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Question to be answered please

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Re:

Postby hashemyemen » Mon Mar 22, 2010 1:36 pm

JackBean wrote: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...


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 :


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Last edited by hashemyemen on Mon Mar 22, 2010 1:43 pm, edited 1 time in total.
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Re:

Postby hashemyemen » Mon Mar 22, 2010 1:43 pm

biohazard wrote: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.



Dear 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......

Thank you
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Re: Re:

Postby hashemyemen » Mon Mar 22, 2010 1:51 pm

JackBean wrote:
hashemyemen wrote: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's my point, you had to transform your cells, so how did you do that, as you had transfered the change to offspring too


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..


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Postby biohazard » Mon Mar 22, 2010 2:07 pm

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 :)
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Postby JackBean » Mon Mar 22, 2010 2:20 pm

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.
http://www.biolib.cz/en/main/

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Re:

Postby hashemyemen » Mon Mar 22, 2010 4:03 pm

biohazard wrote: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 :)


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
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Re:

Postby hashemyemen » Mon Mar 22, 2010 4:16 pm

JackBean wrote: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.



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.
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Postby JackBean » Mon Mar 22, 2010 4:29 pm

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.
http://www.biolib.cz/en/main/

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Re:

Postby hashemyemen » Mon Mar 22, 2010 4:51 pm

JackBean wrote: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.



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....
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Postby JackBean » Mon Mar 22, 2010 4:58 pm

well, you should probably study a little more cell biology, because if you transform somatic cells, it won't be passed to offspring
http://www.biolib.cz/en/main/

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Re:

Postby hashemyemen » Mon Mar 22, 2010 5:13 pm

JackBean wrote:well, you should probably study a little more cell biology, because if you transform somatic cells, it won't be passed to offspring



well, you should improve your knowledge about this topic in much more details..... :wink:

Go here and observe :

http://en.wikipedia.org/wiki/Somatic_cell_nuclear_transfer


Formation of embryo in vitro refers to the production of new generation which is the offsprings....


Go ahead sweetie :lol:
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Re: Question to be answered please

Postby JackBean » Mon Mar 22, 2010 6:15 pm

really?
wiki wrote:The nucleus of the somatic cell is then inserted into the enucleated egg cell.

How did you perform this step w/o the microscope?
http://www.biolib.cz/en/main/

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