p****z
发帖数: 31 | 1 最近很是郁闷，做啥都不顺！又碰上一问题，向大家请教。有一个和癌症相关的蛋白，
它的功能最近被发现能促进cell proliferation，这一现象被好几个group报到了并且
在不同的肿瘤细胞中都是相同的作用。他们用的方法都是使用siRNA knock-down这个基
因，然后用MTT assay 或者是thymidine incoporation assay去测cell proliferation
.我为了方便，就用了lentivirus (pLKO.1 based vector, open biosystem)做了
stable cell line,试了两个细胞系，knock-down 效果都不错，从western 上来看，
估计有80%-90%的reduction. 可是一测cell proliferation ,cell proliferation的抑
制效果都不明显，我用了两种方法：promega的MTS assay 和 BrdU incoporation
assay.这到底是怎么回事啊？我看了他们做的方法，最大的差别就是他们是transient
transfection,而我是stable cell line.这个会导致差别吗？谢谢大家 |
n********k
发帖数: 2818 | 2 This is indeed a typical problem with stable clones/cell lines...not sure
whether you are using clones or using mixtures...for your purpose you likely
better off working with transient infection....With stable lines, one could
virtually get whatever one wants if one is not careful enough and not using
many lines in parallel...
proliferation
transient
【在 p****z 的大作中提到】
: 最近很是郁闷，做啥都不顺！又碰上一问题，向大家请教。有一个和癌症相关的蛋白，
: 它的功能最近被发现能促进cell proliferation，这一现象被好几个group报到了并且
: 在不同的肿瘤细胞中都是相同的作用。他们用的方法都是使用siRNA knock-down这个基
: 因，然后用MTT assay 或者是thymidine incoporation assay去测cell proliferation
: .我为了方便，就用了lentivirus (pLKO.1 based vector, open biosystem)做了
: stable cell line,试了两个细胞系，knock-down 效果都不错，从western 上来看，
: 估计有80%-90%的reduction. 可是一测cell proliferation ,cell proliferation的抑
: 制效果都不明显，我用了两种方法：promega的MTS assay 和 BrdU incoporation
: assay.这到底是怎么回事啊？我看了他们做的方法，最大的差别就是他们是transient
: transfection,而我是stable cell line.这个会导致差别吗？谢谢大家
|
p****z
发帖数: 31 | 3 我用的是mixture,pooled after puromycin selection. 你的意思是还是用transient?
我是觉得很奇怪，这个差别怎么会这么大。谢谢！
likely
could
using
【在 n********k 的大作中提到】
: This is indeed a typical problem with stable clones/cell lines...not sure
: whether you are using clones or using mixtures...for your purpose you likely
: better off working with transient infection....With stable lines, one could
: virtually get whatever one wants if one is not careful enough and not using
: many lines in parallel...
:
: proliferation
: transient
|
L**********O
发帖数: 1761 | 4 我也是做lentivirus (pLKO.1 based vector, open biosystem)。。然后puromycin
select...
你如果用和他们一模一样的测proliferation方法还是得不到相同结果吗？
按理来说，stable cell line,敲除的更稳定，应该结果更加好才对，是不？
proliferation
transient
【在 p****z 的大作中提到】
: 最近很是郁闷，做啥都不顺！又碰上一问题，向大家请教。有一个和癌症相关的蛋白，
: 它的功能最近被发现能促进cell proliferation，这一现象被好几个group报到了并且
: 在不同的肿瘤细胞中都是相同的作用。他们用的方法都是使用siRNA knock-down这个基
: 因，然后用MTT assay 或者是thymidine incoporation assay去测cell proliferation
: .我为了方便，就用了lentivirus (pLKO.1 based vector, open biosystem)做了
: stable cell line,试了两个细胞系，knock-down 效果都不错，从western 上来看，
: 估计有80%-90%的reduction. 可是一测cell proliferation ,cell proliferation的抑
: 制效果都不明显，我用了两种方法：promega的MTS assay 和 BrdU incoporation
: assay.这到底是怎么回事啊？我看了他们做的方法，最大的差别就是他们是transient
: transfection,而我是stable cell line.这个会导致差别吗？谢谢大家
|
p****z
发帖数: 31 | 5 是啊，这就是我百思不得其解的地方。一开始用promega的MTS assay，怕因为要算细胞
数目导致误差，后来就采用BrdU proliferation assay,结果还是相差不大。当时买的
shRNA sets里面有5个clone,选了一个效果最好的。郁闷啊！ |
n********k
发帖数: 2818 | 6 Have you tested proliferation 24-96 hs after infections...do u have cell
death? or how many passages has it been? a fast population could easily take
over and mask the effect with the process/time of making stable cell lines.
