Pronucleus microinjection

microinjection of DNA to murine pronucleus

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[psobies]

Dear lolpvp,

the micropipette on the right is called "holding" the one on the left "injection"

cheers,

Peter

[Brianall7s]

Great video!  Reminds me of the good old days. I miss the lab sometimes...

[Brianall7s]

@mckebb01 Also, the DNA injected is linearized DNA. Plasmids are toxic so the gene of interest is excised and purified. We use a different technique to eliminate a certain gene (called a knockout) or add a functioning gene (called a knockin). Cloning calls for removing the entire nucleus and replacing it with conditioned nucleus from the animal you are cloning. This requires a great deal of work because cells are programmed to be a certain tissue and they don't like being reset.

[Brianall7s]

@mckebb01 They aren't being rejected, just repositioned. The nucleolus is very sticky and will ruin the egg and the needle. So the technician is releasing and repositioning the egg so that the pronuclei are in a better plane of focus and the nucleolus is in a better position away from the path of the needle. Avoiding the polar bodies just helps because they will cause the eggs to shift and that wrecks the position of the nucleus.

[mckebb01]

How are these oocytes being selected? I am noticing the rejection of multiple oocytes, but I am curious as to why? Anything pertaining to polar bodies?

[mckebb01]

How are these oocytes being selected? I am noticing the rejection of multiple oocytes, but I am curious as to why? Anything pertaining to polar bodies?

[lolzpvp]

@psobies Thank you!

[lolzpvp]

What is the thing called that "sucks" the cell to it?

[0sali]

I THINK - if they are injecting DNA into the nucleus to obtain a certain protein, there are either plasmids injected that are producing any gene products or the new DNA is integrated into the genome.

If you want to clone a whole animal then it's of course necessary to remove the other dna first.... or so