4 replies to this topic

[easternblot]

Hi!

I am performing experiments that require me to establish a stable human cell line which will express a protein upon induction. (protein is not toxic and has no endogenous expression in the cell line)

I am currently working on a new system called pTUNE (from Origene vector design here ) I chose this because it requires transfection of only one plasmid and has dual expression control. However the plasmid is quite leaky in my preliminary studies using transient transfection . I wonder if anyone else has experience working with this system? And in case everything fails, any suggestions on other systems with good control? Thanks in advance!

[pmason]

easternblot, on Jun 25 2009, 07:46 PM, said:

Hi!

I am performing experiments that require me to establish a stable human cell line which will express a protein upon induction. (protein is not toxic and has no endogenous expression in the cell line)

I am currently working on a new system called pTUNE (from Origene vector design here ) I chose this because it requires transfection of only one plasmid and has dual expression control. However the plasmid is quite leaky in my preliminary studies using transient transfection . I wonder if anyone else has experience working with this system? And in case everything fails, any suggestions on other systems with good control? Thanks in advance!

HI....I too have been seeing my protein with no IPTG induction  but I am having a terrible time trying to grow up the plasmid in sufficient quantities...what strain do you use and how do you get the plasmid prep....sorry I could not be of more help

[easternblot]

pmason, on Jul 3 2009, 04:15 AM, said:

HI....I too have been seeing my protein with no IPTG induction  but I am having a terrible time trying to grow up the plasmid in sufficient quantities...what strain do you use and how do you get the plasmid prep....sorry I could not be of more help

Its a low copy number plasmid. I grow it in DH5alpha, did 500ml culture and then maxi-prep. Got about 1.5mg of plasmid each time.

[Chit]

Hi all, I am also using the pTUNE plasmid and I believe that it's a good system for on/off experiment. However, I also found that it's leak. Let me tell me why....
This plasmid was developed by James Collin's lab, called LTRi. Actually, he deposited this plasmid, not exactly the same (he broke the plasmid into 2 plasmids), into Addgene. You can search the plasmid using keyword "LTRi" in Addgene's homepage http://www.addgene.org. Then, after choosing the plasmid LTRi, you will find the homepage of the LTRi plasmid. There is a link in the homepage which linked to a pdf file. The pdf file is the protocol of cloning of this plasmid.
In the protocol, the authors stated that there are 2 "introns" in the plasmid. Indeed, the "intron" refered to the lacO repressor binding sites. They also claimed that the "intron" is prone to recombinant with host, and subsequently lost. After sequencing, I found that the original plasmid from Origene lost 2 of the lacO binding sites (initially there should be 3 lacO sites). This caused a significant leakage in this system. I have contacted Origene and asked them to fix it. If you guys have the same experience with this vector, please try the sequence the lacO sites to see if they are deleted. If so, contact Origene immediately.
One more point to note, the authors also claimed that the plasmid is prone to recombine. Therefore, they suggested using "SURE" strain from Stratagene. For more detail, please go to Addgene and Stratagene's homepage.
Hope these help.

[IoannaA]

Chit, on Aug 28 2009, 12:59 AM, said:

Hi all, I am also using the pTUNE plasmid and I believe that it's a good system for on/off experiment. However, I also found that it's leak. Let me tell me why....
This plasmid was developed by James Collin's lab, called LTRi. Actually, he deposited this plasmid, not exactly the same (he broke the plasmid into 2 plasmids), into Addgene. You can search the plasmid using keyword "LTRi" in Addgene's homepage http://www.addgene.org. Then, after choosing the plasmid LTRi, you will find the homepage of the LTRi plasmid. There is a link in the homepage which linked to a pdf file. The pdf file is the protocol of cloning of this plasmid.
In the protocol, the authors stated that there are 2 "introns" in the plasmid. Indeed, the "intron" refered to the lacO repressor binding sites. They also claimed that the "intron" is prone to recombinant with host, and subsequently lost. After sequencing, I found that the original plasmid from Origene lost 2 of the lacO binding sites (initially there should be 3 lacO sites). This caused a significant leakage in this system. I have contacted Origene and asked them to fix it. If you guys have the same experience with this vector, please try the sequence the lacO sites to see if they are deleted. If so, contact Origene immediately.
One more point to note, the authors also claimed that the plasmid is prone to recombine. Therefore, they suggested using "SURE" strain from Stratagene. For more detail, please go to Addgene and Stratagene's homepage.
Hope these help.

Hi
Thank you for explaining this , it was a great help for me. I was wondering if you have this vector available and if you could share it? I am in mass general hospital working on a movement disorder dystonia and the role of torsinA in this disease.
In case you can help me my email address is armata.ioanna@mgh.harvard.edu

thanks a lot