请教鼠基因在人细胞的表达 - 未名空间MITBBS历史存档

请教鼠基因在人细胞的表达# Biology - 生物学

l*p2012-02-08 08:02

1 楼

1.关于处女男对摩羯女上来的接触方式

很多人都认为摩羯女很冷，其实冷只是她的一种面具，初次聊天，她会给你一种神圣不
可侵犯的距离感，这种古典美的气质可能是吸引处女男的极具杀伤力的武器，作为处女
座的男人，你首先要懂得运用自己的优点，温柔，体贴，彬彬有礼，聪明，上进，这是
你手中最基本的牌，如何把一桌蔬菜做成满汉全席的效果，就要看你如何把你的这些优
点放大且不被魔女发现是一种矫揉造作。这确实需要你去和魔女进行巅峰的对战才能发
觉出来。初次聊天，你一定要表现的极度真实，诚恳，摩羯女是那种很容易从语言或者
你其他方面的资料识破你手段的群体，她们对细节的较真程度绝对不亚于处女座，很多
人说处女座吹毛求疵，但是楼主可以肯定的说，处女座吹毛求疵或许会说出来，但是魔
女绝对不会。她们内心对你的评定是从一开始的第一句话就开始断定你这个人未来的发
展方向和在她心里的位置的，这种被看透的恐惧感，我相信追过摩羯的都有。作为处女
座的你，一定要用最真诚和诚恳的态度和她聊天，让她觉得你的内心戏是一类很简单的
桥段，如果你把内心戏做足，摩羯女同样也会和你用更强大的桥段来给予你压力，所以
我认为这种刚开始就要把人逼到窒息的节奏是没有必要的，我以前也试验过，接触摩羯
女是不是应该上来就把这种节奏加快，来测试她对这段感情的接受程度，后来我发现根
本不可能成功。所以循序渐进，你要跟着她的节奏走是非常必要的。
所以作为处女男要富有极强的感受力和分析力，来确定摩羯女给你的台阶和喜欢，这种
细小的差别很可能是摩羯女晚上纠结一个黑夜才会做出的定论，要懂得珍惜和知足，不
要逼她们。逼她们的后果就是黑名单了。

2.关于处女男和摩羯女的相处方式

上面那个是开始的方式，不过那个摩羯女最后我们还是分道扬镳，因为天灾的缘故，只
是生命里一次我很欣赏的繁花雪月，那时候会动心，会冲动的单纯的喜欢她的一眸一笑
，好了继续说相处。

温度是我们处女男必须要掌控，而且要掌控的非常有即视感和节奏感，因为我目前总结
出来的经验，摩羯女普遍存在欠淡的心里，说白了如果你掌控节奏，还是要保留对她的
那种让她虐心的感觉，比如隔三差五的消失一小下，这个对我们处女男来说就是手到擒
来，千万不要被摩羯女说的所谓的安全感让自己打乱了阵脚，摩羯女有一个特别牛逼的
技能叫做观影，她可以在和你热恋的不行的时候，分身出一个观众，来从头到尾审视你
和她整个恋情里的点点滴滴，重新判断一次你们是否合适，永远记住，不要认为摩羯女
对你牵手，拥抱，KISS，乃至上床，她就觉得生是你的魂，死是你的鬼了，说到这里很
多摩女姑娘可能不耐听，我个人认为从处女男的角度去感受和你们的爱情，这些所谓的
肢体语言毫无用处，表达爱意还是走精神的来的更有安全感，荷尔蒙的分泌不会给魔女
和处女男的爱情带来任何的促进作用，相反对摩羯女的重新走一遍的心态可能还会有消
极的影响，天生悲天悯人的她们很可能会觉得这样不是她们的节奏，从而滋生出愧疚感
，觉得你不是她想要的，最终导致分手。所以处女男一定要懂得若即若离欲擒故纵的道
理，对付魔女要时刻规避她的一切荷尔蒙的诱惑，除非风花雪月，对影成双，良辰美景
适合做一些亲密的举动，你再放开了去做，否则不会有任何好下场。相处模式还是以她
为中心，但是要时刻保留自己时不时消失的节奏，让她抓狂的去纠结你不在的那种生活
，只有这样她们才会直勾勾的认为这样是喜欢是爱情，因为她纠结了，动心了。

