So we do NU Nuclear transfer because our group is interested in reprogramming. This is a fascinating process in that the differentiated nucleus can be reprogrammed into an embryonic state. These are the reagents case SUM and HCCV and this is hyaluronidase.
And first I prepare a dish with, and this will go to the incubators to equate with a CO2. These are drops, are several drops for Turing that covered with mineral oil. So they calibrate CO2 atmosphere.
Now make drop of HCZB. This will be used for washing. This is high on days and again covered in this mineral oil.
This high, this drops with hyaluronidase. This part here are just HCCP drops. These are for washing.
This is for removing ulu cells from the O side. This stage is heated so it will warm up until I come back and get the oil signs from the mice and cover everything with mineral oil. This here are PVP drops for cleaning of the needle and for putting the cells into, and this is HCCB plus Cyla B.And here I will make the manipulations.
Okay, this here is a needle color. What I'm doing now is I pull a needle. Alright, now I have a very nice needle, very thin.
And now this is a micro forge. I have to cut the needle at the certain position to have the right size holding pipette. I heat the glass ball, I go down with the needle melts to it, and now it's broken.
I push this away. You see the broken needle is perfect. Now I go down here, I push up the heat.
Now the glass ball will turn red, it will melt, push the glass ball and it, it makes a curve. That's enough. I remove it.
You see, then I insert the needle. We just made the holding pipette sub it into this head. Now insert it back into the microscope.
Micro pipette, capillary and I loaded with mercury. I loaded it into this tip. Mercury load this capillary here with mercury.
It goes inside. Set the needle here in this head. Screw tight, pull it back.
Turning clockwise here will push the mercury here to the front so you can see it. And now the tip into the oil.See? So now I'm moving down the needle to get the vision.
Expel some mercury droplets and suck up some QVP. That's all right. And then move it up again.
I go to the HCCB and move down the hole. My head, I go to P, which is the mouse pipette. I need to prepare a capillary to insert it here.Thanks.Already.Good.
So make some capillary.Okay. And in the pipe. I am ley.
I'm coming from Switzerland and I started this work one year ago and I came to K'S lab and I started learning nuclear transfer. It's really, it's a great place here and it's a great to work here. And, and of course this is a very complex experiment and many problems may arise while doing it.
So when you start, you may encounter problems like many cells lysing while you either nucleate or while you do the transfer. Or then you find even though you transfer successfully, they don't develop. And there are always, can be numerous reasons.
I mean you can start from the culture system of the all sites. So one always needs some controls like the parts of the genetic control or some fertilized emperors to test the culture system, the incubators. And then you also need to improve the, the skills of the microscope.
So it just takes a lot of practice until you reliably can inate the side and then transfer back. This is just a matter of practice and we'll take a few weeks or or months until you are good at it. And otherwise it's ready for lengthy experiments.
So it's good to reserve a whole day for it. And then something tricky in the beginning is also the mouse pipetting. So not to make bubbles to, to control it well and not to lose the oversight or especially you can also damage a clone, particularly because the outer layer, the so of lucita is open, it's actually possible when you mouth, you damage that clone and it kind of leaks out of that sonar lucita.
So when also needs to be careful not to have oil in that mouth pipe. Not to suck too much and not to blow too much, but this is relatively easily acquired, this skill.