..With good training, these are pretty much basics one shall know/watch for
when making stable lines...
transient?
【在 p****z 的大作中提到】
: 我用的是mixture,pooled after puromycin selection. 你的意思是还是用transient?
: 我是觉得很奇怪，这个差别怎么会这么大。谢谢！
:
: likely
: could
: using
|
n********k
发帖数: 2818 | 7 All that said, in no means I am saying it is not possible that the
published results were wrong or somehow not reproducible at your hands for
other reasons...BTW, I would not just use 1shRNA for this kind of in vitro
analysis...it is so easy to work with several...
take
lines.
for
【在 n********k 的大作中提到】
: Have you tested proliferation 24-96 hs after infections...do u have cell
: death? or how many passages has it been? a fast population could easily take
: over and mask the effect with the process/time of making stable cell lines.
: ..With good training, these are pretty much basics one shall know/watch for
: when making stable lines...
:
: transient?
|
p****z
发帖数: 31 | 8 谢谢你的答复。我是在筛选2周后，细胞不怎么死后开始做的。这个期间我传了两次，
避免长太多。你说的对，我应该在感染后不用筛选后直接开始做。对于你说的 “a
fast population could easily take over and mask the effect with the process/
time of making stable cell lines”能在多解释一点吗？谢谢
take
lines.
for
【在 n********k 的大作中提到】
: Have you tested proliferation 24-96 hs after infections...do u have cell
: death? or how many passages has it been? a fast population could easily take
: over and mask the effect with the process/time of making stable cell lines.
: ..With good training, these are pretty much basics one shall know/watch for
: when making stable lines...
:
: transient?
|
p****z
发帖数: 31 | 9 恩，我也在考虑再用一个，不过其他四个reduction的效果都不大怎么好。
【在 n********k 的大作中提到】
: All that said, in no means I am saying it is not possible that the
: published results were wrong or somehow not reproducible at your hands for
: other reasons...BTW, I would not just use 1shRNA for this kind of in vitro
: analysis...it is so easy to work with several...
:
: take
: lines.
: for
|
p****z
发帖数: 31 | 10 我们实验室我是第一个开始做功能的，没人可以问。其他实验室做这方面的也很少，没
有人可以交流，只能瞎做了。 |
|
|
n********k
发帖数: 2818 | 11 it is not a pure population and even if it is, no all cells will respond
equally to the knockdown...so A, B, C, D maybe, so grow slow or die, but E
may be not, grow fast, so it will take over your culture, right?
Anyhow, if you have cell death(sounds u do), in no way, you shall do the
stable line unless you are very experienced/or know what you are dealing
with or death cause...With lentif, it would be better to get high infection
efficiency rather than to go with drug selection if you have the option...
you shall try to find some one experienced around to help you...
process/
【在 p****z 的大作中提到】
: 谢谢你的答复。我是在筛选2周后，细胞不怎么死后开始做的。这个期间我传了两次，
: 避免长太多。你说的对，我应该在感染后不用筛选后直接开始做。对于你说的 “a
: fast population could easily take over and mask the effect with the process/
: time of making stable cell lines”能在多解释一点吗？谢谢
:
: take
: lines.