摩羯女在真的确定你这个人的时候，对爱情执着和付出的热枕基本是其他11个星女无法
比拟的，这点我可以确定肯定以及一定，我交往过天蝎，巨蟹，双鱼，狮子，双子虽然
平时的温度来说天蝎和巨蟹是最热的，可能在和她们在一起过后，你再去和魔女交往会
有温差，但是你一定要清楚的了解魔女一旦确定之后那种一股脑的投入会令人拍案叫绝
的，这种爱情才是所有男人期望的，她们都是那种默默付出的实用派女人，而不是那种
光说不做的，或者是胸大无脑的蠢货。拥有大智慧，平时偶尔犯傻犯二的时候你一定要
懂得包容她的这些举动，跟她一起疯也可以，只要你可以让她发现她最不满意的小自备
你统统都能够包容并且欣赏，你就离成功不远了。我一直偏执的喜欢摩羯女多少也有妈
妈的影响，这种模式的爱情是我需要的，所以从小我的爱情观还是比较正的，虽然接触
的女孩多，我爱过的就那么两个一个巨蟹一个摩羯。其他都只能算是探知，而且我还有
一个这么健康乐观的射手爹，所以脸皮确实不是一般的厚，对魔女的冷比较免疫。。。
。。然后就越发越严重。

我不知道会有多少处女男来看这个帖子，但是其他你可以不看，这段我希望你能好好看
看，或者说现在和处女男在一起的摩羯女也可以把这段发给他，让他知道你们的爱情是
多么的深沉而持久，有句话说的很好么。但当智慧酝酿成红酒，方可一醉自救，摩羯女
就是一个沉香百品不腻的红酒，如果你是一个有品位的人，懂得品尝红酒前的礼仪（红
酒要均匀摇晃，让杯底的沉香散发出来，好的红酒在摇晃的时候是可以挂在杯壁上的）
你一定要试试摩羯女婚后的爱情。

z*e2012-02-08 08:02

2 楼

一个网上的order q9300，今天抽空去pick up，丫的居然在盒子里面装个pentium 4 2.
4GHz的给我

a*f2012-02-08 08:02

3 楼

请教大拿,
用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
I tried two times without success. My boss said it has been done
successfully by others. My gene is not immune-related. It involves in
metabolism. I’m pretty sure that I did not make any technical mistakes.
Anybody can share what may cause it? I do not want to it again if there is
no hope

Thanks you!

M*s2012-02-08 08:02

4 楼

我真不看好处女男，嘿嘿

a*o2012-02-08 08:02

5 楼

MC不会干这事儿，八成他们也是被骗了。

z*62012-02-08 08:02

6 楼

我的老鼠（不是我做的）是表达人基因的...不知道反过来...路过帮顶...

A*y2012-02-08 08:02

7 楼

果然不愧是处女座，这么细致的分析
看来近来大家对分析摩羯的兴趣高涨啊。对天蝎审美疲劳啦？要不咱们提前搞摩羯月活
动得了，呵呵

y*n2012-02-08 08:02

8 楼

mc 的label上写着p4

【在 a*o 的大作中提到】

: MC不会干这事儿，八成他们也是被骗了。

b*n2012-02-08 08:02

9 楼

多大的基因啊
这种外源表达，实在不好说

?
is

【在 a***f 的大作中提到】

: 请教大拿,
: 用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
: I tried two times without success. My boss said it has been done
: successfully by others. My gene is not immune-related. It involves in
: metabolism. I’m pretty sure that I did not make any technical mistakes.
: Anybody can share what may cause it? I do not want to it again if there is
: no hope
: 
: Thanks you!

c*g2012-02-08 08:02

10 楼

我有个摩羯女朋友，虽然现实，但不失为一个很好很好的朋友。

d*t2012-02-08 08:02

11 楼

他们拿错了...

2.

