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stable 3t3l1 transfection:serious help needed!!!


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#1 drevilette_R

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Posted 17 March 2009 - 01:38 AM

Hey Guys,
I m just getting started with stabile transfections of 3t3L1 cells. and i am the only one in my lab to ever do this.i have been having huge problems lately with contamination in the medium containing G418, even though i never ever have contamination problems otherwise. so while reading the forums i decided to add P/S/G to my medium with G418. for the transfektion itself i m gonna keep the medium antibiotic free for 24 h i guess. ok so right now i m using 500g/ml G418 starting 48 h after transfection. what i would like to know is how do i select my colonies exactly? i have some expressing the pIRES-EGFP vector and others the PSCT2 vector. how exactly do i "Screen" the colonies?? and once i screen my colonies and find the perfect one, how exactly do i select it our of the petri?? i know it s a silly question but i am a med student with no experience in this domain and no body at ma lab has worked with this before... :) pleeaaassseee heeellllppp!!!
thanks in advance!
Rima

#2 memo

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Posted 17 March 2009 - 06:58 AM

As I understand you perform a co-transfection with two different plasmids? I have always learned that co-transfection of two plasmids in the same complex (with your reagent) has only a few artefacts, but normally both plasmids will transfect the same cells.

With my reagent SAINT-MIX I use always antibiotics in the medium without affecting my transfections. So transfect your cells with the antibiotics and add G418 48hours later. You will refresh the medium minimal once a week. If you have a lot of transfected cells it is possible you must split the cells. Select for min. 2 weeks and dilute them in a 96 well plate untill you have about 1 cells per well (at first not visible). After about 1 week there will appear small colonies of cells. Select the wells with only one island of cells and grow them further in bigger wells to screen for eGFP positivity and the other protein which is expressed.

Good luck!

#3 drevilette_R

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Posted 19 March 2009 - 09:21 AM

As I understand you perform a co-transfection with two different plasmids? I have always learned that co-transfection of two plasmids in the same complex (with your reagent) has only a few artefacts, but normally both plasmids will transfect the same cells.

With my reagent SAINT-MIX I use always antibiotics in the medium without affecting my transfections. So transfect your cells with the antibiotics and add G418 48hours later. You will refresh the medium minimal once a week. If you have a lot of transfected cells it is possible you must split the cells. Select for min. 2 weeks and dilute them in a 96 well plate untill you have about 1 cells per well (at first not visible). After about 1 week there will appear small colonies of cells. Select the wells with only one island of cells and grow them further in bigger wells to screen for eGFP positivity and the other protein which is expressed.

Good luck!



Thanks a Million!!! this is a great help definitely!! the screening part with the 96 well step just sounded like too much work to be true but i guess it s the only way:D
one question though.. how efficient is a transfection with the 2 plasmids?? the first plasmif carries my construct and the second one carries my Antibiotic resistance. is it really a good idea to transfect with both vectors ? i mean the cell might keep my PKJNeo vector with the G418 resistance and throw the other one out. i mean to me it just seems like a logical thing to do if i were a cell..!!! in that case my transfection is never gonna be stable because over a certain period of time my cells can decide that they just wanna keep the AB resistance and throw the other one out and i wouldnt notice it..(partially because i dont have a fluorescent protein in it and i ll have to do FACS each time..lots of money and time wasted!!)
best regard and thanks again for ur amazing help!!

#4 madrius1

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Posted 19 March 2009 - 10:30 AM

Hi there,

One thing you should know about 3T3-L1 is that they are pretty, pretty hard to transfect. Even adenovirus, at an MOI of 2000, lend about 50% of positively infected cells. If I can give you an advice, I would tell you to go for a lentivirus/retrovirus mediated transduction. I get about 100% positively transduced cells that way.

Also, you can easily select a viral construction carrying both your gene of interest and a puromycin resistance gene. Puromycin kills uninfected cells in two days, rather than one to two weeks with gentamycin.

And you were right when you talked about you cells keeping only the resistance carrying plasmid!

Hope this helps

Madrius

#5 bob1

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Posted 19 March 2009 - 03:49 PM

Regarding the contamination, it sounds like you have a stock of something contaminated with a G418 resistant strain. I would throw out your stocks of G418 and the medium you are using currently, as well as the transfection reagents (including DNA) and start with new stuff.

