Roboticized somatic cell nuclear transfer operation method based on micro-channel

A technology of somatic cell nuclear transfer and operation method, which is applied in the field of micromanipulation at the cell level, can solve the problems of long time and low efficiency, reduce the search time, save the time for objective lens conversion and focusing, and improve efficiency and reliability reproducible effect

Active Publication Date: 2019-08-23
NANKAI UNIV
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Problems solved by technology

[0003] The purpose of the present invention is to solve the problem that the existing operation method of somatic cell nuclear transfer takes a long time and has low efficiency, and provides a microfluidic-base...

Method used

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  • Roboticized somatic cell nuclear transfer operation method based on micro-channel
  • Roboticized somatic cell nuclear transfer operation method based on micro-channel
  • Roboticized somatic cell nuclear transfer operation method based on micro-channel

Examples

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Embodiment 1

[0025] This example is an experiment of robotic somatic cell nuclear transfer based on microfluidics, in which the oocytes used are domestic pig oocytes taken from a local slaughterhouse, and the nuclear donor cells used are "additional" large White pig fetal cells.

[0026] The method of obtaining oocytes is as follows:

[0027] After the ovaries were removed from the slaughterhouse, they were transported to the laboratory within two hours in vacuum flasks filled with saline at 35° to 37°. Immediately wash twice with 37° sterile saline containing 100 IU / L penicillin and 50 mg / L streptomycin. The oocytes were extracted from the small sac with a diameter of 2 to 6 mm on the ovary, and the extracted cells were washed three times with TL-Hepes-PVA, and then matured in vitro (IVM) in an incubator at 39° with a carbon dioxide concentration of 5%42 Hour. After IVM, the cells were deovized with 0.1% hyaluronidase. Finally, the cells were washed three times with M199, and the obta...

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Abstract

The invention provides a roboticized somatic cell nuclear transfer operation method based on a micro-channel. The method is characterized in that the introduced micro-channel is used for storing cells; parallel double needles are introduced and used for cell denucleation and nuclear injection respectively. The method comprises the following key steps of micro-channel storage of the cells, cell positioning, cell holding, cell denucleation, switching between the denucleation needle and the nuclear injection needle, nuclear injection of the cells and release of the operated cells. The experimental results show that compared with a traditional somatic cell nuclear transfer process, time is saved by 41.7% in the process, the surviving rate and the success rate are equivalent to those of manualsomatic cell nuclear transfer, and the somatic cell nuclear transfer efficiency and repeatability are effectively improved.

Description

technical field [0001] The invention belongs to the technical field of cell-level micromanipulation. Background technique [0002] Somatic cell nuclear transfer technology is a key step in cloning technology, mainly including oocyte positioning, holding, poking, piercing, enucleation, somatic cell nuclei injection, and release of reconstituted embryos. Through study, we learned that the current somatic cell nuclear transfer is mainly done manually. Due to long-term and high-intensity manual operations, the operator's operating efficiency and success rate will be greatly reduced. Some existing robotic somatic cell nuclear transfer procedures are consistent with manual procedures. During this operation, the robotic system needs to switch between different operating areas multiple times, and it also needs to perform 4 times of objective lens conversion and refocusing. . In addition, in this operation process, the cell enucleation and injection share a microneedle, which grea...

Claims

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Application Information

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IPC IPC(8): C12N15/873
CPCC12N15/873
Inventor 孙明竹刘曜玮赵新王雪锋赵启立崔茂盛
Owner NANKAI UNIV
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