【在 z*********e 的大作中提到】

: 一个网上的order q9300，今天抽空去pick up，丫的居然在盒子里面装个pentium 4 2.
: 4GHz的给我

s*s2012-02-08 08:02

12 楼

你要知道，BAC里面就是你要的基因 + 调控序列 - epigenetic markers
虽然很多被epigentically repressed的基因很可能表达，但是你缺少必要
的TFs的话就没有办法了。

?
is

【在 a***f 的大作中提到】

: 请教大拿,
: 用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
: I tried two times without success. My boss said it has been done
: successfully by others. My gene is not immune-related. It involves in
: metabolism. I’m pretty sure that I did not make any technical mistakes.
: Anybody can share what may cause it? I do not want to it again if there is
: no hope
: 
: Thanks you!

r*12012-02-08 08:02

13 楼

处女男和摩羯在一起很多的，摩羯女对处女男帮助很大。

m*d2012-02-08 08:02

14 楼

网上怎么order q9300，我怎么搜不到

2.

【在 z*********e 的大作中提到】

: 一个网上的order q9300，今天抽空去pick up，丫的居然在盒子里面装个pentium 4 2.
: 4GHz的给我

w*n2012-02-08 08:02

15 楼

鼠的基因在人细胞表达完全没问题。但是看启动子，不知道你这个基因应该在哪个组织
表达，BAC因为是基因本身的启动子，所以如果不是看家基因，可能需要特定的细胞系
。比如特定在肝脏表达的鼠基因要到对应人的肝脏细胞去表达。

?
is

【在 a***f 的大作中提到】

: 请教大拿,
: 用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
: I tried two times without success. My boss said it has been done
: successfully by others. My gene is not immune-related. It involves in
: metabolism. I’m pretty sure that I did not make any technical mistakes.
: Anybody can share what may cause it? I do not want to it again if there is
: no hope
: 
: Thanks you!

s*r2012-02-08 08:02

16 楼

作者如果就是这个处女男的话，可真矫情啊

z*e2012-02-08 08:02

17 楼

不是拿错了。。。那个小妞指着label跟我说，你看，写的是pentium 4，这个里面就是
pentium 4.但是网上是order的q9300的open box
我问为啥你要装在c2q的盒子里，她说那个不准，得看后面description写的字

【在 d**********t 的大作中提到】

: 他们拿错了...
:
: 2.

l*12012-02-08 08:02

18 楼

why only try BAC vector method only
alternatively you can try PB transposons vector
>Transfection of naked DNA has been used for large-cargo delivery.
Pronuclear injection of bacterial artificial chromosomes (BACs) has been
successful for transgenesis of up to 300 kb. However, the integrity,
integration site and copy number can not be controlled. BAC vectors have
also been used for targeting large cargos to defined genomic positions in ES
cells via homologous recombination (Valenzuela et al., 2003), but the
efficiency is locus-dependent and can be very low.
Recombinases such as Cre have also been used to deliver BACs to a predefined
genomic location by recombination-mediated cassette exchange (Wallace et al
., 2007; Prosser et al., 2008); however, pre-engineering of target sites in
the genome is necessary. While these methods are useful for certain
applications, all have limitations and most of them are not able to revert
the insertion of large DNA fragments.
full text link:
URL address:
f-t-p ://ftp.sanger.ac.uk/pub4/theses/ml8/chapter7.pdf

?
is

【在 a***f 的大作中提到】

: 请教大拿,
: 用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
: I tried two times without success. My boss said it has been done
: successfully by others. My gene is not immune-related. It involves in
: metabolism. I’m pretty sure that I did not make any technical mistakes.
: Anybody can share what may cause it? I do not want to it again if there is
: no hope
: 
: Thanks you!

h*92012-02-08 08:02

19 楼

楼主的分析和我对摩羯女的理解差不多。我也是处女男，可是对摩羯女很难感冒，相反
比较喜欢火向或风向的女生，比如白羊，水瓶。

z*e2012-02-08 08:02

20 楼

有货的才搜的到,oos的不行

【在 m*d 的大作中提到】

: 网上怎么order q9300，我怎么搜不到
:
: 2.

a*f2012-02-08 08:02

21 楼

Thanks a lot for the kind response from Zhang.., Bull..., Lot..., Wak....
and Shak...
My gene is about 2500 aa. I used in kidney cells. Have to use this strategy
as boss want to do it in this way.
:-(
I agree that it is important to have the appropriate transcription factors
in the expression system. In other words, I need to have the right host
cells. If the human orthologue is expressed, can I assume that the mouse
version of the gene will be expressed too, in the human cells?
In addition, besides RT-PCR, is there any dry lab method or database to
check if it is expressed in a given tissue type.
Thanks!

