H*N
6 楼
Just don't give up yet. Try different chimeras. I had one line that I bred
for half a year with several chimeras. I only got one pup that carried the
mutant. That line has since been used by several labs and serveral papers,
including one Nature, and one Nature Medicine, have been published. One the
other hand, I had another line which I could never get germline transmission
. I had to retarget it.
for half a year with several chimeras. I only got one pup that carried the
mutant. That line has since been used by several labs and serveral papers,
including one Nature, and one Nature Medicine, have been published. One the
other hand, I had another line which I could never get germline transmission
. I had to retarget it.
s*a
7 楼
多谢!
继续筛。。。
the
transmission
【在 H****N 的大作中提到】
: Just don't give up yet. Try different chimeras. I had one line that I bred
: for half a year with several chimeras. I only got one pup that carried the
: mutant. That line has since been used by several labs and serveral papers,
: including one Nature, and one Nature Medicine, have been published. One the
: other hand, I had another line which I could never get germline transmission
: . I had to retarget it.
继续筛。。。
the
transmission
【在 H****N 的大作中提到】
: Just don't give up yet. Try different chimeras. I had one line that I bred
: for half a year with several chimeras. I only got one pup that carried the
: mutant. That line has since been used by several labs and serveral papers,
: including one Nature, and one Nature Medicine, have been published. One the
: other hand, I had another line which I could never get germline transmission
: . I had to retarget it.
s*a
8 楼
多谢!
继续筛。。。
the
transmission
【在 H****N 的大作中提到】
: Just don't give up yet. Try different chimeras. I had one line that I bred
: for half a year with several chimeras. I only got one pup that carried the
: mutant. That line has since been used by several labs and serveral papers,
: including one Nature, and one Nature Medicine, have been published. One the
: other hand, I had another line which I could never get germline transmission
: . I had to retarget it.
继续筛。。。
the
transmission
【在 H****N 的大作中提到】
: Just don't give up yet. Try different chimeras. I had one line that I bred
: for half a year with several chimeras. I only got one pup that carried the
: mutant. That line has since been used by several labs and serveral papers,
: including one Nature, and one Nature Medicine, have been published. One the
: other hand, I had another line which I could never get germline transmission
: . I had to retarget it.
b*s
9 楼
我没用过JM8A3,LS讲的对,除外,LZ可以考虑下面几点, my 2 cents:
1. Chimera的百分比高吗?
2. germline transmission的几率跟ES细胞的状态有很大的关系。需要可以germline的
ES line,用Germline Transmission-tested的FBS,跟cell culture整个过程也有关系。
3. 既然有chimera了,这个问题比较次要了 -- ES的karyotyping结果怎样?
4. 要确保提取的DNA质量好,PCR不是假阴性。LZ扩的片段有多大呢?
5. 排除各种低级错误 -- 由于标记错误,用来做injection的ES不是targeted,而是
wild type。
我们用hybrid的ES细胞,筛选50-70只F1之后,没有阳性的,就重新做targeting。这种
情况比较少。
你筛选的数量不多,继续做breeding吧。
1. Chimera的百分比高吗?
2. germline transmission的几率跟ES细胞的状态有很大的关系。需要可以germline的
ES line,用Germline Transmission-tested的FBS,跟cell culture整个过程也有关系。
3. 既然有chimera了,这个问题比较次要了 -- ES的karyotyping结果怎样?
4. 要确保提取的DNA质量好,PCR不是假阴性。LZ扩的片段有多大呢?
5. 排除各种低级错误 -- 由于标记错误,用来做injection的ES不是targeted,而是
wild type。
我们用hybrid的ES细胞,筛选50-70只F1之后,没有阳性的,就重新做targeting。这种
情况比较少。
你筛选的数量不多,继续做breeding吧。
s*a
11 楼
多谢2cents :)
1. 8/14是chimera;
2. 文章里号称JM8A3应该是germline competent系。FBS等用的都是core facility的
SOP;
3. 没有做karyotyping;
4. 是说的筛ES克隆的PCR吗?BAC的阳性对照和阴性对照都很好,应该不会有问题。还
有southern。
5. 复苏之后都做二次validation,出错可能性应该不大。
现在又筛了一窝6个,另一只chimera的,也是都阴性。还有其他chimera,只能再
breeding再筛筛看。
系。
【在 b**********s 的大作中提到】
: 我没用过JM8A3,LS讲的对,除外,LZ可以考虑下面几点, my 2 cents:
: 1. Chimera的百分比高吗?
