h********n
发帖数: 4079 | 1 什么情况下会出现partial KO?
谢谢
mosaic的 |
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n********k
发帖数: 2818 | 2 with my limited expericences, I would say promoters, positioning effects etc
etc, but no one can predict with accuracy until one does the experienment
and found out. as well, cre toxity has been observed in neuron as well... |
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D*a
发帖数: 6830 | 4 不知道你说的partial KO是指什么，大多数时候用cre就是为了partial KO，这个
partial是specific的patial，比如只在特定器官，或者某种特定细胞中造成KO而不涉
及其他细胞，这样就避免了用total KO或者用全身+/-的老鼠，分析实验结果时不能确
定是整体影响，还是特定细胞的特定功能。
但有的partial KO是我们不希望的，比如利用脂肪细胞中的特定promotor建立了一个脂
肪细胞中表达的cre，然后每个器官分别摘出来验证时，发现在肝中也有KO，这就是cre
是leaky的，这个当初做老鼠时很难避免，因为你也不知道你的promotor是不是多用了
一块或者少用了一块，或者旁边一个调控元件根本就是未知的，再加上后面接的又是别
的蛋白，很难预测promotor的行为，比如到底在哪里表达，什么时候表达等等。这时候
paper发表前和发表后就很容易被人critique，不过也不是不能发到好杂志上，这个原
因就不分析了哈。。。但是结果其实有可能是leaky的那部分cre造成的，而跟你本来想
KO的那个器官没有关系。。。
我现在看的一篇nature文章，我 |
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s******e
发帖数: 370 | 5 我们做的是complete KO， 不是tissue specific，所以要从卵子开始就得有active的
cre——跟上面各位不同哈
F1出来，PCR，碰到过既有被cre切过以后产生的小带，又有未切的cKO带，就把这个叫
partial。究其原因，猜想可能是cre被transcribe出来的时间太晚又太短，受精卵分裂
之后有些细胞带了cre有些没有。
但是即使是partial，如果它的生殖细胞是KO，我们还是可以用它来establish line的。
tissue specific暂时还没有做到⋯⋯ |
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s******r
发帖数: 2876 | 6 哪一篇？说来学习学习。
不知道你说的partial KO是指什么，大多数时候用cre就是为了partial KO，这个
partial是specific的patial，比如只在特定器官，或者某种特定细胞中造成KO而不涉
及其他细胞，这样就避免了用total KO或者用全身+/-的老鼠，分析实验结果时不能确
定是整体影响，还是特定细胞的特定功能。
但有的partial KO是我们不希望的，比如利用脂肪细胞中的特定promotor建立了一个脂
肪细胞中表达的cre，然后每个器官分别摘出来验证时，发现在肝中也有KO，这就是cre
是leaky的，这个当初做老鼠时很难避免，因为你也不知道你的promotor是不是多用了
一块或者少用了一块，或者旁边一个调控元件根本就是未知的，再加上后面接的又是别
的蛋白，很难预测promotor的行为，比如到底在哪里表达，什么时候表达等等。这时候
paper发表前和发表后就很容易被人critique，不过也不是不能发到好杂志上，这个原
因就不分析了哈。。。但是结果其实有可能是leaky的那部分cre造成的，而跟你本来想
KO的那个器官没有关系。。。
我现在看 |
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h********n
发帖数: 4079 | 7 我有点不太明白, Cre在每个细胞都表达, 即使第一代不行, 以后应该也会被切掉啊,
能不能详细说说.
谢谢
的。 |
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s******e
发帖数: 370 | 8 还是那句话，看是什么cre
我用的cre是oocyte active，受精后短暂活跃，过了那个阶段就不表达了，所以精子里
来的cre有时就不够effective。beta-actin的话应该会好一点吧
其实我接触这个也才一年多，都是trouble shooting中攒的个人经验而已 |
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S******9
发帖数: 2837 | 11 你这个beta-actin Cre是non inducible的？
我们组研究的target基因就是在胚胎11.5～12.5天的时候死掉。即使是Tie2 Cre内皮特
异性的也是的。所以我们研究的成鼠是用的beta-actin Cre是inducible的，可惜我们
的flox做的不是特别好，有leaky现象，部分老鼠实际出生的时候已经有很大的ko了，
当然用药物诱导以后可以ko掉90%。
叫你老板把我招去吧，我喜欢做老鼠model的活。做atherosclerosis，vasoactivity，
原代血管内皮培养，各种组织切片染色。 |
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D*a
发帖数: 6830 | 12 提供2篇references
Cre-mediated germline mosaicism: a new transgenic mouse for the selective
removal of residual markers from tri-lox conditional alleles.
