Puromycin resistance protein expression and stability - (Jun/30/2014 )

Hi folks,

I'm hoping that someone might be able to answer a problem I'm having. i'm trying to add a GFP tag to my protein of interest in situ in the genome using TALENS and a GFP/puro donor plasmid. 

The problem I'm having is that my puro resistance protein is effectively under the same transcriptional control as my protein of interest as the GFP is replacing the stop codon, and the puro resistance gene is separated from the GFP by a 2A linker. My protein of interest  is expressed only in S-phase though. So my question is, will the puro resistance protein be stable enough in the cell to allow for selection of those cells that have the correct integration?

Thanks in advance for any help or advice you might be able to spare! 

All the best,

Rob

robradford on Mon Jun 30 18:18:09 2014 said:

Hi folks,

 

I'm hoping that someone might be able to answer a problem I'm having. i'm trying to add a GFP tag to my protein of interest in situ in the genome using TALENS and a GFP/puro donor plasmid. 

 

The problem I'm having is that my puro resistance protein is effectively under the same transcriptional control as my protein of interest as the GFP is replacing the stop codon, and the puro resistance gene is separated from the GFP by a 2A linker. My protein of interest  is expressed only in S-phase though. So my question is, will the puro resistance protein be stable enough in the cell to allow for selection of those cells that have the correct integration?

 

Thanks in advance for any help or advice you might be able to spare! 

 

All the best,

 

Rob

Hi Rob!

I would think that the stability of the PAC is only half of the equation.  The other half would be how active the promoter is during the S-Phase and whether the targeting vector that carries the GFP-2A-Puro also expresses on its own.  If the vector is capable of expressing the PAC before recombination at the TALEN's cut site, then you can do a pulse selection of two days to increase the number of stable integrants.  I've seen a couple papers that used Puromycin to select for transient resistance (one included below) but they used a higher lever promoter to do this.  If your targeting vector does not express on its own, then you'll probably have to find out through trial and error.  Worst case scenario, you have to flow-sort for GFP-positive cells.

"Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: A transient gene-integration marker for ES cells"

http://nar.oxfordjournals.org/content/26/2/679.long

Bio-Lad on Tue Jul 1 20:20:20 2014 said:

 

robradford on Mon Jun 30 18:18:09 2014 said:

Hi folks,

 

I'm hoping that someone might be able to answer a problem I'm having. i'm trying to add a GFP tag to my protein of interest in situ in the genome using TALENS and a GFP/puro donor plasmid. 

 

The problem I'm having is that my puro resistance protein is effectively under the same transcriptional control as my protein of interest as the GFP is replacing the stop codon, and the puro resistance gene is separated from the GFP by a 2A linker. My protein of interest  is expressed only in S-phase though. So my question is, will the puro resistance protein be stable enough in the cell to allow for selection of those cells that have the correct integration?

 

Thanks in advance for any help or advice you might be able to spare! 

 

All the best,

 

Rob

Hi Rob!

 

I would think that the stability of the PAC is only half of the equation.  The other half would be how active the promoter is during the S-Phase and whether the targeting vector that carries the GFP-2A-Puro also expresses on its own.  If the vector is capable of expressing the PAC before recombination at the TALEN's cut site, then you can do a pulse selection of two days to increase the number of stable integrants.  I've seen a couple papers that used Puromycin to select for transient resistance (one included below) but they used a higher lever promoter to do this.  If your targeting vector does not express on its own, then you'll probably have to find out through trial and error.  Worst case scenario, you have to flow-sort for GFP-positive cells.

 

 

"Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: A transient gene-integration marker for ES cells"

http://nar.oxfordjournals.org/content/26/2/679.long

 

Thanks Bio-Lad,

Yeah, I suspect that my protein of interest isn't exactly heavily expressed during S-phase either. Unfortunately my donor plasmid doesn't have a promoter upstream of the puro resistance gene - the GFP/2A/Puro sequence replaces the stop codon of my protein of interest. Sorting was something that I was hoping not to have to do, but I guess I might not have a choice. I was also looking into inserting a promoter between the GFP and puro genes but I'm not sure how well that will work (I'm kind of new to this kind of work).

Thanks for the help!

All the best,

Rob

robradford on Tue Jul 1 20:53:55 2014 said:

 

Bio-Lad on Tue Jul 1 20:20:20 2014 said:

 

robradford on Mon Jun 30 18:18:09 2014 said:

Hi folks,
 
I'm hoping that someone might be able to answer a problem I'm having. i'm trying to add a GFP tag to my protein of interest in situ in the genome using TALENS and a GFP/puro donor plasmid. 
 
The problem I'm having is that my puro resistance protein is effectively under the same transcriptional control as my protein of interest as the GFP is replacing the stop codon, and the puro resistance gene is separated from the GFP by a 2A linker. My protein of interest  is expressed only in S-phase though. So my question is, will the puro resistance protein be stable enough in the cell to allow for selection of those cells that have the correct integration?
 
Thanks in advance for any help or advice you might be able to spare! 
 
All the best,
 
Rob

Hi Rob!
 
