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Unicellular fusion method for pig and mouse cells

A fusion method and single-cell technology, applied in the field of cell engineering, can solve the problem of low fusion efficiency of heterogeneous cells and achieve the effect of improving the efficiency of cell fusion

Pending Publication Date: 2018-10-26
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Aiming at the problem of low fusion efficiency of heterogeneous cells at present, the present invention provides a single-cell fusion method of pig and mouse cells, which is to digest mouse embryonic stem cells and pig cells into single cells by trypsin, and use cytohemagglutinin to construct a one-to-one fusion method. single cell pair, the single cell pair is obtained through fusion culture under the action of polyethylene glycol to obtain fusion cells, and the pig cells are porcine pluripotent stem cells or porcine embryonic fibroblasts

Method used

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  • Unicellular fusion method for pig and mouse cells
  • Unicellular fusion method for pig and mouse cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1. Single cell fusion method of pig and mouse cells.

[0077] 1. Cell Culture

[0078] 1) Culture of mouse embryonic stem cells:

[0079] In order to ensure the comparability of the research on the biological characteristics of fusion cells, the mouse embryonic stem cell line R1 used in fusion experiments was first identified for pluripotency.

[0080] ① Alkaline phosphatase activity detection

[0081] The obtained fused cells were washed 3 times with DPBS, the clones were fixed with 4% paraformaldehyde for 90 seconds, the paraformaldehyde was discarded, washed 3 times with DPBS, and detected according to the BCIP / NBT alkaline phosphatase color development kit. Color development can be stopped when the color of the clone turns purple. Remove the staining solution, wash 3 times with DPBS, observe and take pictures under a microscope.

[0082] ② Detection of embryoid body formation

[0083] Use Tryple to digest the fused cell mass for 3 minutes, separate the ...

Embodiment 2

[0120] Example 2. Pig and mouse cell single cell fusion method, repeat Example 1, the difference between this embodiment and Example 1 is that the selection of pig and mouse cells in this example is: mouse embryonic stem cells are R1 line cells, pig cells It is porcine pluripotent stem cell KO, and the culture method of described porcine pluripotent cell KO is: the porcine pluripotent stem cell KO is cultured in KO-DMEM culture medium, and the culture medium is discarded when passaged, DPBS Wash once, add collagenase IV at a final concentration of 1 mg / ml, digest in a 39°C incubator for 4 minutes, discard collagenase, wash once with DPBS, remove the DPBS solution, resuspend the pellet with ES culture medium, and pipette the clone After forming uniform small clumps, passage to the culture plate inoculated with the feeder cells treated with mitomycin C at 1:2. The 1:2 refers to the cells digested from one culture well. Transfer to 2 wells for cultivation. Inhibitor Y27632 was a...

Embodiment 3

[0124] Example 3. Pig and mouse cell single cell fusion method, repeat Example 1, the difference between this embodiment and Example 1 is that the selection of pig and mouse cells in this example is: mouse embryonic stem cells are F1 line cells, pig cells It is porcine embryonic fibroblast PEF, and the cell culture conditions and fusion method refer to Example 1.

[0125] The fusion cell F1-PEF obtained through culture expresses both red fluorescence and green fluorescence, indicating that the single cell fusion of mouse stem cells and pig embryonic fibroblasts is successful. The morphology of the fused cells was not the raised shape of mES, but tended to be flat.

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Abstract

The invention belongs to the technical field of cell engineering and discloses a unicellular fusion method for pig and mouse cells. The unicellular fusion method is provided aiming at the problem of current low xenogenic cell fusion efficiency. The method includes: taking mouse embryonic stem cells and pig pluripotent stem cells or pig fibroblast cells as cellular fusion objects; adopting digestive juice containing pancreatin in mass fraction of 0.25% to digest the pig and mouse cells into single cells, culturing, and adopting cell agglutinin for constructing one-to-one unicellular pairs; culturing for 1min in polyethylene glycol in mass fraction of 50%, and continuing cell culture for seven days to obtain fused cells of pig and mouse single cells. The unicellular fusion method is applicable to animal breeding research or monoclonal antibody production.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a single-cell fusion method of pig and mouse cells. Background technique [0002] Cell fusion experiment started in 1969, which is very important for basic research and has important application value. So far, there are three main methods of cell fusion, Sendai virus, electrofusion and polyethylene glycol (PEG). However, at present, polyethylene glycol is mainly used to mediate cell fusion, and Cowan uses 5×10 7 Individual hESCs with 5 x 10 7 The fusion efficiency of individual cells was only 0.000004%, and 2 clones were obtained. Yu via 1.3×10 7 Individual stem cells and 4.7 x 10 7 The fusion efficiency of a bone marrow precursor cell is only 0.00067%. In 2010, Hasegawa increased the fusion efficiency to 0.005%. By fusing human fibroblasts and human ES, Sumer's fusion efficiency has been qualitatively improved, and the fusion efficiency has been improved...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/08
CPCC12N15/02
Inventor 刘忠华牟彦双郭佳方园厉雪纯
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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