?
is

【在 a***f 的大作中提到】

: 请教大拿,
: 用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
: I tried two times without success. My boss said it has been done
: successfully by others. My gene is not immune-related. It involves in
: metabolism. I’m pretty sure that I did not make any technical mistakes.
: Anybody can share what may cause it? I do not want to it again if there is
: no hope
: 
: Thanks you!

h*92012-02-08 08:02

22 楼

楼主的分析和我对摩羯女的理解差不多。我也是处女男，可是对摩羯女很难感冒，相反
比较喜欢火向或风向的女生，比如白羊，水瓶。

p*r2012-02-08 08:02

23 楼

里面到底是什么？

l*12012-02-08 08:02

24 楼

just try YFP labeling
paper link:
//www.ncbi.nlm.nih.gov/pmc/articles/PMC2889740/pdf/mt201047a.pdf
>besides RT-PCR, is there any dry lab method or database to

strategy

【在 a***f 的大作中提到】

: Thanks a lot for the kind response from Zhang.., Bull..., Lot..., Wak....
: and Shak...
: My gene is about 2500 aa. I used in kidney cells. Have to use this strategy
: as boss want to do it in this way.
: :-(
: I agree that it is important to have the appropriate transcription factors
: in the expression system. In other words, I need to have the right host
: cells. If the human orthologue is expressed, can I assume that the mouse
: version of the gene will be expressed too, in the human cells?
: In addition, besides RT-PCR, is there any dry lab method or database to

A*y2012-02-08 08:02

25 楼

你是什么月座？

【在 h******9 的大作中提到】

: 楼主的分析和我对摩羯女的理解差不多。我也是处女男，可是对摩羯女很难感冒，相反
: 比较喜欢火向或风向的女生，比如白羊，水瓶。

z*e2012-02-08 08:02

26 楼

p4

【在 p**********r 的大作中提到】

: 里面到底是什么？

s*s2012-02-08 08:02

27 楼

我和你说吧，如果有相应的human orthologue表达，那么九成以上你的
老鼠基因能表达，有很小的物种不兼容的可能性你的不表达，很小。
如果相应的Human orthologue不表达，有两种可能，A缺少TF，B分化过
程中被actively epigenetically repress了。如果你得基因是一个重要
的调控基因，那么恭喜，很可能human copy是cis repress了，你得老鼠基
因有比较大的概率仍然可能表达；如果你得是一个下游基因，那么多半不表
达是因为A，那就没辙了

strategy

【在 a***f 的大作中提到】

: Thanks a lot for the kind response from Zhang.., Bull..., Lot..., Wak....
: and Shak...
: My gene is about 2500 aa. I used in kidney cells. Have to use this strategy
: as boss want to do it in this way.
: :-(
: I agree that it is important to have the appropriate transcription factors
: in the expression system. In other words, I need to have the right host
: cells. If the human orthologue is expressed, can I assume that the mouse
: version of the gene will be expressed too, in the human cells?
: In addition, besides RT-PCR, is there any dry lab method or database to

h*92012-02-08 08:02

28 楼

月亮是天平，另外星盘里有好几个在射手。这也许是我喜欢火向和风向的原因。

G*h2012-02-08 08:02

29 楼

那她拿错了啊

【在 z*********e 的大作中提到】

: 不是拿错了。。。那个小妞指着label跟我说，你看，写的是pentium 4，这个里面就是
: pentium 4.但是网上是order的q9300的open box
: 我问为啥你要装在c2q的盒子里，她说那个不准，得看后面description写的字

