j********r
发帖数: 156 | 1 Can i ask a question?
Primary MEFs (p2) were infected with lentivirus (FUW-OSKM, the one made by
the mit group) twice, and on
the third day trypsinized and seeded at low density on rMEF seeder cells.
Cells were then incubated with
knockout-serum replacement (ksr) medium supplemented with LIF and others,
medium was refreshed every
two days. Now it is about 2 wks after the initial infection, there is no ES-
like colonies in the well, but many
round single cells. Having no idea what to do next, app |
o**i
发帖数: 1165 | 2 拿MEF做iPS很容易吧
没做过
你知道你infect的MOI麽？
ES-
【在 j********r 的大作中提到】
: Can i ask a question?
: Primary MEFs (p2) were infected with lentivirus (FUW-OSKM, the one made by
: the mit group) twice, and on
: the third day trypsinized and seeded at low density on rMEF seeder cells.
: Cells were then incubated with
: knockout-serum replacement (ksr) medium supplemented with LIF and others,
: medium was refreshed every
: two days. Now it is about 2 wks after the initial infection, there is no ES-
: like colonies in the well, but many
: round single cells. Having no idea what to do next, app
|
J********3
发帖数: 3151 | 3 消化下来，再种到feeder上，估计几天就可以出客轮啦
ES-
【在 j********r 的大作中提到】
: Can i ask a question?
: Primary MEFs (p2) were infected with lentivirus (FUW-OSKM, the one made by
: the mit group) twice, and on
: the third day trypsinized and seeded at low density on rMEF seeder cells.
: Cells were then incubated with
: knockout-serum replacement (ksr) medium supplemented with LIF and others,
: medium was refreshed every
: two days. Now it is about 2 wks after the initial infection, there is no ES-
: like colonies in the well, but many
: round single cells. Having no idea what to do next, app
|
j********r
发帖数: 156 | 4 Thanks a lot for the valuable information by JiangZM123. If reseeding will
generate ES-like clones, could you share your explanation or theory? Very
appreciating the great help! |
J********3
发帖数: 3151 | 5 我也不能保证一定行哈。俺以前看这个热门，拿MEF练过手，用的非病毒载体，转染后
加药筛克隆，再把挑选的阳性克隆种到feeder上，然后用ES培养基诱导，结果挑选的48
个克隆中11个快得不到一周，慢的两周就长出ES样克隆，AP，SSEA-1染色阳性，也可以
分化为很好的EBs，确实基本是细胞形态发生变化的那些克隆可以诱导出iPS。可能是现
在的几个诱导基因组合远非最佳组合，feeder可以分泌一些细胞因子，维持干细胞状态
，可以促进iPS诱导？俺发现iPS和ES还是有很多不同之处，不如ES好使，就没再关注过
iPS，具体的东东了解有限。
【在 j********r 的大作中提到】
: Thanks a lot for the valuable information by JiangZM123. If reseeding will
: generate ES-like clones, could you share your explanation or theory? Very
: appreciating the great help!
|
j********r
发帖数: 156 | 6 Admiring yi xia! It seems that your efficiency is much higher than the two
NS papers using non-viral vector. Just curious which MEF did you use (to
enable transfection and drug selection)? I also tried plasmid transfection (
via lipids) in primary MEF (at passage 2), however, the transfection is
extremely low. Thanks for the great information! |
J********3
发帖数: 3151 | 7 我用的pCAG2LMKOSimO，MEF确实转染效率很低，但选的是stable lines，也无所谓了，
大不了多用点细胞，俺用0.6mg/ml的G418筛十几天，挑的客轮，效果还可以。MEF是俺
自己做的，immortalized MEF比primary MEF要容易些（俺用p3开始转染成功，p2应该
没问题），但个人感觉immortalized MEF诱导的iPS没多大用，因为在漫长的
immortalize过程中发生了n多的事情，细胞基因组本身已不大稳定，iPS估计很容易癌
变。
(
【在 j********r 的大作中提到】
: Admiring yi xia! It seems that your efficiency is much higher than the two
: NS papers using non-viral vector. Just curious which MEF did you use (to
: enable transfection and drug selection)? I also tried plasmid transfection (
: via lipids) in primary MEF (at passage 2), however, the transfection is
: extremely low. Thanks for the great information!
|
j********r
发帖数: 156 | 8 多谢帮助！巧的是我最初从addgene买的两个质粒其中一个就是pCAG2LMKOSimO。在293T
中满眼的红色，在primary MEF中啥荧光都没有。请问你G418拿到的克隆显微镜下有荧
光吗？原来primary MEF也能长十几天，我也来试试。Thanks a lot, |
J********3
发帖数: 3151 | 9 我们也是从addgene买的，因为没认真做这个，就是大概齐建立个方法以备将来用，还
真没观测过荧光，不过293T跟MEF表达水平差太远，肉眼几乎看不见荧光也正常，俺以
前用U2OS做GFP-tag的稳定细胞株，初期能看见很绿，到挑克隆时也基本看不清了，但
用FACS能sort。俺的primary MEF是3/48的iPS，比永生MEF少几倍，俺们lab另一个人是
0/96，俺也不大清楚他为啥就不灵。
实在不行，可以把培养基里加点GSK-3 inhibitor，俺加了2i，90%以上的胚ICM能诱导
出ES来，不加的话，也就30-50%的胚ICM能诱导出ES来，这个不是简单的两三倍差别，
应该是非常大的差别。
293T
【在 j********r 的大作中提到】
: 多谢帮助！巧的是我最初从addgene买的两个质粒其中一个就是pCAG2LMKOSimO。在293T
: 中满眼的红色，在primary MEF中啥荧光都没有。请问你G418拿到的克隆显微镜下有荧
: 光吗？原来primary MEF也能长十几天，我也来试试。Thanks a lot,
|
f*******e
发帖数: 354 | 10 is your virus OK?
【在 o**i 的大作中提到】
: 拿MEF做iPS很容易吧
: 没做过
: 你知道你infect的MOI麽？
:
: ES-
|
j********r
发帖数: 156 | 11 An update - 10 days after seeding infected cell on feeder MEF (or 13 days
after initial infection), I found one ES-like clone. If it is IPS, the
efficiency is extremely low (one clone from 50000 cells seeded).
The virus might be the reason for that, but I am not sure about the quality
or titer of the virus supernatant I made from 293T cells (the ratio of OSKM
to CMV delta 8.91 to pMD2-VSVG is 4:3:1). Also this time I used frozen sup.
I will try fresh one next time.
The purpose of this experiment |
