Question to be answered please
March 20, 2010 at 5:48 pm #12982
I would like to know what type of microscope should be used during performing " animals’ cloning ", I mean in that step of nucleus isolation and injection !!
Can i use Light microscope or I must use other type than that??
March 20, 2010 at 5:52 pm #98445
You, evolution blamer, wants to clone animals?
Some fine light microscope should be fine
I thought, that you have already cloned animals… 🙄 :-O
March 20, 2010 at 6:06 pm #98447quote JackBean:
No, actually I usually use Electron Microscope due to its high magnification, but I want to try this time via light microscope….
By the way, How can you say " I thought, that you have already cloned animals "..
Sweetie, my previous project about blocking transofmration of genetic information was via PCR technology, and it was not at all dealing with cloning. Means i was dealing with molecular biology not cell biology…
one thing els, I hope that you guide me to the best way of microinjection as well…. 😀
and thank you very much
March 20, 2010 at 7:59 pm #98450
Well, IMHO if you changed their DNA, you had to transform their cells, didn’t you?
March 20, 2010 at 10:11 pm #98458david23Participant
If you are using it for the injection then it’s live sample, something that EM cant do.
March 21, 2010 at 1:58 am #98463quote JackBean:
Yes, Of coure.
But that procedure is simple, because I did changed their DNA via rDNA technology, then after the induction of cellular division, I tranformed the changed DNA again to the animal……
That experiment was something other than what I’m about to do now. By that way, L want to know what are the suitable conditions for maintains Somatic cell under the terms of Lab ??
I mean who much Ph, and temperature?
March 21, 2010 at 2:00 am #98465quote david23:
I think E.M can do it, because the cells are placed in the culture medium while transforming the nucleus from cell to another cell…
March 22, 2010 at 8:01 am #98505quote hashemyemen:
You cannot use EM in microinjection for several reasons: it kills cells, which is a pretty obvious reason. Depending on whether you use SEM or TEM or something else, you must fix and then coat or stain the cells/target organisms in order to get the image. Furthermore the cells are typically placed in a high vacuum, something which live cells do not like. You also cannot manipulate the cells, that is, perform the injection under the EM. Lastly, you do not even need an EM for this because a light microscope is sufficient to visualise animal cells, just like JackBean already said.
March 22, 2010 at 11:47 am #98508quote biohazard:
Hmmmmmm, You are right up to some extent because most of my trying in this protocol was so much success, yet I injected the nucleus and transformed it, but no good results !!
Hmmm, I hope that you give full idea about utilizing L.Microscope for micro-injection procedure…
My question is:
Do we need this high developed microscope :
Or we can use this simple as well??
March 22, 2010 at 12:21 pm #98509
IMHO in the simple one you
1) don’t have enough space
2) your hand is not from stone, so it would be probably better, if the injection was hold by machine…
March 22, 2010 at 12:25 pm #98510
It is mostly about the technical skill of the operator. A skilled person with good injection tools might be able to carry out the procedure even under a fairly basic microscope. Actually the only required thing from the microscope is that it allows at least around x400 magnification and has enough light output to give clear vision and there is enough space to operate the petri dish and the injector on the sample holding space. Actually much bigger magnification is not even practically possible, since immersion oil would be required. A skilled person can perform the injection even with smaller magnifications than x400.
March 22, 2010 at 12:29 pm #98511quote hashemyemen:
that’s my point, you had to transform your cells, so how did you do that, as you had transfered the change to offspring too
March 22, 2010 at 1:36 pm #98514quote JackBean:
Dear Mr JackBean, I think your point is right, 🙂
Of course. machine will provide you a good handler to do such job, but the problem is that, there is no such machine in our Lab rather than we other equipments like PCR and E.M and so further… Yet we do not have have such advanced machine to handle the process…..
I think we can do it under simple light microscope but after much more practice in handling and constant performance to do it like this :
March 22, 2010 at 1:43 pm #98515quote biohazard:
from your comment I read that it is not such an impossible job to perform the procedure under simple light microscope, but I need to work hard and practice more…..
I shall try it ASAP using that simple equipments…
If you have much more information to share with, I will be much more pleased……
March 22, 2010 at 1:51 pm #98516quote JackBean:
fist, I ensured that DNA is completely changed in that exact sequences and I did that via PCR analysis of DNA sequences…
Then, I used gene gun to insert that modified DNA into the exact cell, at that time cell was placed in the petri dish in culture medium..
The process via Gene Gun was so simple…
But, I told you, that was regarding molecular biology, and it now about cellular biology, and procedure is different as we are dealing with cell rather than molecules..
