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How to produce transgenic mice?


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10 replies to this topic

#1 Curtis

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Posted 24 January 2015 - 06:44 AM

One of my friends is asking me to help her with production of a poilovirus-receptor-expressing transgenic mice. I read many papers. All of them use the Tg line that Koike et al 1991 produced more than 22 years ago. Is it so difficult to produce a transgenic mice that only Koike provides this line to people in need?

 

Moreover, I read Vincent Racaniello's (Baltimore's student) blog. He said he was the first person to do this before Koike et al. but he killed the last line just some time ago. He produced it using cosmids. Now the technology has advanced and there might be other systems to produce such lines. What do I do? Where to find info about production of such transgenic lines?



#2 Curtis

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Posted 24 January 2015 - 10:15 PM

found it

http://www.ncbi.nlm....les/PMC3119260/



#3 Trof

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Posted 25 January 2015 - 04:27 AM

Well there are many transgenic mice, and many ways how to do it now, but home-making is getting less and less used. It takes to much time, companies make it in months. But keeping a mouse line already made alive is another thing.

 

Depends how big your modification is, but modern methods of manipulations incude Zinc-Finger Nucleases or CRISPR (but this one probably only for small changes and is much more unspecific). I know nothing about that specific thing, but targeted modifications by forementioned methods can be used to create a recombination sites for a larger DNA exchange.

 

But so far I'm only met a knockouts or mouse with a human gene with high homology or so, nothing so complicated.

 

 

If you have cells ready with the change you want, then the microinjections and mouse fertilizations.. this is usually very far from what usual lab has, and it's tricky.. (colleague had to go di a different country for this, for several years) if you need just the mouse and have a money, companies will do it now as a service, they have optimized methods.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#4 Curtis

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Posted 25 January 2015 - 11:09 AM

I need to insert the PVR gene into a vector. Don't know what vector to use, don't know which PVR receptor to amplify, don't know what promoter I need. I'm new to this and I'm still learning. They only want the plasmid from me. I think I need to go for lenti. the link above doesn't actually explain how the vector was prepared.



#5 Trof

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Posted 25 January 2015 - 01:22 PM

LOL, with this amout of information you shouldn't do anything.

Either you all sit together and they tell you everything they plan and together you decide what use and how, or if they themselves don't know it, then back off. You just can't work like that, it's always backwards, first how you make a mouse, then what cells to modify, then how to mdify them, then vector selection and for god's sake what gene they want. You wouldn't want to get stuck with a wrong gene, or in a wrong vector.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#6 Curtis

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Posted 25 January 2015 - 01:48 PM

When you start a project, many things are not crystal clear. I am not sure why you are telling me to back off. I like this project. I wouldn't lose anything if it doesn't work. At least we're trying.

 

They already have the Tg mice. They bring it from Japan. But it is costly and troublesome. That's why they must generate it here. They need it.



#7 CPRES

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Posted 25 January 2015 - 01:56 PM

There are so many ways to do transgenics with dozens of twists to the technique. There is also this consideration of what promoter you want your gene to be expressed under, do you want the gene to be tagged, do you want a bicistronic construct, do you want it in plasmid, bac, viral construct, do you want just transgenic or knock-in transgenic line, etc. Not to mention conditional expression. If you have not done transgenic for a year or two, do NOT plan anything yourself. Meet experts, meet your collaborators, meet all together. Otherwise you are heading towards a certain disaster.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#8 Curtis

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Posted 25 January 2015 - 02:01 PM

we are about 10 people. thanks for the link.



#9 Trof

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Posted 25 January 2015 - 02:07 PM

No, I was just saying that project that doesn't have defined strategy or backbone before you start working on it, is a bad one from start.

It doesn't have to be crystal clear, but it has to be at least transparent enough to see from the start to the end.

Because you can lose, time, effort and nerves also.

 

 

There are funny projects and there are difficult projects, and challenging, and then there are nightmare project where you don't even know why are you trying anymore and if anthing goes wrong you are to blame, because someone has to be. And those later often start as a projects without proper study design or goal or something. That's why I warn everyone to beware of the fish that stinks already the first day. but that was not the main point. The main point was that you SHOULD have project outline before you start cloning something.

 

 

And I don't know the prices of mice and transport from Japan, but I would seriously doubt that it's more costly than to generate a knockin anew. Mice you can breed to get more of them (you would need to anyway from the transfected hybrid). If the mouse line exists I really think making a new one and starting a new line is neither cheaper nor quicker. If you do it in-house and are not a mouse-generating lab, then definitelly not quicker.

 

 

But maybe services today gone very cheap and you only need a kick-ass vector for that. But I don't think this is the case. Making knockin mouse by vector and homologous recombination was a several year project in the past, sometimes as a proof of concent too, but there was no other option. Now you can buy it.
So, maybe you have several years to startt from the scratch, it's a nice side project, you know, to "build a mouse". But if they need the mouse now, for testing a hypothesis, I really thing it's better to buy the mouse (also, if you make a new knockin, it has to be characterized, so to prove that only has this transgene, existing mouse lines has already been characterized, so more work again).


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#10 Trof

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Posted 25 January 2015 - 02:10 PM

Also another thing to consider.. you should also check whether the transgenic mice wasn't patented (I'm not sure if people do that but nowadays you can patent almost anything). If was, then making the same trangenic animal would risk possible penalty or something.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#11 Curtis

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Posted 26 January 2015 - 02:26 PM

I designed it already. I designed primers to amplify the CDS of the PVR (isoform 1) from its cDNA, and then clone into pLVX-Puro by Clontech. You dont even need to use microinjection with this system. 






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