ES cell targeting - (May/11/2008 )
Is there anyone who can tell me the positive rate of the ES homologous recombination? After gene targeting, I got 200 ES cell strains. But by now, I can't find any positive clone. I am doing the PCR screening. I can't understand why I got some bands which size is bigger than the WT genomic band but is smaller than recombination band? Is it possible the neo gene recombinating with the ES genome partly? Any suggestion is very welcome! Thank you very much.
-wuyu-
QUOTE (wuyu @ May 11 2008, 05:31 PM)
Is there anyone who can tell me the positive rate of the ES homologous recombination? After gene targeting, I got 200 ES cell strains. But by now, I can't find any positive clone. I am doing the PCR screening. I can't understand why I got some bands which size is bigger than the WT genomic band but is smaller than recombination band? Is it possible the neo gene recombinating with the ES genome partly? Any suggestion is very welcome! Thank you very much.
The rate for ES cell HR is generally very law. With positive selection also it comes to about 1-10% of growing clones. No one can tell you precise rate, it depends upon a lot of factors.
1. ES cell quality
2. Handling
3. SIze of homologous arms
4. Isogenic or non-isogenic targeting DNA
5. Expression of ab resistance protein
6. Number of repeats in targeted sequence
Most of the clones you have are random integrants, it is not only possible, but it is more common than HR, so you need to do southern using 5 and 3' external probes (external to your 5 and 3' HR arms). PCR screening after HR is risky, not at all advisable. Southern has to be done. (less preferable --loss of allele real-time PCR)
hth/
-cellcounter-
QUOTE (cellcounter @ May 11 2008, 07:31 PM)
QUOTE (wuyu @ May 11 2008, 05:31 PM)
Is there anyone who can tell me the positive rate of the ES homologous recombination? After gene targeting, I got 200 ES cell strains. But by now, I can't find any positive clone. I am doing the PCR screening. I can't understand why I got some bands which size is bigger than the WT genomic band but is smaller than recombination band? Is it possible the neo gene recombinating with the ES genome partly? Any suggestion is very welcome! Thank you very much.
The rate for ES cell HR is generally very law. With positive selection also it comes to about 1-10% of growing clones. No one can tell you precise rate, it depends upon a lot of factors.
1. ES cell quality
2. Handling
3. SIze of homologous arms
4. Isogenic or non-isogenic targeting DNA
5. Expression of ab resistance protein
6. Number of repeats in targeted sequence
Most of the clones you have are random integrants, it is not only possible, but it is more common than HR, so you need to do southern using 5 and 3' external probes (external to your 5 and 3' HR arms). PCR screening after HR is risky, not at all advisable. Southern has to be done. (less preferable --loss of allele real-time PCR)
hth/
Thank you very much! But what do you mean the random integrants? Did the random integrants lost part DNA sequence when HR? I still don't understand why you say I need to do southern. Can you explain it in detail? Thank you.
-wuyu-