l*12012-02-08 08:02

30 楼

Still you can try HAC Human artificial chromosome not BAC bacterial artificial chromosome
with that mice gene with labeled YFP knock in HAC
then expressed in Human kidney cells.
please refer:
//www.ncbi.nlm.nih.gov/pubmed/21750534
RE:
>如果你得是一个下游基因，那么多半不表
[回复]
发信人: amysf (kdfa), 信区: Biology
标题: Re: 请教鼠基因在人细胞的表达
发信站: BBS 未名空间站 (Thu Feb 9 10:03:15 2012, 美东)
...
My gene is about 2500 aa. I used in kidney cells.
-----------

A*y2012-02-08 08:02

31 楼

可能是，见过不少日月分别处在水土，风火两大阵营中的有这种情况。

【在 h******9 的大作中提到】

: 月亮是天平，另外星盘里有好几个在射手。这也许是我喜欢火向和风向的原因。

d*t2012-02-08 08:02

32 楼

太悲剧了...直接退了吧

【在 z*********e 的大作中提到】

: 不是拿错了。。。那个小妞指着label跟我说，你看，写的是pentium 4，这个里面就是
: pentium 4.但是网上是order的q9300的open box
: 我问为啥你要装在c2q的盒子里，她说那个不准，得看后面description写的字

w*n2012-02-08 08:02

33 楼

如果RT-PCR阳性，蛋白手段检测不到的话，可能表达量比较低，调整检测手段。最近做
几个蛋白也是这样，提高WB一抗浓度后，有改观，可以试一下。

strategy

【在 a***f 的大作中提到】

: Thanks a lot for the kind response from Zhang.., Bull..., Lot..., Wak....
: and Shak...
: My gene is about 2500 aa. I used in kidney cells. Have to use this strategy
: as boss want to do it in this way.
: :-(
: I agree that it is important to have the appropriate transcription factors
: in the expression system. In other words, I need to have the right host
: cells. If the human orthologue is expressed, can I assume that the mouse
: version of the gene will be expressed too, in the human cells?
: In addition, besides RT-PCR, is there any dry lab method or database to

A*t2012-02-08 08:02

34 楼

楼主爱过的巨蟹和磨女，我表示都是很腹黑可怕的两类人，反正我是要防备的。
处女男果真龟毛阿，名不虚传，没有恶意哦:-)。

s*r2012-02-08 08:02

35 楼

label 写错啦。就是个P4. 要不然也不会是卖32.96.

a*f2012-02-08 08:02

36 楼

Thanks to Lot..., Wak.... and Shak... again!
Sorry for not responding sooner. I did RT-PCR, as expected, the human
endogenous gene is expressed. But the mouse gene (BAC)is not.
I guess it is correct to say that the mouse transgene should have decent
chance to be expressed if the human orthorlogue is expressed, as a general
rule. But for a given gene, it is very hard to be sure. At least, the
organization of CpG elements and regulatory sequence of gene are very
different from mouse to human, right?
In my case, I used a known gene as a technical control. The mouse version of
this gene is expressed, but not gene-of-interest. Both this gene and my
gene-of-interest are all expressed in human kidney cell.
Could I then safely conclude the promoter of the mouse version of my gene-of
-interest does not work in human cell system?
What do you guys think? Thanks!

【在 w******n 的大作中提到】

: 如果RT-PCR阳性，蛋白手段检测不到的话，可能表达量比较低，调整检测手段。最近做
: 几个蛋白也是这样，提高WB一抗浓度后，有改观，可以试一下。
:
: strategy

