neo, hygro, puro, and zeo. which is better for stable transfection marker? - 未名空间MITBBS历史存档

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neo, hygro, puro, and zeo. which is better for stable transfection marker?

neo, hygro, puro, and zeo. which is better for stable transfection marker?# Biology - 生物学

b*l2011-02-09 08:02

1 楼

http://finance.yahoo.com/news/Congress-scrutinizes-problems-apf-3498429273.html?x=0&sec=topStories&pos=5&asset=&ccode=
不是extension本身。而是有人在质疑这个tax credit是不是得不偿失。
现在的数据是大概有1.5M买房者申请了这个credit。但是政府发现其中有超过11万的
cases可能有欺诈行为。冻结了refund程序，并进行调查（frozen more than 110,000
refunds pending civil or criminal examinations）。
其中有1.9万人甚至还没有买房子就已经申请了。另外有7.4万人被怀疑不是第一次买房
者。还有一经发现的580个人不是成年。甚至有4岁的儿童申请credit.
根据NAR(一贯以扭曲数据制造有利realtor的消息著称，其首席分析师更是业界著名小
丑）总共由于这个credit而买房的人是35万。
也就是说taxpayer总共花了100亿刀，换得35万人由旁观改为购买。每个人的成本

B*Z2011-02-09 08:02

2 楼

真实天赋也交代了，该填的坑也都填了，比亵渎强
烟雨江南现在已经熟练掌握了打脸流和兽血沸腾的土豪流，日后就不停的流水线生产吧

g*52011-02-09 08:02

3 楼

thanks.

a*t2011-02-09 08:02

4 楼

Good!

000
金3

【在 b*****l 的大作中提到】

: http://finance.yahoo.com/news/Congress-scrutinizes-problems-apf-3498429273.html?x=0&sec=topStories&pos=5&asset=&ccode=
: 不是extension本身。而是有人在质疑这个tax credit是不是得不偿失。
: 现在的数据是大概有1.5M买房者申请了这个credit。但是政府发现其中有超过11万的
: cases可能有欺诈行为。冻结了refund程序，并进行调查（frozen more than 110,000
: refunds pending civil or criminal examinations）。
: 其中有1.9万人甚至还没有买房子就已经申请了。另外有7.4万人被怀疑不是第一次买房
: 者。还有一经发现的580个人不是成年。甚至有4岁的儿童申请credit.
: 根据NAR(一贯以扭曲数据制造有利realtor的消息著称，其首席分析师更是业界著名小
: 丑）总共由于这个credit而买房的人是35万。
: 也就是说taxpayer总共花了100亿刀，换得35万人由旁观改为购买。每个人的成本

y*n2011-02-09 08:02

5 楼

花了好几年挖坑，一天就都打算填了。填坑时候不小心又挖了个坑。老龙那个大坑一直
没填，填别的坑的时候没填流砂的坑，结果老龙的坑又挖大了一点。

H*s2011-02-09 08:02

6 楼

It depends on your purpose and the cell line to be transfected. Generally
speaking, if you cell line is tough enough, puromycin is much easier to work
with. You just need to add a relevant amount of puromycin to the media and
it starts killing.
For neomycin and hygromycin, you have to determine optimal killing
concentration specific to the cell line you will use. To initiate killing,
you have to split cells and supplement with neo/hygro containing media.
Every selective marker has its specific problem. So be careful with the
trade offs. For example, if you use puromycin and your target gene does not
share promoter with puromycin resistance gene, there is good possibility
that some puromycin resistant clones will not express your protein of
interest. Even worse, if you use pooled clones, those non-expressing clones
will outgrow clones that express your protein of interest.

【在 g*********5 的大作中提到】

: thanks.

B*Z2011-02-09 08:02

7 楼

流沙的算填了啊，无面就是流砂的化身
老龙的没填，不过作为幕后黑手留着给续集也是普遍现象

【在 y*****n 的大作中提到】

: 花了好几年挖坑，一天就都打算填了。填坑时候不小心又挖了个坑。老龙那个大坑一直
: 没填，填别的坑的时候没填流砂的坑，结果老龙的坑又挖大了一点。

q*n2011-02-09 08:02

8 楼

My personal experience:
neo and puro are good, quick selection.
Hygro is OK, but kill cells slowly.
Zeo is the worst, even zeo resistant cells have funny shapes. After a few
days of selection, you have to trypsinize cells off the plates and replated
cells without zeocin. Only zeo resistant cells will survive and grow.

g*52011-02-09 08:02

9 楼

非常感谢。我准备用两个lentivirus质粒转两个基因，so neo and puro.

z*62011-02-09 08:02

10 楼

I don't think the selection marker matters a lot. I think it is the cell
line and the way of transfection that matters more. If you don't care the
expression level tooooo much, lentivirus works really good. But last time I
was doing A549 and I need the expression level to be really high to secrete
a lot of soluble proteins for ELISA detection, none of methods (
electroporation, nucleous transfection, lentivirus) or selection markers (I
tried multiple ones) worked.

g*52011-02-09 08:02

11 楼

how is the blast?
this marker work well?