l**********n 发帖数: 240 | 1 我在这方面是新手,先谢大伙帮忙。
用pGIPZ shRNA plasmid 转染细胞后,用puromycin select, 刚开始细胞有GFP表达,但是传代几
次后,细胞就没有GFP荧光了。但是仍然对puromycin 抵抗。
这种情况下 shRNA 还表达吗?
plasmid contains: CMV promotor followed by GFP, IRES, PuroResistance, and
shRNA.
http://www.openbiosystems.com/RNAi/shRNAmirLibraries/GIPZLentiviralshRNAmir/
是不是因为有IRES,PuromycinResistance 蛋白被高效翻译,而影响GFP的翻译? |
l**********n 发帖数: 240 | 2 自己顶。
,但是传代几
【在 l**********n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我在这方面是新手,先谢大伙帮忙。 : 用pGIPZ shRNA plasmid 转染细胞后,用puromycin select, 刚开始细胞有GFP表达,但是传代几 : 次后,细胞就没有GFP荧光了。但是仍然对puromycin 抵抗。 : 这种情况下 shRNA 还表达吗? : plasmid contains: CMV promotor followed by GFP, IRES, PuroResistance, and : shRNA. : http://www.openbiosystems.com/RNAi/shRNAmirLibraries/GIPZLentiviralshRNAmir/ : 是不是因为有IRES,PuromycinResistance 蛋白被高效翻译,而影响GFP的翻译?
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s******y 发帖数: 28562 | 3 很多细胞会变异而产生对puro的抵抗力的,尤其如果你用的是癌细胞的话
我做类似试验的时候,都是在用药一个星期后必须用荧光sorting
一此
,但是传代几
【在 l**********n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我在这方面是新手,先谢大伙帮忙。 : 用pGIPZ shRNA plasmid 转染细胞后,用puromycin select, 刚开始细胞有GFP表达,但是传代几 : 次后,细胞就没有GFP荧光了。但是仍然对puromycin 抵抗。 : 这种情况下 shRNA 还表达吗? : plasmid contains: CMV promotor followed by GFP, IRES, PuroResistance, and : shRNA. : http://www.openbiosystems.com/RNAi/shRNAmirLibraries/GIPZLentiviralshRNAmir/ : 是不是因为有IRES,PuromycinResistance 蛋白被高效翻译,而影响GFP的翻译?
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l**********n 发帖数: 240 | 4 我用的是从成年大鼠分离的血管平滑肌细胞, Passage 1-3. 这种原代细胞变异可能性
大吗?
【在 s******y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 很多细胞会变异而产生对puro的抵抗力的,尤其如果你用的是癌细胞的话 : 我做类似试验的时候,都是在用药一个星期后必须用荧光sorting : 一此 : : ,但是传代几
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s******y 发帖数: 28562 | 5 No, those cells should not have big probability to mutate
Anyway I would still suggest you to do a sorting if possible (if your cells
can survive sorting).
【在 l**********n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我用的是从成年大鼠分离的血管平滑肌细胞, Passage 1-3. 这种原代细胞变异可能性 : 大吗?
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n********k 发帖数: 2818 | 6 promotor silencing is more common than what people might appreciate, it is
very common for one to have
resistent clones but without overexpression, in ur case would be shRNA, so u
would have to do western/rt-
pcr to check it out, as well, it is rare to keep viral infected cells as
stable line because it is widely
recoginzed/believed that the expression goes down dramatically after a
couple of passages...the same goes
with tranfected cells...so the rule of thumb in general is to not to use
them a
【在 l**********n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我在这方面是新手,先谢大伙帮忙。 : 用pGIPZ shRNA plasmid 转染细胞后,用puromycin select, 刚开始细胞有GFP表达,但是传代几 : 次后,细胞就没有GFP荧光了。但是仍然对puromycin 抵抗。 : 这种情况下 shRNA 还表达吗? : plasmid contains: CMV promotor followed by GFP, IRES, PuroResistance, and : shRNA. : http://www.openbiosystems.com/RNAi/shRNAmirLibraries/GIPZLentiviralshRNAmir/ : 是不是因为有IRES,PuromycinResistance 蛋白被高效翻译,而影响GFP的翻译?
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l**********n 发帖数: 240 | 7 非常感谢你的帮助!
cells
【在 s******y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : No, those cells should not have big probability to mutate : Anyway I would still suggest you to do a sorting if possible (if your cells : can survive sorting).
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l**********n 发帖数: 240 | 8 Thanks very much for your help!
I will prepare new batch of cells for infection and avoid passaging
cells after infection.
u
【在 n********k 的大作中提到】![](/moin_static193/solenoid/img/up.png) : promotor silencing is more common than what people might appreciate, it is : very common for one to have : resistent clones but without overexpression, in ur case would be shRNA, so u : would have to do western/rt- : pcr to check it out, as well, it is rare to keep viral infected cells as : stable line because it is widely : recoginzed/believed that the expression goes down dramatically after a : couple of passages...the same goes : with tranfected cells...so the rule of thumb in general is to not to use : them a
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s******y 发帖数: 28562 | 9 我被你的其中一个说法吓了一跳,你说病毒转染的细胞不适合于做stable
cell line? 那大家都是怎么作stable cell line 的呀?
