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Method for separating cells from blood and cultivating the cells and method for cloning non-human animal

A technology for culturing cells and neutrophils, which is applied in the field of cloning non-human animals, can solve the problems such as the need to improve the source of nuclear donor cells, and achieve the effects of improving animal welfare, increasing cell survival rate, and simple collection operation

Active Publication Date: 2015-04-15
深圳华大基因农业控股有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the source of nuclear donor cells currently used for somatic cell transplantation still needs to be improved

Method used

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  • Method for separating cells from blood and cultivating the cells and method for cloning non-human animal
  • Method for separating cells from blood and cultivating the cells and method for cloning non-human animal
  • Method for separating cells from blood and cultivating the cells and method for cloning non-human animal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Obtaining mononuclear cells from blood

[0068] Add 125IU heparin to the 15mL conical centrifuge tube in advance, and make it evenly distributed on the tube wall of the conical centrifuge tube by shaking. About 12 mL of the peripheral blood of the miniature pig was collected by directly drawing blood from the porcine anterior vena cava with a 50 mL syringe into the conical centrifuge tube added with 15 mL of heparin. The conical centrifuge tube loaded with peripheral blood was transported to the laboratory in a 4°C ice box.

[0069] Take another 15mL conical centrifuge tube, add 5mL of neutrophil separation solution (containing 57g / L polysucrose and 90g / L sodium diatrizoate) to each of them, and carefully pipette the 15mL Slowly add 5mL of the collected anticoagulated peripheral blood to the wall of the conical centrifuge tube close to the surface of the neutrophil separation medium, and ensure that the anticoagulant peripheral blood does not mix with the neu...

Embodiment 2

[0071] Embodiment 2 Mononuclear cells cultured in vitro

[0072] Add 2 mL of low sugar-MEM, 15% FBS, [1×] non-essential amino acids, 10 ng / mL bFGF and 30 IU / mL of the first adherent cell culture medium of heparin, and use a Pasteur pipette to evenly disperse the mononuclear cell pellet in the first adherent cell culture medium. The first adherent cell culture medium containing mononuclear cells was transferred to a gelatin-coated 35 mm culture dish, and then placed in a carbon dioxide incubator at 37 degrees Celsius for cultivation.

[0073] After 48 hours of culturing, the 35mm culture dish loaded with mononuclear cells was taken out of the incubator, and after discarding the culture medium of the first adherent cells, PBS was added for rinsing. After discarding the PBS, add a second adherent cell medium containing MEM-α, 15% FBS, [1×] non-essential amino acids, and 10 ng / mL bFGF to the 35 mm culture dish loaded with mononuclear cells, and culture The dish was returned to ...

Embodiment 3

[0074] Subculture of embodiment 3 mononuclear cells

[0075] When the adherent mononuclear cells are observed under the microscope as clumps of contact inhibitors (see Figure 4 ), discard the second culture medium for adherent cells in the culture dish; then add PBS buffer to wash the cells twice; after discarding the PBS buffer, add 200 μL of 0.05 % trypsin-200mg / L EDTA, and place the cells in a carbon dioxide incubator at 37 degrees Celsius for digestion. When observed under the microscope, the adherent cells gradually become round and bright, and gradually break away from the culture dish and float in the trypsin. , add 1mL of the second adherent cell culture medium to the culture dish, and pipette through a Pasteur pipette to mix the mononuclear cells with the added fresh second adherent cell culture medium evenly, so as to obtain a cell suspension; The cell suspension was transferred to a gelatin-coated culture device for subculture. The adherent mononuclear cells that...

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Abstract

The invention provides a method for separating cells from blood and cultivating the cells and a method for cloning non-human animals. The method for separating cells from blood and cultivating the cells comprises following steps: (1) adding the blood to an upper layer of a separation liquid and performing centrifugation in a manner of density gradient centrifugation for obtaining mononuclear cells; (2) inoculating the mononuclear cells onto an in-vitro cultivation device containing an adherent cell culture medium to obtain adherent cells; and (3) performing serial subcultivation to the adherent cells to obtain somatic cell nuclear transplanted donor cells. The somatic cell nuclear transplanted donor cells obtained through the method can be directly used for manually cloning the non-human animals. The method is simple in operation processes, is easy to carry out, is low in cost, is less in stress stimulation and harm in animals, and can ensure a complete appearance of the animals and improve animal welfare.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for isolating and culturing cells from blood and a method for cloning non-human animals. Background technique [0002] Somatic cell nuclear transfer, also known as cloning, is the most commonly used technique for animal cell engineering. The highly differentiated somatic cells are transferred to oocytes by electric shock or direct microinjection, and then chemically activated to reprogram them to develop into embryos, which are transplanted back into the surrogate mother to give birth to a complete individual. The traditional somatic cell nuclear transfer technology requires expensive and sophisticated micromanipulation system, which has high technical requirements for operators and is not suitable for large-scale production of cloned animals. [0003] In 2001, the Danish scientist Gabor Vajta first proposed the manual cloning technology. The biggest key point of this t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12N15/877
Inventor 杨珍珍徐颖林木飞杜玉涛
Owner 深圳华大基因农业控股有限公司
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