking about 5' RACE to let the product ligate into a loop? then
: you will be able to design anther primer using known sequence. However in
: the case of a transgene, it is no as simple as 5'RACE since very often the
: transgene construct forms concatemers which greatly increase the complexity
: of real products. there are several of these similar techniques such as "Pan
: PCR", "selective circularization" or sth etc.
: There are also commercial kits available.
:
: Cre
: Cre
|
c****l
发帖数: 1086 | 58 You can try. But you have to have restriction site inside your construct and
either 5' upstream or 3' downstream and not too far from your insertion
sites (say not more than a couple of kb. the bigger the size of your product
, the lower the efficiency of PCR)
In the simplest situation, say the concatemers are all head to tail and only
one restriction site in the construct and it is very close to your
insertion sites in the genomic DNA, you will still have 2 products.
【在 n********k 的大作中提到】
: thanks, I c....so I guess that's not the case for insertional mutagenesis,
: then? I see your points, however, still can the concatemers problem be
: overcame with introducing an extra cloning step with primers designed
: containing specific rare enzyme cut sites...
:
: complexity
: Pan
|
D*a
发帖数: 6830 | 59 想到下面几个问题:
cre周围的序列测出来之后怎么办？现在有老鼠全基因组图了？别忘了不同background
基因组不同
有计算机工具可以通过一段序列搜索全基因组（或者某染色体）来对比么？
测多长的片段可以确定说是这个位点？ |
D*a
发帖数: 6830 | 60 曾经为了考试，做过一个寻找mutation的project作业，用的是两只不同background老
鼠交配，然后通过pcr不同background的Polymorphism的marker，来计算后代的重组率
来找的。
不知道能不能用在这里，是什么的Polymorphism已经忘了。。。snp还是微卫星？你要
感兴趣我回头找找
want
【在 c****l 的大作中提到】
: Sometimes, like in my case, homo cre cause neurological symptom, do you want
: to figure out which gene is disrupted? Could be a potential CNS paper. :-)
|
|
|
n********k
发帖数: 2818 | 61 good points...nonetheless, for the most, this won't be a concern and if you
look at the whole genome mutagenesis in 129 or alike, they just sequence
about 100bp or even 30 bp, enough to determine the locus for most...
background
【在 D*a 的大作中提到】
: 想到下面几个问题:
: cre周围的序列测出来之后怎么办？现在有老鼠全基因组图了？别忘了不同background
: 基因组不同
: 有计算机工具可以通过一段序列搜索全基因组（或者某染色体）来对比么？
: 测多长的片段可以确定说是这个位点？
|
n********k
发帖数: 2818 | 62 this is very classical genome mapping...those ENU people do this all the
time before genome era and still does quite a bit too...
【在 D*a 的大作中提到】
: 曾经为了考试，做过一个寻找mutation的project作业，用的是两只不同background老
: 鼠交配，然后通过pcr不同background的Polymorphism的marker，来计算后代的重组率
: 来找的。
: 不知道能不能用在这里，是什么的Polymorphism已经忘了。。。snp还是微卫星？你要
: 感兴趣我回头找找
:
: want
|
c****l
发帖数: 1086 | 63 I am interested but sure if the approach you mention will be suitable for my
purpose. Pls let me know the principle at least. Thanks.
【在 D*a 的大作中提到】
: 曾经为了考试，做过一个寻找mutation的project作业，用的是两只不同background老
: 鼠交配，然后通过pcr不同background的Polymorphism的marker，来计算后代的重组率
: 来找的。
: 不知道能不能用在这里，是什么的Polymorphism已经忘了。。。snp还是微卫星？你要
: 感兴趣我回头找找
:
: want
|
D*a
发帖数: 6830 | 64 thanks, 请问对照有软件什么的么？还是手工去数据库比？
you
【在 n********k 的大作中提到】
: good points...nonetheless, for the most, this won't be a concern and if you
: look at the whole genome mutagenesis in 129 or alike, they just sequence
: about 100bp or even 30 bp, enough to determine the locus for most...
:
: background
|
d****0
发帖数: 90 | 65 feels like virtually impossible. I had discussion on similar operation long
time ago with some expert, he told me it would be very hard, if not
impposible, to get your positive line. just dont remember the detail he gave
. |
D*a
发帖数: 6830 | 66 用的是QTL，quantitative trait loci
pubmed搜qtl mapping 可以搜出来一堆
找了找我上课的几张图，看看能不能发到你邮箱
工作量很大啊。。整天pcr好无聊
my
【在 c****l 的大作中提到】
: I am interested but sure if the approach you mention will be suitable for my
: purpose. Pls let me know the principle at least. Thanks.
|
c****l
发帖数: 1086 | 67 Thank you very much. I understand QTL, very traditional gene mapping
approach. Do we really have to to this in a post-genomic era plus we know
the insertion sequence?
【在 D*a 的大作中提到】
: 用的是QTL，quantitative trait loci
: pubmed搜qtl mapping 可以搜出来一堆
: 找了找我上课的几张图，看看能不能发到你邮箱
: 工作量很大啊。。整天pcr好无聊
:
: my
|
D*a
发帖数: 6830 | 68 如果ls几位说的测序能做的话还是测序快吧
很久没有追踪这方面的进展了，我也不知道发展到什么程度了:)
【在 c****l 的大作中提到】
: Thank you very much. I understand QTL, very traditional gene mapping
: approach. Do we really have to to this in a post-genomic era plus we know
: the insertion sequence?
|
h********n
发帖数: 4079 | 69 design primer for homo vs hetero?
background
【在 D*a 的大作中提到】
: 想到下面几个问题:
: cre周围的序列测出来之后怎么办？现在有老鼠全基因组图了？别忘了不同background
: 基因组不同
: 有计算机工具可以通过一段序列搜索全基因组（或者某染色体）来对比么？
: 测多长的片段可以确定说是这个位点？
|
h********n
发帖数: 4079 | 70 I agree, spend some money and get sequence.
【在 D*a 的大作中提到】
: 如果ls几位说的测序能做的话还是测序快吧
: 很久没有追踪这方面的进展了，我也不知道发展到什么程度了:)
|
|
|
c****l
发帖数: 1086 | 71 Do you mean whole genome sequencing, r u insane?
【在 h********n 的大作中提到】
: I agree, spend some money and get sequence.
|
c****l
发帖数: 1086 | 72 How do you sequence? Where to start? I have spent so many posts on this, no
easy way sequencing unless you are talking about whole genome sequencing.
【在 D*a 的大作中提到】
: 如果ls几位说的测序能做的话还是测序快吧
: 很久没有追踪这方面的进展了，我也不知道发展到什么程度了:)
|
c********o
发帖数: 135 | 73 即使是一个copy的Cre转基因也应该有control。记得nature发过一篇关于Cre的
toxicity的paper，大致是因为很多转基因的Cre都是很强的promoter驱动的，会影响插
入位点附近的基因表达。所以floxed/floxed并不是floxed/floxed;Cre的最好control
，应该是floxed/wt,Cre。
现在有一些cre是knockin在promoter下游的，至少位置是清楚的。另外一个办法是
knockin在Rosa26的位置。
Homo Cre有phenotype是挺有意思的，原来隔壁实验室就有过，但花了很多时间想搞清
楚插入位点，但最后是不了了之了。 |
h********n
发帖数: 4079 | 74 not whole genome. I am talking with the facility and try to have them work
for me.
【在 c****l 的大作中提到】
: Do you mean whole genome sequencing, r u insane?
|