I would think that the stability of the PAC is only half of the equation.  The other half would be how active the promoter is during the S-Phase and whether the targeting vector that carries the GFP-2A-Puro also expresses on its own.  If the vector is capable of expressing the PAC before recombination at the TALEN's cut site, then you can do a pulse selection of two days to increase the number of stable integrants.  I've seen a couple papers that used Puromycin to select for transient resistance (one included below) but they used a higher lever promoter to do this.  If your targeting vector does not express on its own, then you'll probably have to find out through trial and error.  Worst case scenario, you have to flow-sort for GFP-positive cells.
 
 
"Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: A transient gene-integration marker for ES cells"
http://nar.oxfordjournals.org/content/26/2/679.long

 

Thanks Bio-Lad,
 
Yeah, I suspect that my protein of interest isn't exactly heavily expressed during S-phase either. Unfortunately my donor plasmid doesn't have a promoter upstream of the puro resistance gene - the GFP/2A/Puro sequence replaces the stop codon of my protein of interest. Sorting was something that I was hoping not to have to do, but I guess I might not have a choice. I was also looking into inserting a promoter between the GFP and puro genes but I'm not sure how well that will work (I'm kind of new to this kind of work).
 
Thanks for the help!
 
All the best,
 
Rob

 

 
You could conceivably make an Expression/Targeting construct as diagrammed below.  I've made a similar one for a gene I was targeting a while back and it worked great.  You'd go with the first if your 5' homology arm includes a portion of an intron or the lower one if your 5' homology arm is straight coding sequence (you'd have to make sure to keep it in frame with the starter sequence, obviously) or has intact introns.
 HA = Homology Arm

pA = Poly A signal

Bio-Lad on Tue Jul 1 21:45:44 2014 said:

 

robradford on Tue Jul 1 20:53:55 2014 said:

 

Bio-Lad on Tue Jul 1 20:20:20 2014 said:

 

robradford on Mon Jun 30 18:18:09 2014 said:

Hi folks,
 
I'm hoping that someone might be able to answer a problem I'm having. i'm trying to add a GFP tag to my protein of interest in situ in the genome using TALENS and a GFP/puro donor plasmid. 
 
The problem I'm having is that my puro resistance protein is effectively under the same transcriptional control as my protein of interest as the GFP is replacing the stop codon, and the puro resistance gene is separated from the GFP by a 2A linker. My protein of interest  is expressed only in S-phase though. So my question is, will the puro resistance protein be stable enough in the cell to allow for selection of those cells that have the correct integration?
 
Thanks in advance for any help or advice you might be able to spare! 
 
All the best,
 
Rob

Hi Rob!
 
I would think that the stability of the PAC is only half of the equation.  The other half would be how active the promoter is during the S-Phase and whether the targeting vector that carries the GFP-2A-Puro also expresses on its own.  If the vector is capable of expressing the PAC before recombination at the TALEN's cut site, then you can do a pulse selection of two days to increase the number of stable integrants.  I've seen a couple papers that used Puromycin to select for transient resistance (one included below) but they used a higher lever promoter to do this.  If your targeting vector does not express on its own, then you'll probably have to find out through trial and error.  Worst case scenario, you have to flow-sort for GFP-positive cells.
 
 
"Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: A transient gene-integration marker for ES cells"
http://nar.oxfordjournals.org/content/26/2/679.long

 

Thanks Bio-Lad,
 
Yeah, I suspect that my protein of interest isn't exactly heavily expressed during S-phase either. Unfortunately my donor plasmid doesn't have a promoter upstream of the puro resistance gene - the GFP/2A/Puro sequence replaces the stop codon of my protein of interest. Sorting was something that I was hoping not to have to do, but I guess I might not have a choice. I was also looking into inserting a promoter between the GFP and puro genes but I'm not sure how well that will work (I'm kind of new to this kind of work).
 
Thanks for the help!
 
All the best,
 
Rob

 

 
You could conceivably make an Expression/Targeting construct as diagrammed below.  I've made a similar one for a gene I was targeting a while back and it worked great.  You'd go with the first if your 5' homology arm includes a portion of an intron or the lower one if your 5' homology arm is straight coding sequence (you'd have to make sure to keep it in frame with the starter sequence, obviously) or has intact introns.
 HA = Homology Arm

pA = Poly A signal

 

Thanks for that Bio-Lad. So would the plasmids you have detailed above express the PAC protein once they are transfected? In other words, they don't need integration? Did that make selection difficult for you, or was it just a case of selecting for the cells that had transfected 1st, then selecting cells that had integrated the construct at a later time?

Once again, thanks for the help, I really appreciate the advice,

Rob

You would be selecting for initial transfected cells. If properly made, the construct should express the Pac at a high level from the cmv promoter after transaction and then lose the promoter during homologous recombination with your HAs. 

Another alternative which is used more often is a Frt or LoxP-flanked resistance cassette. This may be a better choice if you want longer term, high level expression for better selection. You can get the sequence easily by pcr amplifying it from vectors such as this one from Addgene (http://www.addgene.org/22073/) and then ligating it after the stop codon and the polyA signal of your construct.