急问，一个包子 mES cell里用的loxp－stop－loxp －reporter# Biology - 生物学
g*n
1 楼
中了快半年了。还是光秃秃的。这是算没种活，还是再等等？邻居家的桃树中了四年才
开始张叶子。
开始张叶子。
y*u
2 楼
请问有哪些commercial available的质粒好用的？
有人用过如下这个质粒么？
Generation of a double-fluorescent double-selectable Cre/loxP indicator
vector for monitoring of intracellular recombination events.
Pfannkuche K, Wunderlich FT, Doss MX, Spitkovsky D, Reppel M, Sachinidis A,
Hescheler J.
Source
Institute for Neurophysiology, Robert Koch Str. 39, 50931 Cologne, Germany.
Abstract
Here we describe the generation of a double-fluorescent Cre/loxP indicator
system. This protocol involves (i) all cloning steps to generate the plasmid
vector (3-5 months); (ii) a guide to prepare high-efficiency transformation
competent E. coli; (iii) generation of double-fluorescent reporter cell
lines (3-4 weeks); and (iv) the functional testing of the indicator cell
lines by application of cell-permeable Cre recombinase. The indicator is
designed to monitor recombination events by switching the fluorescence light
from red to green. The red fluorescence, indicating the nonrecombined state
, is accompanied by the expression of a resistance gene against the
antibiotic blasticidin. Appearance of green fluorescence concomitantly with
the activation of puromycin-acetyltransferase monitors the recombination of
the indicator construct by the Cre recombinase. In summary, we have
developed a plasmid vector allowing a fast, stable and straightforward
generation of transgenic clones. The expression of red fluorescent protein
enables the selection of positive clones upon transfection and significantly
shortens the time for identification of stable clones. This feature and the
option to select for recombined cells by puromycin application are
advantages compared with other alternative methods. Moreover, we developed a
method utilizing cell-permeable Cre protein to validate the transgenic
clones. Ultimately, this powerful methodology facilitates Cre/loxP-based
applications such as cell lineage tracking or monitoring of cell fusion.
有人用过如下这个质粒么？
Generation of a double-fluorescent double-selectable Cre/loxP indicator
vector for monitoring of intracellular recombination events.
Pfannkuche K, Wunderlich FT, Doss MX, Spitkovsky D, Reppel M, Sachinidis A,
Hescheler J.
Source
Institute for Neurophysiology, Robert Koch Str. 39, 50931 Cologne, Germany.
Abstract
Here we describe the generation of a double-fluorescent Cre/loxP indicator
system. This protocol involves (i) all cloning steps to generate the plasmid
vector (3-5 months); (ii) a guide to prepare high-efficiency transformation
competent E. coli; (iii) generation of double-fluorescent reporter cell
lines (3-4 weeks); and (iv) the functional testing of the indicator cell
lines by application of cell-permeable Cre recombinase. The indicator is
designed to monitor recombination events by switching the fluorescence light
from red to green. The red fluorescence, indicating the nonrecombined state
, is accompanied by the expression of a resistance gene against the
antibiotic blasticidin. Appearance of green fluorescence concomitantly with
the activation of puromycin-acetyltransferase monitors the recombination of
the indicator construct by the Cre recombinase. In summary, we have
developed a plasmid vector allowing a fast, stable and straightforward
generation of transgenic clones. The expression of red fluorescent protein
enables the selection of positive clones upon transfection and significantly
shortens the time for identification of stable clones. This feature and the
option to select for recombined cells by puromycin application are
advantages compared with other alternative methods. Moreover, we developed a
method utilizing cell-permeable Cre protein to validate the transgenic
clones. Ultimately, this powerful methodology facilitates Cre/loxP-based
applications such as cell lineage tracking or monitoring of cell fusion.
z*n
3 楼
我买的，当年冬天就桃花了，第二年就吃小毛桃了，现在是第三年，又吃了好多好多小
桃子
买来就是个小枯枝子。现在已经相当大了
【在 g******n 的大作中提到】
: 中了快半年了。还是光秃秃的。这是算没种活，还是再等等？邻居家的桃树中了四年才
: 开始张叶子。
桃子
买来就是个小枯枝子。现在已经相当大了
【在 g******n 的大作中提到】
: 中了快半年了。还是光秃秃的。这是算没种活，还是再等等？邻居家的桃树中了四年才
: 开始张叶子。
g*n
4 楼
你是来气人的吗？
【在 z*********n 的大作中提到】
: 我买的，当年冬天就桃花了，第二年就吃小毛桃了，现在是第三年，又吃了好多好多小
: 桃子
: 买来就是个小枯枝子。现在已经相当大了
【在 z*********n 的大作中提到】
: 我买的，当年冬天就桃花了，第二年就吃小毛桃了，现在是第三年，又吃了好多好多小
: 桃子
: 买来就是个小枯枝子。现在已经相当大了
f*8
5 楼
你是不是水浇太多了？我在STAKBROS 买的光脚桃树，下地3天就看到发芽了
【在 g******n 的大作中提到】
: 你是来气人的吗？
【在 g******n 的大作中提到】
: 你是来气人的吗？
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