: 2. germline transmission的几率跟ES细胞的状态有很大的关系。需要可以germline的
: ES line,用Germline Transmission-tested的FBS,跟cell culture整个过程也有关系。
: 3. 既然有chimera了,这个问题比较次要了 -- ES的karyotyping结果怎样?
: 4. 要确保提取的DNA质量好,PCR不是假阴性。LZ扩的片段有多大呢?
: 5. 排除各种低级错误 -- 由于标记错误,用来做injection的ES不是targeted,而是
: wild type。
: 我们用hybrid的ES细胞,筛选50-70只F1之后,没有阳性的,就重新做targeting。这种
: 情况比较少。
1. 8/14是chimera;
2. 文章里号称JM8A3应该是germline competent系。FBS等用的都是core facility的
SOP;
3. 没有做karyotyping;
4. 是说的筛ES克隆的PCR吗?BAC的阳性对照和阴性对照都很好,应该不会有问题。还
有southern。
5. 复苏之后都做二次validation,出错可能性应该不大。
现在又筛了一窝6个,另一只chimera的,也是都阴性。还有其他chimera,只能再
breeding再筛筛看。
系。
【在 b**********s 的大作中提到】
: 我没用过JM8A3,LS讲的对,除外,LZ可以考虑下面几点, my 2 cents:
: 1. Chimera的百分比高吗?
: 2. germline transmission的几率跟ES细胞的状态有很大的关系。需要可以germline的
: ES line,用Germline Transmission-tested的FBS,跟cell culture整个过程也有关系。
: 3. 既然有chimera了,这个问题比较次要了 -- ES的karyotyping结果怎样?
: 4. 要确保提取的DNA质量好,PCR不是假阴性。LZ扩的片段有多大呢?
: 5. 排除各种低级错误 -- 由于标记错误,用来做injection的ES不是targeted,而是
: wild type。
: 我们用hybrid的ES细胞,筛选50-70只F1之后,没有阳性的,就重新做targeting。这种
: 情况比较少。
s*a
13 楼
刚筛了第一窝老鼠,11个都是阴性。失望啊!!
H*N
18 楼
Just don't give up yet. Try different chimeras. I had one line that I bred
for half a year with several chimeras. I only got one pup that carried the
mutant. That line has since been used by several labs and serveral papers,
including one Nature, and one Nature Medicine, have been published. One the
other hand, I had another line which I could never get germline transmission
. I had to retarget it.
for half a year with several chimeras. I only got one pup that carried the
mutant. That line has since been used by several labs and serveral papers,
including one Nature, and one Nature Medicine, have been published. One the
other hand, I had another line which I could never get germline transmission
. I had to retarget it.
s*a
19 楼
多谢!
继续筛。。。
the
transmission
【在 H****N 的大作中提到】
: Just don't give up yet. Try different chimeras. I had one line that I bred
: for half a year with several chimeras. I only got one pup that carried the
: mutant. That line has since been used by several labs and serveral papers,
: including one Nature, and one Nature Medicine, have been published. One the
: other hand, I had another line which I could never get germline transmission
: . I had to retarget it.
继续筛。。。
the
transmission
【在 H****N 的大作中提到】
: Just don't give up yet. Try different chimeras. I had one line that I bred
: for half a year with several chimeras. I only got one pup that carried the
: mutant. That line has since been used by several labs and serveral papers,
: including one Nature, and one Nature Medicine, have been published. One the
: other hand, I had another line which I could never get germline transmission
: . I had to retarget it.
b*s
20 楼
我没用过JM8A3,LS讲的对,除外,LZ可以考虑下面几点, my 2 cents:
1. Chimera的百分比高吗?
2. germline transmission的几率跟ES细胞的状态有很大的关系。需要可以germline的
ES line,用Germline Transmission-tested的FBS,跟cell culture整个过程也有关系。
3. 既然有chimera了,这个问题比较次要了 -- ES的karyotyping结果怎样?
4. 要确保提取的DNA质量好,PCR不是假阴性。LZ扩的片段有多大呢?
5. 排除各种低级错误 -- 由于标记错误,用来做injection的ES不是targeted,而是
wild type。
我们用hybrid的ES细胞,筛选50-70只F1之后,没有阳性的,就重新做targeting。这种
情况比较少。
你筛选的数量不多,继续做breeding吧。
1. Chimera的百分比高吗?
2. germline transmission的几率跟ES细胞的状态有很大的关系。需要可以germline的
ES line,用Germline Transmission-tested的FBS,跟cell culture整个过程也有关系。
3. 既然有chimera了,这个问题比较次要了 -- ES的karyotyping结果怎样?
