m******5
发帖数: 1383 | 1 如标题，这是第一个问题
第二个问题是，大家用在mES cell里通过转染Flpe表达载体的方式delete过FRT
flanked 区域么？ | f*********6
发帖数: 94 | 2 Yes, you can derive ES cell lines from the mixed genetic bg, but the
efficiency is pretty low. You need to be patient and persistent. It took me
three months to get my 1st ES cell line. After that, things become really
easy. My boss almost fired me..., but then she found out the mouse line was
on a mixed genetic bg (we found one white offspring) and she apologized to
me.
You may want to add the ERK inhibitor PD184352 to improve efficiency. ref: http://dev.biologists.org/content/137/20/3351.full.pdf
For the 2nd question, i have not tried the method, but i know others use it
and it works fine. | m******5
发帖数: 1383 | 3 Thank you !!!! for the ERK inhibitor, I supposed you were talking about 2i
medium? I thought I would need more than ERK, maybe also p38 inhibitor?
Could you coomment more on which part is the most tricky part?
Thank you very very much!
me
was
http://dev.biologists.org/content/137/20/3351.full.pdf
it
【在 f*********6 的大作中提到】
: Yes, you can derive ES cell lines from the mixed genetic bg, but the
: efficiency is pretty low. You need to be patient and persistent. It took me
: three months to get my 1st ES cell line. After that, things become really
: easy. My boss almost fired me..., but then she found out the mouse line was
: on a mixed genetic bg (we found one white offspring) and she apologized to
: me.
: You may want to add the ERK inhibitor PD184352 to improve efficiency. ref: http://dev.biologists.org/content/137/20/3351.full.pdf
: For the 2nd question, i have not tried the method, but i know others use it
: and it works fine.
| f*********6
发帖数: 94 | 4 You are the most welcome.
I only used the ERK inhibitor, and that was several years ago. The inhibitor
did improve the derivation efficiency. Having the p38 inhibitor in the
medium might help. You may also want to be sure that LIF is working well,
and i think this is very important.
The most tricky part to me is the patience. If you don't see any colony at
the first a few days, don't give up and just wait for a few more days.
Meanwhile set up more crossing in case that batch does not work, and then
try it again. When the inner cell mass hatched, use trypsine to break the
epiblasts into clusters of cells (2-4cell cluster), not single cell, and
then transfer them to a new 48-well/24-well plate without any extraembryonic
tissue. The extraembryonic tissue promotes epiblast differentiation.
Good luck and have fun! | m******5
发帖数: 1383 | 5 Thank you!!! I am having fun.
BTW, I was a little confused when you say epiblast
I thought epiblast cells are already further differentiated than ICM cells
inhibitor
【在 f*********6 的大作中提到】
: You are the most welcome.
: I only used the ERK inhibitor, and that was several years ago. The inhibitor
: did improve the derivation efficiency. Having the p38 inhibitor in the
: medium might help. You may also want to be sure that LIF is working well,
: and i think this is very important.
: The most tricky part to me is the patience. If you don't see any colony at
: the first a few days, don't give up and just wait for a few more days.
: Meanwhile set up more crossing in case that batch does not work, and then
: try it again. When the inner cell mass hatched, use trypsine to break the
: epiblasts into clusters of cells (2-4cell cluster), not single cell, and
| f*********6
发帖数: 94 | 6 Epiblast is a loose term to define undifferentiated pluripotent stem cells
in ICM.
Here is an excerpt from the review I suggested you to read:
...It is expressed in the 8- to 16-cell morula but becomes
restricted to the epiblast cells of the ICM in the expanded
blastocyst, and has been shown to be under the direct regulation of
the pluripotency factors Oct4 (Pou5f1) and Sox2 (Yuan et al.,
1995).
I will strongly suggest you to read the review if you have not done so. It
is a very well-written review, and you can find useful references within. |
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