Leneuve P, Colnot S, Hamard G, Francis F, Niwa-Kawakita M, Giovannini M,
Holzenberger M.
Nucleic Acids Res. 2003 Mar 1;31(5):e21.
36.
Cre-mediated germline mosaicism: a method allowing rapid generation of
several alleles of a target gene.
Holzenberger M, Lenzner C, Leneuve P, Zaoui R, Hamard G, Vaulont S, Bouc YL.
Nucleic Acids Res. 2000 Nov 1;28( |
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D*a
发帖数: 6830 | 13 pdf淹没在茫茫paper大海中了 -_-lll
是关于ap2 cre和obesity / IR的一篇......
cre |
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p*f
发帖数: 77 | 14 You current boss is less smart than you.
As a way out, you can get 3 mice in a group and repeat 3 times, which should
only cost you two more mice to get your dumb boss satisfied. |
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m*****s
发帖数: 2227 | 17 我也是在网上查的。
With brown or black mice and rats, an additional light source may be
necessary to aid in visualizing the tail veins.
It is more difficult to visualize the tail vein in black mice. In these
cases, we have found that spraying 70% isopropanol on the tail increases the
contrast. |
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p*****m
发帖数: 7030 | 18 You can do forward screens in mice (like in Susan Ackerman? and Bruce
Beutler labs). Actually you can even do behavioral screens in mice (like in
Joe Takahashi lab)
Sorry if I mis spell any name... |
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b*****1
发帖数: 17 | 19 我还是一年级的研究生，做rotation。一直疑惑这个问题。
虽然过去书本上学了不同organism的优劣，但是没做过相关的research还是体会不到。
但是rotation也没那么多机会把各种模式生物都做一下。
1。我个人其实是想做genomics，RNA，gene regulation，还有systems biology这方面
的。我觉得是不是对于希望往genome啊，系统生物学啊，数理模型发展的话，就尽量选
择简单的生物？比如一般的cell line，yeast，最多C elegans？
2。是不是以后的趋势都是做mice呢？如果都是这个趋势，那做什么C elegans，或者
yeast的岂不都要关门了？这也牵扯出一个问题，就是postdoc和phd做的东西差别有多
大呢？比如我phd做C elegans，那么postdoc的时候做mice可以吗？我觉得是可以的，
但是这么多年的模式动物的经验，也不是一天两天就学来的，postdoc老板还是喜欢能
立刻上手干活的。
或者说，比如C elegans（和drosophila）有没有什么unique的优势，是别的生物代替
不了的？
3 |
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w***j
发帖数: 88 | 20 前辈们指教一下：
我有一个A flox/flox 的mice，需要和Tek-Cre杂交，得到血液系统敲除的A -/- mice。
Breeding程序如下：
1. A flox/flox mating with Tek+ 得到 "A wt/lox Tek+" and "A wt/lox Tek-"
2. "A flox/flox" mating with "A wt/flox Tek+" 得到以下四种：
"Rictor wt/flox Tek+"
"Rictor flox/flox Tek+"
"Rictor flox/flox Tek-"
"Rictor flox/wt Tek-"
"Rictor flox/flox Tek+"是我要的KO, 称为Hem_A -/-, 那么"Rictor flox/flox Tek-
"是我的对照,称为Hem_A +/+，是不是这样？
做实验是不是最好还要wild-type？ 那么wild-type是哪个？
叩谢！ |
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n****n
发帖数: 165 | 21 Usually mate wt/lox cre+ each other and then use wt/wt cre+ as control to
compare lox/lox cre+ one. One question: how can you get wt/lox Tek- mice if
you just mate lox/lox with Tek one?
mice。
Tek- |
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b****u
发帖数: 903 | 22 it's very interested paper and i am curious how to set up a ER model for
mice, since the birth day the mice have been raised in cage, more feed, more
clean, or broadcast the Mozart that's the ER? |
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D****g
发帖数: 275 | 23 The plasma should be around 55-60% if you spin down at 10,000g for 5-10 min,
if you get more than that, your mice may have anemia, if you get less than
that, your mice may have polycythemia. |
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w******e
发帖数: 1187 | 24 I learned from others. don't quote me on that:) I'm very new to mice work.
for the past few exps, we typically were able to draw <2ml when sacrificing
the mice. Is there a reason why some blood can't be drawn? just curious |
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P*****l
发帖数: 438 | 25 http://www.nytimes.com/2010/10/15/science/15retract.html?_r=1
3 Harvard Researchers Retract a Claim on the Aging of Stem Cells
By NICHOLAS WADE
Published: October 14, 2010
Harvard researchers have retracted a far-reaching claim they made in January
that the aging of stem cells might be reversible.