4. 要确保提取的DNA质量好,PCR不是假阴性。LZ扩的片段有多大呢?
5. 排除各种低级错误 -- 由于标记错误,用来做injection的ES不是targeted,而是
wild type。
我们用hybrid的ES细胞,筛选50-70只F1之后,没有阳性的,就重新做targeting。这种
情况比较少。
你筛选的数量不多,继续做breeding吧。
s*a
22 楼
多谢2cents :)
1. 8/14是chimera;
2. 文章里号称JM8A3应该是germline competent系。FBS等用的都是core facility的
SOP;
3. 没有做karyotyping;
4. 是说的筛ES克隆的PCR吗?BAC的阳性对照和阴性对照都很好,应该不会有问题。还
有southern。
5. 复苏之后都做二次validation,出错可能性应该不大。
现在又筛了一窝6个,另一只chimera的,也是都阴性。还有其他chimera,只能再
breeding再筛筛看。
系。
【在 b**********s 的大作中提到】
: 我没用过JM8A3,LS讲的对,除外,LZ可以考虑下面几点, my 2 cents:
: 1. Chimera的百分比高吗?
: 2. germline transmission的几率跟ES细胞的状态有很大的关系。需要可以germline的
: ES line,用Germline Transmission-tested的FBS,跟cell culture整个过程也有关系。
: 3. 既然有chimera了,这个问题比较次要了 -- ES的karyotyping结果怎样?
: 4. 要确保提取的DNA质量好,PCR不是假阴性。LZ扩的片段有多大呢?
: 5. 排除各种低级错误 -- 由于标记错误,用来做injection的ES不是targeted,而是
: wild type。
: 我们用hybrid的ES细胞,筛选50-70只F1之后,没有阳性的,就重新做targeting。这种
: 情况比较少。
1. 8/14是chimera;
2. 文章里号称JM8A3应该是germline competent系。FBS等用的都是core facility的
SOP;
3. 没有做karyotyping;
4. 是说的筛ES克隆的PCR吗?BAC的阳性对照和阴性对照都很好,应该不会有问题。还
有southern。
5. 复苏之后都做二次validation,出错可能性应该不大。
现在又筛了一窝6个,另一只chimera的,也是都阴性。还有其他chimera,只能再
breeding再筛筛看。
系。
【在 b**********s 的大作中提到】
: 我没用过JM8A3,LS讲的对,除外,LZ可以考虑下面几点, my 2 cents:
: 1. Chimera的百分比高吗?
: 2. germline transmission的几率跟ES细胞的状态有很大的关系。需要可以germline的
: ES line,用Germline Transmission-tested的FBS,跟cell culture整个过程也有关系。
: 3. 既然有chimera了,这个问题比较次要了 -- ES的karyotyping结果怎样?
: 4. 要确保提取的DNA质量好,PCR不是假阴性。LZ扩的片段有多大呢?
: 5. 排除各种低级错误 -- 由于标记错误,用来做injection的ES不是targeted,而是
: wild type。
: 我们用hybrid的ES细胞,筛选50-70只F1之后,没有阳性的,就重新做targeting。这种
: 情况比较少。
a*a
32 楼
进germline和es状态关系非常大,给你个极端的例子,我们有一阵子怎么都不进,过了
两年换了个养细胞的技术员就好了
【在 s******a 的大作中提到】
: 多谢2cents :)
: 1. 8/14是chimera;
: 2. 文章里号称JM8A3应该是germline competent系。FBS等用的都是core facility的
: SOP;
: 3. 没有做karyotyping;
: 4. 是说的筛ES克隆的PCR吗?BAC的阳性对照和阴性对照都很好,应该不会有问题。还
: 有southern。
: 5. 复苏之后都做二次validation,出错可能性应该不大。
: 现在又筛了一窝6个,另一只chimera的,也是都阴性。还有其他chimera,只能再
: breeding再筛筛看。
两年换了个养细胞的技术员就好了
【在 s******a 的大作中提到】
: 多谢2cents :)
: 1. 8/14是chimera;
: 2. 文章里号称JM8A3应该是germline competent系。FBS等用的都是core facility的
: SOP;
: 3. 没有做karyotyping;
: 4. 是说的筛ES克隆的PCR吗?BAC的阳性对照和阴性对照都很好,应该不会有问题。还
: 有southern。
: 5. 复苏之后都做二次validation,出错可能性应该不大。
: 现在又筛了一窝6个,另一只chimera的,也是都阴性。还有其他chimera,只能再
: breeding再筛筛看。
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