m*82012-02-08 08:02

37 楼

双鱼也很腹黑，是想不到的那种黑。

【在 A********t 的大作中提到】

: 楼主爱过的巨蟹和磨女，我表示都是很腹黑可怕的两类人，反正我是要防备的。
: 处女男果真龟毛阿，名不虚传，没有恶意哦:-)。

a*i2012-02-08 08:02

38 楼

open box以后换了个box装
估计是哪个环节除了问题
退了完事

s*s2012-02-08 08:02

39 楼

normally, there are multiple BACs for the same gene. You can try more

of

【在 a***f 的大作中提到】

: Thanks to Lot..., Wak.... and Shak... again!
: Sorry for not responding sooner. I did RT-PCR, as expected, the human
: endogenous gene is expressed. But the mouse gene (BAC)is not.
: I guess it is correct to say that the mouse transgene should have decent
: chance to be expressed if the human orthorlogue is expressed, as a general
: rule. But for a given gene, it is very hard to be sure. At least, the
: organization of CpG elements and regulatory sequence of gene are very
: different from mouse to human, right?
: In my case, I used a known gene as a technical control. The mouse version of
: this gene is expressed, but not gene-of-interest. Both this gene and my

w*n2012-02-08 08:02

40 楼

再找小鼠细胞验证一下，确认BAC没问题。

of

【在 a***f 的大作中提到】

: Thanks to Lot..., Wak.... and Shak... again!
: Sorry for not responding sooner. I did RT-PCR, as expected, the human
: endogenous gene is expressed. But the mouse gene (BAC)is not.
: I guess it is correct to say that the mouse transgene should have decent
: chance to be expressed if the human orthorlogue is expressed, as a general
: rule. But for a given gene, it is very hard to be sure. At least, the
: organization of CpG elements and regulatory sequence of gene are very
: different from mouse to human, right?
: In my case, I used a known gene as a technical control. The mouse version of
: this gene is expressed, but not gene-of-interest. Both this gene and my

l*12012-02-08 08:02

41 楼

Congratulation!
If you try that successful others same BAC and same protocol and same
materials
then you try again and again it is still negative.
And not only you but your college PD does it again and also it is negative=
It seems that paper might had misconduct
likes below blog shown:
//md-anderson-cc.blogspot.com/
or some trigger around it.
That is more interesting than you get positive data from that paper.
回复]
发信人: amysf (kdfa), 信区: Biology
标题: 请教鼠基因在人细胞的表达
发信站: BBS 未名空间站 (Tue Feb 7 23:13:13 2012, 美东)
请教大拿,
用bacterial artificial chromosome 带的鼠基因在人细胞进行表达通常是否会成功?
I tried two times without success. My boss said it has been done
successfully by others

of

【在 a***f 的大作中提到】

: Thanks to Lot..., Wak.... and Shak... again!
: Sorry for not responding sooner. I did RT-PCR, as expected, the human
: endogenous gene is expressed. But the mouse gene (BAC)is not.
: I guess it is correct to say that the mouse transgene should have decent
: chance to be expressed if the human orthorlogue is expressed, as a general
: rule. But for a given gene, it is very hard to be sure. At least, the
: organization of CpG elements and regulatory sequence of gene are very
: different from mouse to human, right?
: In my case, I used a known gene as a technical control. The mouse version of
: this gene is expressed, but not gene-of-interest. Both this gene and my

a*f2012-02-08 08:02

42 楼

Thanks all for input.
I guess I did not make it cleary enough. What I meant is that a BAC
containing my mouse gene was not expressed. But a BAC containing another
known gene was. I did both in parallel and the latter wss used as a control.

【在 l**********1 的大作中提到】

: Congratulation!
: If you try that successful others same BAC and same protocol and same
: materials
: then you try again and again it is still negative.
: And not only you but your college PD does it again and also it is negative=
: It seems that paper might had misconduct
: likes below blog shown:
: //md-anderson-cc.blogspot.com/
: or some trigger around it.
: That is more interesting than you get positive data from that paper.

l*12012-02-08 08:02

43 楼

Agree with
shakuras said:
[回复]
发信人: shakuras (doskey), 信区: Biology
标题: Re: 请教鼠基因在人细胞的表达
发信站: BBS 未名空间站 (Sat Feb 11 17:13:35 2012, 美东)
normally, there are multiple BACs for the same gene. You can try more

control.

【在 a***f 的大作中提到】

: Thanks all for input.
: I guess I did not make it cleary enough. What I meant is that a BAC
: containing my mouse gene was not expressed. But a BAC containing another
: known gene was. I did both in parallel and the latter wss used as a control.