The retraction was published in Thursday’s issue of Nature and is signed by
the senior author, Amy J. Wagers, and two others. They say that serious
concerns, which they did not specify, have undermined... 阅读全帖 |
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a*****g
发帖数: 543 | 26 Jumping to a deep water hole...
I would caution the widely "credited" potential of CSC(technically here refe
ring to the tumor-inititating cells identified in immune-deficient mice) ass
ociated with relapse, drug resistance and metastasis.
Can anyone point out an original research paper with some experimental data
showing these cells have any of the three major capabilities? My impression
is sadly none...But we read it everywhere...
Second, CSCs are defined vaguely. The standards mentioned by ht... 阅读全帖 |
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h********n
发帖数: 4079 | 27 are you making Cre mice or making LoxP mice |
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m*****s
发帖数: 156 | 28 this is paired sample design, used to minimize variation from difference
mice.
One analysis method could be to calculate the difference (d=weight1-weight2)
first. Then you could do a non-parametric sign test on d1, d2, ..., dn (n
is sample size or number of mice). Whether there is difference from the
material is equivalent to test whether d is greater than 0.
g |
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h*******g
发帖数: 711 | 29 I don't think that paired-test can be used in LZ's situation unless u have
demonstrated that there are no significant difference between two kidneys in
one mice, which I would say is not that easy. I guess your experiment can
be settled in two ways: first, you have to increase your sample size to
narrow down the deviation of the data. The variations of mice experiments
are always to be quite high. To my experience, the sample size should at
least be over 100. Second, I think the wilcoxon signed-... 阅读全帖 |
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D*a
发帖数: 6830 | 30 1
Expression of insulin-like growth factor I in transgenic mice with elevated
levels of growth hormone is correlated with growth.
Mathews LS, Hammer RE, Brinster RL, Palmiter RD.
Endocrinology. 1988 Jul;123(1):433-7.
2
Growth enhancement of transgenic mice expressing human insulin-like growth
factor I.
Mathews LS, Hammer RE, Behringer RR, D'Ercole AJ, Bell GI, Brinster RL,
Palmiter RD.
Endocrinology. 1988 Dec;123(6):2827-33.
多谢!
x********[email protected] |
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l**********n
发帖数: 240 | 31 Josef Martin Penninger
Most significant scientific contributions:
Our basic approach is to genetically manipulate and change genes in mice and
to
determine the effects of these mutations in development of the whole
organism and in
diseases. From these mutations we are trying to establish basic principles
of
development and basic mechanisms of disease pathogenesis. My laboratory
focuses on
heart diseases, autoimmune diseases and cancers, and bone diseases. On all
the listed
contributions below I ... 阅读全帖 |
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g*********d
发帖数: 233 | 32 人们都知道锻炼身体可以促进新陈代谢，有益于心血管健康，但此前科学家对运动究竟如何影响心脏
却所知甚少。美国哈佛大学医学院的研究人员日前报告说，他们首次从分子水平发现运动有益心脏健
康的机理，这一发现将有助于开发出治疗心血管疾病的新疗法。
研究人员通过小鼠实验发现，经常运动可以使小鼠体内的C/EBPβ转录因子水平显著下降，其结果会促
进小鼠心脏肌肉细胞增殖，有益于心脏生长。此外，研究人员还发现，体内C/EBPβ水平较低的小鼠对
心力衰竭具有抵抗能力。
研究人员表示，这项研究对心脏肌肉再生的潜力有了深入理解。参与研究的哈佛大学医学院教授安东
尼·罗森茨魏希表示，通过这项研究可以开发出针对那些无法运动的心脏病患者的疗法。
这项研究成果发表在新一期美国《细胞》杂志上。
C/EBPβ Controls Exercise-Induced Cardiac Growth and Protects against
Pathological Cardiac Remodeling
Cell, Volume 143, Issue 7, 1072-1083, 23 December 2010
10.1016/j... 阅读全帖 |
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c*********t
发帖数: 340 | 33 No, Fisher's Exact test is not necessarily better
The "exact" methods are usually too conservative
a rule of thumb is that if all cells in the contingency table (mice with B
in each group, mice without B in each
group) are greater than 5 then you don't have to use fisher's exact. Chi-sq
is good enough |
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M*****n
发帖数: 16729 | 34 inducing human iPS in mice or other animals? that will be cool, and I will
be surprised to see that human iPS can receive sufficient instructions in
mice to become the right cell type: this bridges/breaks the boundary between
species!