Stable transfection takes a long time and is tricky to do. For the single colonies, I seeded mine in 6 well plates with about 1000 cells per well and left to grow for quite some time, until colonies of 50+ cells had established, then picked the colonies by trypsinising gently (1:1 trypsin:PBS) and then sucking the colonies off the plate with a 20ul pipette tip, and placing into 96 well plates. From there I grew them up until I had enough to freeze a tube and be able to assay each clone for the desired characteristics. I had about a 70% survival rate for the picking colonies thing.

I did this after trying to seed 1 cell per well in 96 well plates and having no successful clones. Cells typically need some other cells around to provide various metabolites for good growth. You could try conditioned medium (take a flask of cells and add fresh medium, leave for 24 hours, then remove medium and filter - voila conditioned medium) which will contain these factors, and should help with cell growth.

You could screen your colonies for each plasmid by PCR (will still detect any plasmids that haven't integrated though, not that there should be any after about 2 weeks) or by Southern blot for detection of integrated DNA.

#6 drevilette_R

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Posted 20 March 2009 - 01:30 AM

Hi there,

One thing you should know about 3T3-L1 is that they are pretty, pretty hard to transfect. Even adenovirus, at an MOI of 2000, lend about 50% of positively infected cells. If I can give you an advice, I would tell you to go for a lentivirus/retrovirus mediated transduction. I get about 100% positively transduced cells that way.

Also, you can easily select a viral construction carrying both your gene of interest and a puromycin resistance gene. Puromycin kills uninfected cells in two days, rather than one to two weeks with gentamycin.

And you were right when you talked about you cells keeping only the resistance carrying plasmid!

Hope this helps

Madrius



Hey Madrius!
thanks! i have so little insight into the subject..right now i feel like it s growing into a loop and sucking me in!
ok, so is there a certain lentivirus vector that you can recommand, that had worked well for you? or even a database of vectors that i can use for
my 3t3L1 cells?
i m sure u can probably buy the vector itself with a Antibiotic resistance (thanks for the tip with puromycin!) and just clone ur gene of interest in it like any other vector right?? or is it a lot trickier??
u know.. the thing with this cotransfection is that i m supposed to do it according to Evans et al. from 1993 :cotransfection with PKJ-neo.
first of all i can t seem to find this publication ANYWHERE..just other publication citing it. and i can t find PKJ-Neo! i mean i guess it has a neomycin resistance. but i dont see why i should be using it..i have my gene cloned on pIRES2-eGFP which ITSELF carries a neomycin resistance.. what s the point of throwing PKJ-neo in anyway??!!
have u heard about this kind of cotransfection?
thanks a lot!
Rima

#7 drevilette_R

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Posted 20 March 2009 - 01:43 AM

Regarding the contamination, it sounds like you have a stock of something contaminated with a G418 resistant strain. I would throw out your stocks of G418 and the medium you are using currently, as well as the transfection reagents (including DNA) and start with new stuff.

Stable transfection takes a long time and is tricky to do. For the single colonies, I seeded mine in 6 well plates with about 1000 cells per well and left to grow for quite some time, until colonies of 50+ cells had established, then picked the colonies by trypsinising gently (1:1 trypsin:PBS) and then sucking the colonies off the plate with a 20ul pipette tip, and placing into 96 well plates. From there I grew them up until I had enough to freeze a tube and be able to assay each clone for the desired characteristics. I had about a 70% survival rate for the picking colonies thing.

I did this after trying to seed 1 cell per well in 96 well plates and having no successful clones. Cells typically need some other cells around to provide various metabolites for good growth. You could try conditioned medium (take a flask of cells and add fresh medium, leave for 24 hours, then remove medium and filter - voila conditioned medium) which will contain these factors, and should help with cell growth.

You could screen your colonies for each plasmid by PCR (will still detect any plasmids that haven't integrated though, not that there should be any after about 2 weeks) or by Southern blot for detection of integrated DNA.


Hey!
uurrgghh..throw everything out??? it took me a half a year to get my bastard gene cloned into my vector!! but the thing is , i thought that G418 kills mammalian cells..ONLY.. so i guess which ever bacteria is growing in there probably doesnt care that i have geneticin in.. and is especially delighted that i have no other antibiotic in there (such as Pen/strep) that i always use.. so right now i added these to my NEW medium (of course) and i am just gonna see what happens.
i dont get the thing with the 6 well plates so well.. so u splitt ur cells onto the 6 well plate with a density of 1000/ well and selected them with the Antibiotic for donno..2 weeks lets say.. so they grew..and they formed colonies? isnt possible that the cells just clump together (even at 1000?well) or can u really tell that it s a cleam clone?? ..and then u trypsinized (but i guess u did that with a minimal volume of trypsin so that cells arent flowing around .. ) and did u pick the clones under the microscope or what???
i think u re also right about the cells growing better when they re together.. i noticed that when i started working with 3t3l1 ! it s almost cute :)
thanks again for ur reply and let me know if i understood u right on those points.