尤其是很多细胞不是那么容易直接用质粒转染的,比方说我们实验室用的
fibroblast, B cell等等,不用病毒几乎就没有其他办法了。
而我们又特别不喜欢挑单克隆,因为我们一般来讲,转染细胞是为了做
phenotype rescue的,如果用单克隆的话不要说reviewer, 连我们自己
都不能说服。
我们的确注意到retro virus 转染的细胞过一阵子后荧光会变少。
我们的解决办法是把头几批细胞经过sorting 后再养一天,然后统统冻起来,
只用那几批细胞。不知道这么行不?能不能说服reviewer? 或者你们实验室
有更好的方法?
u
【在 n********k 的大作中提到】![](/moin_static193/solenoid/img/up.png) : promotor silencing is more common than what people might appreciate, it is : very common for one to have : resistent clones but without overexpression, in ur case would be shRNA, so u : would have to do western/rt- : pcr to check it out, as well, it is rare to keep viral infected cells as : stable line because it is widely : recoginzed/believed that the expression goes down dramatically after a : couple of passages...the same goes : with tranfected cells...so the rule of thumb in general is to not to use : them a
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p**i 发帖数: 3525 | 10 不是说用几个独立的单克隆最有说服力吗?
【在 s******y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我被你的其中一个说法吓了一跳,你说病毒转染的细胞不适合于做stable : cell line? 那大家都是怎么作stable cell line 的呀? : 尤其是很多细胞不是那么容易直接用质粒转染的,比方说我们实验室用的 : fibroblast, B cell等等,不用病毒几乎就没有其他办法了。 : 而我们又特别不喜欢挑单克隆,因为我们一般来讲,转染细胞是为了做 : phenotype rescue的,如果用单克隆的话不要说reviewer, 连我们自己 : 都不能说服。 : 我们的确注意到retro virus 转染的细胞过一阵子后荧光会变少。 : 我们的解决办法是把头几批细胞经过sorting 后再养一天,然后统统冻起来, : 只用那几批细胞。不知道这么行不?能不能说服reviewer? 或者你们实验室
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n********k 发帖数: 2818 | 11 unless in special occasions/needed/desired(say u don't want the high
expression etc), what's the point making stable cell lines if you use
retroviral approach...I(many if not most) rarely keep them as stable lines..
..Nolan's lab is an excellent place for viral stuff...Yes, u can keep them
as stable cell lines,but you would have to take much precautions...single
clones is an old old(updated too) approach...of course u cannot do one
clones, u need at least dozens of clones intially and then narro
【在 s******y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我被你的其中一个说法吓了一跳,你说病毒转染的细胞不适合于做stable : cell line? 那大家都是怎么作stable cell line 的呀? : 尤其是很多细胞不是那么容易直接用质粒转染的,比方说我们实验室用的 : fibroblast, B cell等等,不用病毒几乎就没有其他办法了。 : 而我们又特别不喜欢挑单克隆,因为我们一般来讲,转染细胞是为了做 : phenotype rescue的,如果用单克隆的话不要说reviewer, 连我们自己 : 都不能说服。 : 我们的确注意到retro virus 转染的细胞过一阵子后荧光会变少。 : 我们的解决办法是把头几批细胞经过sorting 后再养一天,然后统统冻起来, : 只用那几批细胞。不知道这么行不?能不能说服reviewer? 或者你们实验室
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p**i 发帖数: 3525 | 12 直接用质粒转的stable line呢,好些吗?
u
【在 n********k 的大作中提到】![](/moin_static193/solenoid/img/up.png) : promotor silencing is more common than what people might appreciate, it is : very common for one to have : resistent clones but without overexpression, in ur case would be shRNA, so u : would have to do western/rt- : pcr to check it out, as well, it is rare to keep viral infected cells as : stable line because it is widely : recoginzed/believed that the expression goes down dramatically after a : couple of passages...the same goes : with tranfected cells...so the rule of thumb in general is to not to use : them a
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n********k 发帖数: 2818 | 13 BTW, the way u are doing is likely fine...within several passage is fine or
as long as they are well-chaterized, it is fine...but is it that hard to go
retroviral and treat them as transient approach...I rarely keep any stable
cell lines now...And for many studies, it is impossible or not right to keep
stable cell lines because the manipulation cause them to change...for
example differentiation...so u are screening against the correct phenotypes.
..anyway, nothing is absolute and it all depends.
