seeking help in stable cell line generation - (Jul/28/2009 )
Hi, All
I am encounting problems in generating stable cell line that overexpresses the gene XXX. The host cells are B16F10, vector I used is pSecTag2B from invitogen with CMV as promoter, selection marker is Zeocin. However, all the stable clones I selected under zeocin are not expressing my gene XXX. By troubleshooting, I exclude the following factors that may cause the failure:
1. the concentration of zeocin I used for selection of stable cell lines is OK because the same concentration of zeocin kills all wildtype B16F10 cells within 2 weeks.
2. the protein expression can be detected after transient transfection, which means that the plasmid is OK as well
3. the gene XXX has been overexpressed in other cell lines by other people although the vector they used is different, therfore my protein should not be very toxic to cells.
20 clones were picked up and cultured for few passages, BUT none of them give me positive signal by Western Blot. I am looking for your comments and advice. Thank you very much in advance !
-jueduimeinv-
jueduimeinv on Jul 28 2009, 10:30 AM said:
Hi, All
I am encounting problems in generating stable cell line that overexpresses the gene XXX. The host cells are B16F10, vector I used is pSecTag2B from invitogen with CMV as promoter, selection marker is Zeocin. However, all the stable clones I selected under zeocin are not expressing my gene XXX. By troubleshooting, I exclude the following factors that may cause the failure:
1. the concentration of zeocin I used for selection of stable cell lines is OK because the same concentration of zeocin kills all wildtype B16F10 cells within 2 weeks.
2. the protein expression can be detected after transient transfection, which means that the plasmid is OK as well
3. the gene XXX has been overexpressed in other cell lines by other people although the vector they used is different, therfore my protein should not be very toxic to cells.
20 clones were picked up and cultured for few passages, BUT none of them give me positive signal by Western Blot. I am looking for your comments and advice. Thank you very much in advance !
I am encounting problems in generating stable cell line that overexpresses the gene XXX. The host cells are B16F10, vector I used is pSecTag2B from invitogen with CMV as promoter, selection marker is Zeocin. However, all the stable clones I selected under zeocin are not expressing my gene XXX. By troubleshooting, I exclude the following factors that may cause the failure:
1. the concentration of zeocin I used for selection of stable cell lines is OK because the same concentration of zeocin kills all wildtype B16F10 cells within 2 weeks.
2. the protein expression can be detected after transient transfection, which means that the plasmid is OK as well
3. the gene XXX has been overexpressed in other cell lines by other people although the vector they used is different, therfore my protein should not be very toxic to cells.
20 clones were picked up and cultured for few passages, BUT none of them give me positive signal by Western Blot. I am looking for your comments and advice. Thank you very much in advance !
I have encountered similar problems before. Depending on the number of genomic integrations, your expression may just be low. Cells generally don't need more than 1 copy of a resistance gene to survive selection, but one copy of your target gene may yield low expression. Check your cells by qRT-PCR to see if your transcript is being made and how much transcript is being made relative to other genes. Compare this to your transient transfection results as this can clue you in on the general expression level.
If the expression is there, but low, try repeating with increased zeocin concentration or a higher multiplicity of infection or higher plasmid concentration depending on how you are making your stables.
-Dr Teeth-
Dr Teeth on Jul 28 2009, 06:57 AM said:
jueduimeinv on Jul 28 2009, 10:30 AM said:
Hi, All
I am encounting problems in generating stable cell line that overexpresses the gene XXX. The host cells are B16F10, vector I used is pSecTag2B from invitogen with CMV as promoter, selection marker is Zeocin. However, all the stable clones I selected under zeocin are not expressing my gene XXX. By troubleshooting, I exclude the following factors that may cause the failure:
1. the concentration of zeocin I used for selection of stable cell lines is OK because the same concentration of zeocin kills all wildtype B16F10 cells within 2 weeks.
2. the protein expression can be detected after transient transfection, which means that the plasmid is OK as well
3. the gene XXX has been overexpressed in other cell lines by other people although the vector they used is different, therfore my protein should not be very toxic to cells.
20 clones were picked up and cultured for few passages, BUT none of them give me positive signal by Western Blot. I am looking for your comments and advice. Thank you very much in advance !
I am encounting problems in generating stable cell line that overexpresses the gene XXX. The host cells are B16F10, vector I used is pSecTag2B from invitogen with CMV as promoter, selection marker is Zeocin. However, all the stable clones I selected under zeocin are not expressing my gene XXX. By troubleshooting, I exclude the following factors that may cause the failure:
1. the concentration of zeocin I used for selection of stable cell lines is OK because the same concentration of zeocin kills all wildtype B16F10 cells within 2 weeks.