March 22, 2010 at 2:07 pm #98517
There might be many systems for microinjection, but the one I’ve seen in action was quite a straightforward one: the "blunt needle" / cell holder has a verly mild suction applied to it, which keeps the target cell in place. Then the injection needle is guided there manually (by stabilising help of the needle holder, though) and simply pressed against the target cell. Requires a steady hand but no sophisticated tools. Any decent light microscope with enough room for the injection system and its attachment should be enough 🙂
March 22, 2010 at 2:20 pm #98518
cell or molecular biology, that doesn’t make much difference.
You wrote, that you got offspring, so I don’t see much, how could you achieve that only with gene gun.
March 22, 2010 at 4:03 pm #98524quote biohazard:
In such case, we can’t do it with simple light microscope and manual way of microinjection as we can’t be that much accurate in handling both the needle and the blunt needle and the pipette…
I shall get advanced machine to perform it…..
Hmmmmm, Thank you
March 22, 2010 at 4:16 pm #98525quote JackBean:
How can you that " cell or molecular biology, that doesn’t make much difference " !!!
It make so much differences, because in molecular biology we are only dealing with too tiny macromolecules which are smaller than the cell like DNA, Proteins, enzymes, and so further. While in cell biology we dealing with large components of the cell like its nucleus, and its mitochondria or other organelles…
Hmmmmm, about how could i achieved my work with gene gun that was not that tough job, you know I just injected my modified DNA without removal of the nucleus, while in mitochondria and cloning procedure, removal of the nucleus is prime thing.
March 22, 2010 at 4:29 pm #98529
so, if I assemble some vector with PCR, restriction enzymes and ligase; put it into bacteria, which I cultivate and isolate some protein, I’m using molecular, cell and molecular biology respectively?
OK, but if you used gene gun, you were not able to pass your trait to the offspring.
March 22, 2010 at 4:51 pm #98531quote JackBean:
No dear JackBean,
1- if you assemble some vector with PCR, restriction enzymes and ligase; put it into bacteria, which you cultivate and isolate some protein, then you are dealing with molecular biology as 80% of your work related to that field.
2- As you know, when we are injected the cell manually with our modified DNA, the cell will reject it and it will be concedred as non-self or foreign molecule, and the cell will degradation of that DNA via its enzymotic endonuclease activity before it reaches in the nucleus, therefor your DNA will not function..
But, with gene gun will exert high energy for DNA to be thrown directly inside the nucleus passing all the endonuclease enzymes which are located in cytoplasm… That’s why gene gun helped so much to modify the offsprings….
March 22, 2010 at 4:58 pm #98532
well, you should probably study a little more cell biology, because if you transform somatic cells, it won’t be passed to offspring
March 22, 2010 at 5:13 pm #98535quote JackBean:
well, you should improve your knowledge about this topic in much more details….. 😉
Go here and observe :
Formation of embryo in vitro refers to the production of new generation which is the offsprings….
Go ahead sweetie 😆
March 22, 2010 at 6:15 pm #98538
How did you perform this step w/o the microscope?
March 22, 2010 at 6:26 pm #98539quote JackBean:
That’s what I’m projecting to do in my upcoming experiments….
March 22, 2010 at 6:33 pm #98541
but you said in the thread I have linked before, that you have already done it. So you were lying?
March 22, 2010 at 6:41 pm #98543quote JackBean:
Don’t be that far away from what i said before Mr JackBean,
I have done the process of transfer of genetic material ( Molecular biology ), not transfer of the nucleus from somatic cell to an egg ( Cell biology ), and I that what made me create this topic to get more knowledge about the procedure !!
March 22, 2010 at 7:21 pm #98545quote you:
The thing was, that you got white mice as offsprings, so how did you get them?
March 23, 2010 at 1:46 am #98556quote JackBean:
A segment of DNA has been removed……….
March 23, 2010 at 8:10 am #98574
Oh mein Got, that all time again and again.
You said, that you got offspring by transfer of somatic cell nucleus to egg. How did you do this transfer?
March 23, 2010 at 8:31 am #98576quote JackBean:
wait until I publish this work, and you will get all the knowledge about it….
Hmmmm, It is not good to spread information I tired my self to prove it practically….
So, go ahead and keep waiting…
March 23, 2010 at 8:39 am #98579
You said, you have done the work already; you said you got offspring; you did thing, which is pretty obvious.
March 23, 2010 at 8:49 am #98583quote JackBean:
Hmmm, It will better if go back to check my first topic written in this forum
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