【在 s******y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我被你的其中一个说法吓了一跳,你说病毒转染的细胞不适合于做stable : cell line? 那大家都是怎么作stable cell line 的呀? : 尤其是很多细胞不是那么容易直接用质粒转染的,比方说我们实验室用的 : fibroblast, B cell等等,不用病毒几乎就没有其他办法了。 : 而我们又特别不喜欢挑单克隆,因为我们一般来讲,转染细胞是为了做 : phenotype rescue的,如果用单克隆的话不要说reviewer, 连我们自己 : 都不能说服。 : 我们的确注意到retro virus 转染的细胞过一阵子后荧光会变少。 : 我们的解决办法是把头几批细胞经过sorting 后再养一天,然后统统冻起来, : 只用那几批细胞。不知道这么行不?能不能说服reviewer? 或者你们实验室
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n********k 发帖数: 2818 | 14 I would go for either inducible clones or go viral approach but as transient
approaches...单克隆 is out of dated generally speaking though. and in many
occasions, to have stable clones is not right or simply impossible. and it
depends on reviewers too...I would say most of reviewers will buy viral
approach as transient approach while some hates single clones unless it is
very well characterized with many clones...if one is not careful enough, one
could get single clones of whatever desired phenotyp
【在 p**i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 不是说用几个独立的单克隆最有说服力吗?
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n********k 发帖数: 2818 | 15 likely yes, but never compare that(besides the normal silencing, retroviral
has extra silencing effects)...but for single clones, u are picking those
with good/reasonable expressions, so there isn't a real issue in term of
promoter silencing because you already exclude those when u pick the clones..
【在 p**i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 直接用质粒转的stable line呢,好些吗? : : u
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p**i 发帖数: 3525 | 16 xiexie,很有道理。那如何做才是现代的最有说服力的细胞实验呢? 我们一般是加例如
GFP-tag,
然后用retroviruses转染建立几个细胞系,然后看接受信号后的蛋白移动。
inducible clones 是否比较不过时有说服力?
transient
many
one
【在 n********k 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I would go for either inducible clones or go viral approach but as transient : approaches...单克隆 is out of dated generally speaking though. and in many : occasions, to have stable clones is not right or simply impossible. and it : depends on reviewers too...I would say most of reviewers will buy viral : approach as transient approach while some hates single clones unless it is : very well characterized with many clones...if one is not careful enough, one : could get single clones of whatever desired phenotyp
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n********k 发帖数: 2818 | 17 don't know, I haven't followed closely lately with this as I do mostly in
vivo now...in vitro is just for confirmation or mechanistic probing...for ur
case, it is likely less a problem...I am more talking about in term of
phenotypical changes...BTW, as I said earlier, I don't see why you have to
use stable cell lines if with retroviral...
【在 p**i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : xiexie,很有道理。那如何做才是现代的最有说服力的细胞实验呢? 我们一般是加例如 : GFP-tag, : 然后用retroviruses转染建立几个细胞系,然后看接受信号后的蛋白移动。 : inducible clones 是否比较不过时有说服力? : : transient : many : one
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s******y 发帖数: 28562 | 18 很多场合下我们没有别的选择阿
另外,逆转录病毒载体也有高表达的promoter的。比方说pQC vector
我们有时候不喜欢选克隆,是因为我们做的是癌症,而癌症细胞本身就是
一个很复杂的群体,如果不是整个群体都得到一定程度的rescue我们会
感到害怕下结论,如果只挑几十个克隆我们会担心的。
..
experiment,
【在 n********k 的大作中提到】![](/moin_static193/solenoid/img/up.png) : unless in special occasions/needed/desired(say u don't want the high : expression etc), what's the point making stable cell lines if you use : retroviral approach...I(many if not most) rarely keep them as stable lines.. : ..Nolan's lab is an excellent place for viral stuff...Yes, u can keep them : as stable cell lines,but you would have to take much precautions...single : clones is an old old(updated too) approach...of course u cannot do one : clones, u need at least dozens of clones intially and then narro
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p**i 发帖数: 3525 | 19 好可怜,要挑几百个?
【在 s******y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 很多场合下我们没有别的选择阿 : 另外,逆转录病毒载体也有高表达的promoter的。比方说pQC vector : 我们有时候不喜欢选克隆,是因为我们做的是癌症,而癌症细胞本身就是 : 一个很复杂的群体,如果不是整个群体都得到一定程度的rescue我们会 : 感到害怕下结论,如果只挑几十个克隆我们会担心的。 : : .. : experiment,
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s******y 发帖数: 28562 | 20 我们的策略就是不挑克隆。
转染一个10cm 碟的细胞,然后药选一个星期(一般能得到至少几万个不同的隆),sorting, 然后看整个群体的性状。
但是我们本身也不是做癌症研究出身的,所以不知道这个是否被同行认可。
【在 p**i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 好可怜,要挑几百个?
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s******y 发帖数: 28562 | 21 inducible clones 也有很多麻烦,特别是很多inducible line 并不是很严格
的inducible.或者inducer 对细胞毒害很大
【在 p**i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : xiexie,很有道理。那如何做才是现代的最有说服力的细胞实验呢? 我们一般是加例如 : GFP-tag, : 然后用retroviruses转染建立几个细胞系,然后看接受信号后的蛋白移动。 : inducible clones 是否比较不过时有说服力? : : transient : many : one
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