: for
|
p****z
发帖数: 31 | 12 恩，这个解释确实很合理。非常感谢，我用transient 试试看。
E
infection
【在 n********k 的大作中提到】
: it is not a pure population and even if it is, no all cells will respond
: equally to the knockdown...so A, B, C, D maybe, so grow slow or die, but E
: may be not, grow fast, so it will take over your culture, right?
: Anyhow, if you have cell death(sounds u do), in no way, you shall do the
: stable line unless you are very experienced/or know what you are dealing
: with or death cause...With lentif, it would be better to get high infection
: efficiency rather than to go with drug selection if you have the option...
: you shall try to find some one experienced around to help you...
:
: process/
|
d*p
发帖数: 534 | 13 Do you have a empty vector as control? I don't like puromycin. All of my
shRNAs were cloned into plko.3G. GFP will tell you the infection rate. And
you may sort the GFP positive cells. |
S****r
发帖数: 982 | 14 I think the sequence of shRNA you were using differs from reported siRNA,
and it's often that some sequences of sh/siRNA affect on cell proliferation,
presumbly targeting other proteins. |
w*****n
发帖数: 310 | 15 你挑单克隆,想要什么样的结果都能做到.stable cell line应该用来做pathway看上下
游protein的变化。
proliferation
transient
【在 p****z 的大作中提到】
: 最近很是郁闷，做啥都不顺！又碰上一问题，向大家请教。有一个和癌症相关的蛋白，
: 它的功能最近被发现能促进cell proliferation，这一现象被好几个group报到了并且
: 在不同的肿瘤细胞中都是相同的作用。他们用的方法都是使用siRNA knock-down这个基
: 因，然后用MTT assay 或者是thymidine incoporation assay去测cell proliferation
: .我为了方便，就用了lentivirus (pLKO.1 based vector, open biosystem)做了
: stable cell line,试了两个细胞系，knock-down 效果都不错，从western 上来看，
: 估计有80%-90%的reduction. 可是一测cell proliferation ,cell proliferation的抑
: 制效果都不明显，我用了两种方法：promega的MTS assay 和 BrdU incoporation
: assay.这到底是怎么回事啊？我看了他们做的方法，最大的差别就是他们是transient
: transfection,而我是stable cell line.这个会导致差别吗？谢谢大家
|
h********n
发帖数: 4079 | 16 It is possible that the stable clone/pools of cells were already selected.
Those grow-advanced cells become donimate |
l********7
发帖数: 175 | 17 you can pick up 5-10 single clones and check the proliferation of these
clones. or you can rescue the knowdown cell line with the method that
Xiaodong has used in their cell paper. |
i*****g
发帖数: 11893 | 18 用siRNA吧，
stable cell line 很难说，或许integration site effect，考虑Rosa， Flipin之类
的细胞系site specific
以前我做过stable cell line immunostaning，发现里面明显几个表达程度不一的克隆 |
p****z
发帖数: 31 | 19 恩，是啊，我原本的想法也是这样，但是phenotype重复不出来，还能继续做吗？
【在 w*****n 的大作中提到】
: 你挑单克隆,想要什么样的结果都能做到.stable cell line应该用来做pathway看上下
: 游protein的变化。
:
: proliferation
: transient
|
p****z
发帖数: 31 | 20 恩。我觉得这个是最有可能的原因。
【在 h********n 的大作中提到】
: It is possible that the stable clone/pools of cells were already selected.
: Those grow-advanced cells become donimate
|
p****z
发帖数: 31 | 21 我觉得rescue暂时没有必要了，wild type 和knock-down都长一样快了。
【在 l********7 的大作中提到】
: you can pick up 5-10 single clones and check the proliferation of these
: clones. or you can rescue the knowdown cell line with the method that
: Xiaodong has used in their cell paper.
|
p****z
发帖数: 31 | 22 因为knock-down后，用的细胞要比较多，siRNA比较不现实。
【在 i*****g 的大作中提到】
: 用siRNA吧，
: stable cell line 很难说，或许integration site effect，考虑Rosa， Flipin之类
: 的细胞系site specific
: 以前我做过stable cell line immunostaning，发现里面明显几个表达程度不一的克隆
|