2. the protein expression can be detected after transient transfection, which means that the plasmid is OK as well
3. the gene XXX has been overexpressed in other cell lines by other people although the vector they used is different, therfore my protein should not be very toxic to cells.
20 clones were picked up and cultured for few passages, BUT none of them give me positive signal by Western Blot. I am looking for your comments and advice. Thank you very much in advance !
I have encountered similar problems before. Depending on the number of genomic integrations, your expression may just be low. Cells generally don't need more than 1 copy of a resistance gene to survive selection, but one copy of your target gene may yield low expression. Check your cells by qRT-PCR to see if your transcript is being made and how much transcript is being made relative to other genes. Compare this to your transient transfection results as this can clue you in on the general expression level.
If the expression is there, but low, try repeating with increased zeocin concentration or a higher multiplicity of infection or higher plasmid concentration depending on how you are making your stables.
Thank you very much ! I will try to increase the plamisd amount when doing transfection next time. I had tried reselecting using much higher concentration of zeocin in culture media when I could not found any postive clones. However, I still failed to detect expression in reselected stable cells by WB. Does it mean that I need to increase the zeocin concentration in the beginning of stable cells culturing?
moreover, do you have any suggestions what gene I select as comparison in qRT-PCR?
Sorry for all the questions I asked and thank you for your help again.
-jueduimeinv-
jueduimeinv on Jul 29 2009, 07:44 AM said:
Dr Teeth on Jul 28 2009, 06:57 AM said:
jueduimeinv on Jul 28 2009, 10:30 AM said:
Hi, All
I am encounting problems in generating stable cell line that overexpresses the gene XXX. The host cells are B16F10, vector I used is pSecTag2B from invitogen with CMV as promoter, selection marker is Zeocin. However, all the stable clones I selected under zeocin are not expressing my gene XXX. By troubleshooting, I exclude the following factors that may cause the failure:
1. the concentration of zeocin I used for selection of stable cell lines is OK because the same concentration of zeocin kills all wildtype B16F10 cells within 2 weeks.
2. the protein expression can be detected after transient transfection, which means that the plasmid is OK as well
3. the gene XXX has been overexpressed in other cell lines by other people although the vector they used is different, therfore my protein should not be very toxic to cells.
20 clones were picked up and cultured for few passages, BUT none of them give me positive signal by Western Blot. I am looking for your comments and advice. Thank you very much in advance !
I am encounting problems in generating stable cell line that overexpresses the gene XXX. The host cells are B16F10, vector I used is pSecTag2B from invitogen with CMV as promoter, selection marker is Zeocin. However, all the stable clones I selected under zeocin are not expressing my gene XXX. By troubleshooting, I exclude the following factors that may cause the failure:
1. the concentration of zeocin I used for selection of stable cell lines is OK because the same concentration of zeocin kills all wildtype B16F10 cells within 2 weeks.
2. the protein expression can be detected after transient transfection, which means that the plasmid is OK as well
3. the gene XXX has been overexpressed in other cell lines by other people although the vector they used is different, therfore my protein should not be very toxic to cells.
20 clones were picked up and cultured for few passages, BUT none of them give me positive signal by Western Blot. I am looking for your comments and advice. Thank you very much in advance !
I have encountered similar problems before. Depending on the number of genomic integrations, your expression may just be low. Cells generally don't need more than 1 copy of a resistance gene to survive selection, but one copy of your target gene may yield low expression. Check your cells by qRT-PCR to see if your transcript is being made and how much transcript is being made relative to other genes. Compare this to your transient transfection results as this can clue you in on the general expression level.
If the expression is there, but low, try repeating with increased zeocin concentration or a higher multiplicity of infection or higher plasmid concentration depending on how you are making your stables.
Thank you very much ! I will try to increase the plamisd amount when doing transfection next time. I had tried reselecting using much higher concentration of zeocin in culture media when I could not found any postive clones. However, I still failed to detect expression in reselected stable cells by WB. Does it mean that I need to increase the zeocin concentration in the beginning of stable cells culturing?
moreover, do you have any suggestions what gene I select as comparison in qRT-PCR?
Sorry for all the questions I asked and thank you for your help again.
Compare the expression of your target to a few housekeeping genes and some with lower expression (you can probably use primer sets already in your lab) just to get a general idea of the expression level. I still think your best bet is to compare the transcript level in your stables to a transient transfection (just make sure the plasmid DNA doesn't carry over by doing a DNase treatment on your RNA prep) and get an idea of the relative expression level that way.
As for the zeocin concentration, increasing it during the initial selection is best.
-Dr Teeth-