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Needle-like microelectrode for karyon transplanting/ clonal electrical fusion

An electrofusion and microelectrode technology, applied in the field of agricultural biology, can solve the problems of low magnification, difficult cell-membrane contact surface, decreased vitality of nuclear transfer embryos, etc.

Inactive Publication Date: 2009-06-03
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of the electrofusion cell method is that electrofusion is performed under an ordinary stereomicroscope. Due to the low magnification of the stereomicroscope, in the actual operation process, if the electrofusion is performed with smaller cells such as granulosa cells and fibroblasts, When used as a donor cell, it is very difficult to judge whether the contact surface of the cell membrane is parallel to the electrode, and it is necessary to repeatedly operate between two microscopes during operation, which greatly prolongs the in vitro operation time of the recombinant, resulting in a decrease in the vitality of the nuclear transfer embryo , thus affecting the overall efficiency of nuclear transfer
[0003] According to the data retrieval conducted by the applicant, there are no relevant reports at home and abroad on the use of needle-shaped microelectrodes for cell nuclear transfer (cloning) electrofusion

Method used

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  • Needle-like microelectrode for karyon transplanting/ clonal electrical fusion
  • Needle-like microelectrode for karyon transplanting/ clonal electrical fusion
  • Needle-like microelectrode for karyon transplanting/ clonal electrical fusion

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Experimental program
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Effect test

Embodiment 1

[0024] The length of section A of the two electrodes is 25mm and the diameter is 1mm; the length of section B is 25mm and the diameter is 0.6mm; the length of section C is 7mm and the diameter is 150μm; The diameter is 100 microns, and the segment C of the second electrode has a conical front end.

Embodiment 2

[0026] Section A of the two electrodes has a length of 20mm and a diameter of 1mm; section B has a length of 20mm and a diameter of 0.9mm; section C has a length of 5mm and a diameter of 100μm; The diameter is 50 microns, and the C segment of the second electrode is a conical front end.

[0027] Of course, the size of the two electrodes can be adjusted according to the needs of different researches. For example, when cloning large animals such as cattle and sheep, the electrode with a larger diameter should be selected, while the electrode with a smaller diameter should be selected for mouse cloning. Appropriate, if cloning is not carried out, but only the fusion between two somatic cells, just choose two thinner electrodes.

experiment Embodiment 1

[0029] Rough estimation of output voltage and optimization of conditions when using the microelectrode of the present invention for electrofusion:

[0030] (1) Rough estimation of the output voltage

[0031] Goat enucleated oocytes were used as recipients, goat fetal fibroblasts were used as nuclear donors for nuclear transfer, and the constructed recombinants were randomly divided into groups. In this experiment, the fusion voltage when using the electric fusion tank is 2.4kv / cm, and the diameter of goat egg cells is 140-160μm. Put these two values ​​into the above formula:

[0032]

[0033] (2) Optimization of output voltage

[0034] Calculated from (1), the output voltage is about 0.34 ~ 0.38kv, and the following experiments are designed to optimize the output voltage, see Table 1 for details.

[0035] Table 1 Optimization of output voltage

[0036] Output voltage (kv) total number of electrofusions fusion success Fusion success rate / % 0.32 69 35...

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Abstract

This invention discloses a needle-like microelectrode for nuclei transplantation / clone electric fusion. The needle-like microelectrode comprises two electrodes, both of which are composed of A, B and C segments with different dismeters. The length of segment A is 20-30 mm, and the diameter is 1 mm. The length of segment B is 20-30 mm, and the diameter is 0.4-1 mm. The length of segment C is 5-10 mm, and the diameter is 100-180 mums. The segment C of one electrode has a cylindrical front end, and that of the other electrode has a cone front end. The microelectrode can be placed at the locations of the two manipulation needles of a micromanipulation device, and can be manipulated by the manipulating handles, which has such advantages as simple manipulation, high accuracy, rapid manipulation and high utility. For electric fusion, the recombinant obtained by injecting the donor cell into perivitelline space are placed between two microelectrodes, and the pericellular membrane contact surface is perpendicular to the connector line of the two microelectrodes by manipulating the recombinant. The electric fusion time can be 20-50 times shorter than that of conventional fusion cell method, and the success rate can be higher than 20%.

Description

technical field [0001] The invention belongs to the technical field of agricultural biology, and relates to an electric fusion operation auxiliary device used in the process of cell nucleus transplantation (cloning), in particular to a needle-shaped microelectrode used for cell nucleus transplantation / cloning electric fusion. Background technique [0002] With the development of biotechnology, the horizontal combination of various disciplines and technologies is becoming more and more extensive. Cell nucleus transfer (cloning) technology has become an irreplaceable technology in frontier fields such as the production of transgenic animals, the production of mammary gland bioreactors, and therapeutic cloning. The platform, its value and its status in the field of biotechnology are self-evident. But so far, the overall efficiency of nuclear transplantation is relatively low, which is attributed to the cumulative result of the low efficiency of each link in the entire nuclear t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87C12M1/42
Inventor 刘凤军张涌郑月茂张玉玲安志兴赵晓娥
Owner NORTHWEST A & F UNIV
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