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Transfecting Trypanosome without electroporator

trypanosome transfection

Best Answer phage434, 14 March 2015 - 07:14 AM

The only item missing from that electroporator on Ebay is the unit holding the cuvette. You can easily make one of these (although you have to be careful of the high voltage). Enlist help from one of your electrical engineering colleagues.

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#1 mgp

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Posted 12 March 2015 - 09:11 PM

Hi everybody!

 

I work in a very small and new lab focused on Trypanosoma cruzi (needless to say, with a very tight budget, which seems to be an epidemic lately).

 

We need to get the molecular part of the research center going (it has been focused on epidemiology research and chemistry). The molecular lab is a recent addition and in order to make it grow we need to have an ongoing project with the few things we have.

 

I basically have PCR and electrophoresis capabilities, a mass spec, sonicator, incubators and centrifuges. There is a rather "simple" project that focuses on new PCR methods to KO certain proteins to determine if they are essential or not (for future use as drug targets).

 

Problem is, we don't have an electroporator and I don't think we are getting one any time soon. I am trained in bacterial genetics and in some immunology, but I have no previous experience with protozoans.

 

So the question: is it possible to transfect trypanosomes chemically (i.e. calcium phosphate)? Or any other way that doesn't require equipment? I am willing to try chemical transfection, but I don't want to waste money we barely have in something that has been proven as ineffective.

 

Any suggestion (transfection methods or research with the few things I have) will be highly appreciated!

 



#2 phage434

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Posted 13 March 2015 - 06:43 AM

So, electroporators are quite cheap on Ebay. For example:

http://www.ebay.com/...=item2edbc13ef3

 

Alternatively, Cullen Buie at MIT has shown that mechanically squeezing cells can induce transfection. I believe you could do this at low efficiency by mixing cells and DNA and passing the result through a filter sized to barely pass the cells.



#3 pito

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Posted 14 March 2015 - 12:15 AM

I briefly worked on protozoans and I have not really encountered papers on transfection using a chemical method... The first breakthrough in protozoans transfection actually happened when they were able to use an electroporation system.

 

Nowadays they actually use a novel system that is even better: nucleofection. It is even more expensive.

Transfection protozoans is not that easy!

 

My best guess for you would be to use an electroporation device.. you would waste too much time otherwise.

 

You could buy one , second hand, sometimes labs sell theirs and they can still be good.

Another option is to write to companies that sell these machines and ask them if they would let you use their machine for a while to test it.... labs often do this too.. you do not need to buy it in the end so you are just using it for a short while.

Of you try the latter you of course need a good protocol and hope it works right away.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#4 mgp

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Posted 14 March 2015 - 06:57 AM

Thank you both of you for your answers.

Phage434: the unit on ebay is incomplete. But still, I will ask if we can buy a second hand one.

Pito: I live and work in a developing nation. The equipment testing will not be possible. Even buying a second hand will not be that easy because of the burocratic system involved in buying equipment. In order to make things with more "transparency" and "offer benefits for local businesses" the system actually stalls this kind of purchases and promotes monopoly (with the subsequent ridiculous prices we have to pay for the stuff we need). It may work in other areas, but not for biomedical research. But there's always a workaround. I hope I find one :D

 

Oh the extra challenges a reasearcher can find in life! As if the experiments by itself were not enough!



#5 pito

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Posted 14 March 2015 - 07:07 AM

well, try to find one nearby that you can use? from another university?

 

I can see it will be a difficult task then!


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#6 mgp

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Posted 14 March 2015 - 07:14 AM

No electroporators are found (death or alive) in this country :(

I think I should change research. Try to find something that we can do without genetically manipulate trypanosome. Using E. coli or the likes instead?

I also have an idea with recombinant antibodies and phage display. It is more complicated that the project needing an electroporator, but we can work with E. coli and ELISA for validation....

 

I tell you, my life is hard :P



#7 phage434

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Posted 14 March 2015 - 07:14 AM   Best Answer

The only item missing from that electroporator on Ebay is the unit holding the cuvette. You can easily make one of these (although you have to be careful of the high voltage). Enlist help from one of your electrical engineering colleagues.







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