the first example is certainly useful for modeling idiopathic diseases in
dish, but further scrutinization is warranted to determine how
representative the invitro model is. for example is the ALS neurons in vitro
the same type of neuron in vivo that is afflicted b... 阅读全帖 |
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w******e
发帖数: 1187 | 35 Tan group striked again...
http://www.pnas.org/content/early/2011/02/15/1016197108.abstrac
1. construct a conformation switch-based aptamer sensor which elicits
signal upon binding to specific target on cell(have been done many times);
2. use the sensor to quantify cancer cells down to a few hundred cells,
in both saline & serum, with a huge dynamic range (new to me, but
shouldn't be too hard)
3. use the sensor for in vivo imaging of cancer mouse model induced by
SC injected cancer cells. imagin... 阅读全帖 |
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m***f
发帖数: 1622 | 36
扯淡呢，看看高最近两年发表的文章吧
CELL STEM CELL, JBC, STEM CELL, PNAS，都是很不错的
就这样还不能PROMOTION? 在美国都可以tennure了
1. Wu T., Wang H., He J., Kang L., Jiang Y., Liu J., Zhang Y., Kou
Z., Liu L., Zhang X., Gao S. (2011) Reprogramming of trophoblast stem
cells into pluripotent stem cells by Oct4. Stem Cells 2011 Feb 8.
[Epub ahead of print].
2. Kang L., Wu T., Tao Y., Yuan Y., He J., Zhang Y., Luo T., Kou Z.,
Gao S. (2010) Viable mice produced from 3-factor induced pluripotent
stem (iPS) cells through tetrapl... 阅读全帖 |
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s******y
发帖数: 220 | 37 I am trying to breed some mice, I put a 1.5 months old male with a 1
month (or even younger)female,
after 2 weeks or so, the female died.
It could be because of the disease phenotype, but I am just concerned
could it be that the female is too young,the male somehow is too aggressive?
My boss had this concern too, but neither of us are mice
experts.
Thanks for any advice. |
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s****9
发帖数: 932 | 38 I would recommend colitis model. Both Fiona's one (needs adaptive transfer)
and DSS treated model (no transfer requirement) are fine.
For EAE, CIA, Th17 cells mediated disease pathogenesis as well. IFNgKO mice
have exacerbated EAE and CIA, whereas IFNgKO or TbetKO mice do not develop
colitis. |
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D*a
发帖数: 6830 | 39 我刚做过。。。不是故意的阿-_-!!!
GTT 和ITT基本程序差不多，不知道lz用哪种glucometre; 我用的只需要一滴血，剪尾
巴尖就够了。
我刚写的protocol.
Put one mouse per cage, aline all 15 cages. (like: I I I I I I…I).Then put
each seringe and post-it near each cage. prepare also several scissors and
pens. Inject ??U/kg insulin solution in fed mice, cut the tail to measure
glucose at T0, T15, T30 and T60.
- T0: First i.p. inject insulin, then start the timer from 0' right after,
then cut 1 mm the tail immediately and immediately measure the glucose. (
wi... 阅读全帖 |
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s****9
发帖数: 932 | 40 I am sure it is not a pleasant process for the mice. You are welcome to
submit this procedure for your institutional animal procedure committee for
review and let us know if it can be officially approved. It is not my
personal invention. Although I did not see people spray ethanol and bleach
on their fur to combine male mice, I did see animal staff spray 0.04% bleach
on the young pulps to remove their smell for the fostering process (e.g.
mom eat up puppies. They get ride of puppies' smell b... 阅读全帖 |
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s******r
发帖数: 2876 | 41 对pups来说，不用spray，也可以foster。
I am sure it is not a pleasant process for the mice. You are welcome to
submit this procedure for your institutional animal procedure committee for
review and let us know if it can be officially approved. It is not my
personal invention. Although I did not see people spray ethanol and bleach
on their fur to combine male mice, I did see animal staff spray 0.04% bleach
on the young pulps to remove their smell for the fostering process (e.g.