#8 madrius1

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Posted 23 March 2009 - 09:27 AM

Hi there,

One thing you should know about 3T3-L1 is that they are pretty, pretty hard to transfect. Even adenovirus, at an MOI of 2000, lend about 50% of positively infected cells. If I can give you an advice, I would tell you to go for a lentivirus/retrovirus mediated transduction. I get about 100% positively transduced cells that way.

Also, you can easily select a viral construction carrying both your gene of interest and a puromycin resistance gene. Puromycin kills uninfected cells in two days, rather than one to two weeks with gentamycin.

And you were right when you talked about you cells keeping only the resistance carrying plasmid!

Hope this helps

Madrius



Hey Madrius!
thanks! i have so little insight into the subject..right now i feel like it s growing into a loop and sucking me in!
ok, so is there a certain lentivirus vector that you can recommand, that had worked well for you? or even a database of vectors that i can use for
my 3t3L1 cells?
i m sure u can probably buy the vector itself with a Antibiotic resistance (thanks for the tip with puromycin!) and just clone ur gene of interest in it like any other vector right?? or is it a lot trickier??
u know.. the thing with this cotransfection is that i m supposed to do it according to Evans et al. from 1993 :cotransfection with PKJ-neo.
first of all i can t seem to find this publication ANYWHERE..just other publication citing it. and i can t find PKJ-Neo! i mean i guess it has a neomycin resistance. but i dont see why i should be using it..i have my gene cloned on pIRES2-eGFP which ITSELF carries a neomycin resistance.. what s the point of throwing PKJ-neo in anyway??!!
have u heard about this kind of cotransfection?
thanks a lot!
Rima


Hi there, sorry about the delay.

For the cloning of your gene in a retroviral/lentiviral vector, it goes as you said : you just find a retroviral (pBabe.puro) or a lentiviral (pLVTHM) and clone your gene in it. The virus production is not that hard neither, you just cotransfect 2 (for retrovirus) or 3 (for lentivirus) plasmids into 293T cells and wait two days. The ready-to-use viruses will be in the supernatant.

You can check the litterature for plasmids you may want to try : there are plenty of them! As for the resistance, I know pBabe has the puro gene in it. pLVTHM has a IRES-GFP, I'm not shure about antibiotics. But as I said, lentivirus mediated transduction works with almots 100% or my 3T3-L1.

Not sure I get your method for co-transfection.. I would really go with virus-mediated transduction :) Make sure your facilty has the right bio-security level for using such viruses!

Madrius

#9 memo

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Posted 25 March 2009 - 08:13 AM

Hi I don't have a lot of time on my hands at the moment, but I see you have get a lot answers.
I prefer to do a single plasmid transfection instead of co-transfection as you mentioned the problem which could arise.
I transfect 3T3-NIH cells stably with a vector containing also the GFP gene. After stabilization (96-well selection) I did get a lot of resistant clones with G418 resitance, but no GFP expression. Because I could screen a lot of clones there were also positive clones in my experiment, giving 80-95% GFP (strange????) expression!

#10 drevilette_R

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Posted 25 March 2009 - 12:40 PM

Thanks for all the help! really , i appreciate this immensely. i ve never done anything with viruses before and it would be definitely very interesting to master a new method. i d have to ask my supervisor about her opinion and i ll probably have to ask u some more question if i were to start off with this new Field!
so thanks again! i ll keep u updated!

#11 drevilette_R

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Posted 25 March 2009 - 12:44 PM

Hi I don't have a lot of time on my hands at the moment, but I see you have get a lot answers.
I prefer to do a single plasmid transfection instead of co-transfection as you mentioned the problem which could arise.
I transfect 3T3-NIH cells stably with a vector containing also the GFP gene. After stabilization (96-well selection) I did get a lot of resistant clones with G418 resitance, but no GFP expression. Because I could screen a lot of clones there were also positive clones in my experiment, giving 80-95% GFP (strange????) expression!


yes! i really thought that would be smarter, so this is the first thing i m planning to try . that s to clone my gene in pIRES2 EGFP. i m doing a killing curve actually and u re right about those crazy resistant cells! i mean a cell has its reasons. :)
i ll make sure to screen hundreds and hundreds of clones then!
so thanks for the input! and good for u that u had such high expression levels all in all!
have a great rest of the week and again thank u for ur help! ^_^




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