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Transplantation method for nucleus

A cell nucleus and oocyte technology, applied in the field of cell engineering, can solve the problems of wasting too much time, removing the target nucleus and cytoplasm, and controlling the force poorly, and achieves the effects of reducing time, easy operation and improving survival rate.

Inactive Publication Date: 2006-07-26
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, when the perforation is performed, the cytoplasm will undergo a large deformation and displacement, so the initial alignment may not be very accurate later; it is difficult to control the strength of the cytoplasm by squeezing the zona pellucida with negative pressure. If the strength is too small, the target nucleus cannot be removed. If it is too large, too much cytoplasm will be removed at once, and it is difficult to find the right strength.
Another very important weakness is that the needle needs to be changed and the opening re-aligned when the nucleus is transferred, which will waste too much time during the operation, and the operation time has a great impact on the survival rate of eggs
[0007] To sum up, most of the currently used micromanipulation nuclear transfer methods need the help of special instruments (such as Piezoimpact device or Spindle-View system), which cause great damage to eggs and are quite time-consuming. A Simple, Practical, Less Injury and Rapid Nuclear Transfer Method

Method used

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  • Transplantation method for nucleus
  • Transplantation method for nucleus
  • Transplantation method for nucleus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (1) Use an injection needle with a diameter of 8 μm to absorb granule cells, repeatedly blow and blow the cells to rupture the cell membrane, and then suck them into the injection needle. leave appropriate gaps;

[0048] (2) Get the mice that have undergone superovulation treatment, kill them by necking, and take out the oviducts;

[0049] (3) Pull out the oocytes with granulosa cells in the ampulla of the oviduct with a fine needle, then put them into the culture medium containing hyaluronidase, and treat them in a 37°C incubator for 5 minutes to completely remove the granulosa cells;

[0050] (4) Take oocytes with complete polar bodies in good condition and fix them at 9 o’clock with a 15 μm ovum-holding needle ( figure 1 );

[0051] (5) Move the tip of the injection needle on the other side to directly below the oocyte along the opposite direction of the egg-holding needle ( figure 2 );

[0052] (6) Move the injection needle upwards, squeeze the oocytes to form ...

Embodiment 2

[0060] (1) Use an injection needle with a diameter of 8 μm to absorb granule cells, repeatedly blow and blow the cells to rupture the cell membrane, and then suck them into the injection needle. leave appropriate gaps;

[0061] (2) Get the mice that have undergone superovulation treatment, kill them by necking, and take out the oviducts;

[0062] (3) Pull out the oocytes with granulosa cells in the ampulla of the oviduct with a fine needle, then put them into the culture medium containing hyaluronidase, and treat them in a 37°C incubator for 5 minutes to completely remove the granulosa cells;

[0063] (4) Take oocytes with complete polar bodies in good condition and fix them in the direction of 9 o’clock with a 20 μm ovum-holding needle ( figure 1 );

[0064] (5) Move the tip of the injection needle on the other side to directly below the oocyte along the opposite direction of the egg-holding needle ( figure 2 );

[0065] (6) Move the injection needle upwards, squeeze the...

Embodiment 3

[0073] (1) Use an injection needle with a diameter of 8 μm to absorb granule cells, repeatedly blow and blow the cells to rupture the cell membrane, and then suck them into the injection needle. leave appropriate gaps;

[0074] (2) Get the mice that have undergone superovulation treatment, kill them by necking, and take out the oviducts;

[0075] (3) Pull out the oocytes with granulosa cells in the ampulla of the oviduct with a fine needle, then put them into the culture medium containing hyaluronidase, and treat them in a 37°C incubator for 5 minutes to completely remove the granulosa cells;

[0076] (4) Take oocytes with complete polar bodies in good condition and fix them at the 9 o’clock direction with a 30 μm ovum-holding needle ( figure 1 );

[0077] (5) Move the tip of the injection needle on the other side to directly below the oocyte along the opposite direction of the egg-holding needle ( figure 2 );

[0078] (6) Move the injection needle upwards, squeeze the oo...

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Abstract

This invention discloses a simple and effective microoperation nucleolus transplant method. This invention has the following steps: extruding the inject pin that has absorbed biology substance before on the ovum cell to form the transparent strip gauffer between the inject pin and ovum holding pin, then sticking and penetrating into the transparent strip gauffer with the inject pin to enter the opening of the ovum holing pin, then pulling the inject pin out from the surrounding gap of the ovum and adding the negative pressure in the ovum holing pin to absorb the pole body and object karyoplasms into the ovum holing pin, and then adjusting the place of the ovum cell with ovum holing pin to make its center at the same horizontal line with the needle-point of the inject pin, fixing up the ovum cell again, injecting the biology substance in the inject pin into the cell cytoplast or the surrounding gap of the ovum. This invention can complete the micro stoning and nucleus injecting processing at one time rapidly without special device; it has little damage to the ovum and can be used extensively in the cell project such as animal clone and transfer gene and so on.

Description

technical field [0001] The invention relates to a method for cell engineering by using a micromanipulator, in particular to a method for nuclear transplantation by using a micromanipulator. Background technique [0002] Cell engineering is an important aspect of bioengineering. Currently, the main technical fields involved in cell engineering include cell culture, cell fusion, cell disassembly, chromosome manipulation, and gene transfer. Cell transformation by means of nuclear transfer is playing an increasingly important role in the field of cell engineering. At present, the main methods of cell nuclear transfer are as follows: [0003] (1) A method of direct denucleation and injection with the help of a pulse (Piezoimpact) device. Wakayama et al. used the Piezoimpact device to complete the enucleation and enucleation of MII stage oocytes, and first obtained cloned mice derived from somatic cells, and had a high nuclear transfer efficiency. However, very few people can r...

Claims

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Application Information

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IPC IPC(8): C12N15/09
Inventor 时永香吴克良田海滨白增亮
Owner SHANDONG UNIV
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