mom eat up puppies. They ... 阅读全帖 |
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m******5
发帖数: 1383 | 42 长过，是后来掉了的
不是transgenic mice，是knock in Mice, chimera没问题 |
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j*****d
发帖数: 787 | 43 //地地道道的理想主义者
而且他这篇文章不知道要得罪多少体制内 有共产党头衔的 红色科学家
http://blog.sciencenet.cn/home.php?mod=space&uid=2237
共产党员 精选
已有 8220 次阅读 2011-6-28 09:01 |系统分类:观点评述|关键词:剑桥大学 共产党员
诺贝尔奖 科学家
加入执政党的党员，原因不易单一化地叙述。
加入还在奋斗期间的党、或永远不能执政的党，其党员绝大多数是为了理念。
英国共产党（1920-1991）恐怕从来没有执政的机会。英共党员多彩多姿，如结构生物
学先驱（被人称Sage/智者的）JD Bernal（他的学生诺贝尔奖得主Hodgkin不是党员，
但她丈夫曾是）、著名遗传学家JBS Haldane、进化论学者John Maynard Smith，其中
不乏有剑桥大学、牛津大学的教授。他们入党，对自己的学术生涯有影响，可以说基本
是负面的。最简单的是，号称无政治歧视的美国，禁止共产党员入境，无论党员是英国
剑桥大学教授、是世界著名科学家、还是是美国需要征求特定领域意见的皇家学会会员
，都被这样对待。
英国著... 阅读全帖 |
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W*****o
发帖数: 1780 | 44 去查了一下，张旭总共发表了90多篇英文文章（不算中文）。回国之后作为联系作者也
有快20篇了。其中有cell, neuron, Journal of neuroscience, PNAS 好多篇。而且他
1995年就回国了，那个时候国内的环境有多差不用说了吧。这也快20年了。比饶益2007
年才全职回去多了12年。比起培养的中国学生也多多了，算国内贡献肯定不会小。如果
他一直留在国外，成就未必就比饶差。谁把饶益的文章列一列。国内多少篇，总共多少
篇，培养了多少中国学生？
1. Li KC, Wang F, Zhong YQ, Lu YJ, Wang Q, Zhang FX, Xiao HS, Bao L, Zhang X
. (2011) Reduction of follistatin-like 1 in primary afferent neurons
contributes to neuropathic pain hypersensitivity. Cell Res, 21: 697-699.
2. Li KC, Zhang FX, Li CL, Wang F, Yu MY, Z... 阅读全帖 |
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s**u
发帖数: 9035 | 46 Dr. Samuel M Cohen(Chairman of Department of Pathology, UNMC) wrote a letter to "Toxicological Science" to criticize a misleading from Chengfeng Yang(Michigan State University)lab's a recent paper published in this journal, see below:
Reply to “Reversal and Prevention of Arsenic-Induced Human Bronchial
Epithelial Cell Malignant Transformation by MicroRNA-200b”
1. Samuel M. Cohen1
+ Author Affiliations
1. Department of Pathology and Microbiology, University of Nebraska
Medical Center, Omaha... 阅读全帖 |
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i***R
发帖数: 663 | 47 UT Southwestern Scientist Shares 2011 Nobel Prize in Physiology or Medicine
Dr. Bruce A. Beutler, the new Director of the Center for the Genetics of
Host Defense at UT Southwestern Medical Center, today shared the Nobel Prize
in Physiology or Medicine with two other scientists for their discoveries
in how the immune system works.
Dr. Beutler and Dr. Jules A. Hoffmann of Strasbourg University’s Institut
de Biologie Moléculaire et Cellulaire in France shared half the prize for
their discovery of r... 阅读全帖 |
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c******a
发帖数: 267 | 48 I think the mouse line with tlr4 mutation is C3H/HeJ line, not C57/BL6 mice.
I did all my endotoxic experiments using LPS on C57/BL6 mice, how come they
are TLR4 deficient? |
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b****r
发帖数: 17995 | 49 Science. 1998 Dec 11;282(5396):2085-8.
Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4
gene.
Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos
E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B,
Beutler B.
Source
Howard Hughes Medical Institute and the Department of Internal Medicine,
University of Texas Southwestern Medical Center, Dallas, TX 75235-9050, USA.
mice.
they |
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c******a
发帖数: 267 | 50 I think the mouse line with tlr4 mutation is C3H/HeJ line, not C57/BL6 mice.
I did all my endotoxic experiments using LPS on C57/BL6 mice, how come they
are TLR4 deficient? |
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