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Method for improving chimeric capability of ES cells of mouse to epiblasts of early embryos

A cell and chimera technology, applied in the field of improving the chimeric ability of mouse ES cells to the epiblast of early embryos, can solve problems such as unfavorable production scale

Active Publication Date: 2017-11-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although injection of 4-cell embryos or 8-cell embryos can obtain higher chimeric mouse individuals, this technique requires injection of a certain number of ES cells, which is not conducive to the expansion of production scale

Method used

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  • Method for improving chimeric capability of ES cells of mouse to epiblasts of early embryos
  • Method for improving chimeric capability of ES cells of mouse to epiblasts of early embryos
  • Method for improving chimeric capability of ES cells of mouse to epiblasts of early embryos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, embryo acquisition and in vitro culture

[0038] At around 4:00 p.m. on the first day, female mice were injected intraperitoneally with PMSG (5IU / rat, serum gonadotropin of pregnant horse, purchased from Ningbo Second Hormone Factory), and injected HCG (5IU / rat, human Chorionic gonadotropin (purchased from Ningbo No.2 Hormone Factory) was injected into cages with male mice. On the third day at about 8 o'clock in the morning, all female mice were checked for thrombus, and the female rats (that is, 0.5dpc) that saw the thrombus were marked. On the afternoon of the fourth day, the embryos at the 2-cell stage were washed out from the oviducts of the marked mice, cleaned with mouse embryo operating solution M2 (Millipore, MR-015-D), and then transferred to mouse embryos for in vitro culture. cultured in liquid KSOM (Millipore, MR-121-D).

Embodiment 2

[0039] Embodiment 2, Activin A hinders the development of EPI

[0040] 1. Use mouse embryo in vitro culture medium KSOM to prepare Activin A as Activin A working solution with a concentration of 500ng / ml, and make micro-droplets with a standard of 10-20μl per drop, cover with paraffin oil (Sigma, M8410) Equilibrate in a 37°C incubator for at least 2 hours to obtain culture drops containing Activin A.

[0041]2. Place the embryos cultured to the 4-cell stage in Example 1 in acidic bench-top solution (Millipore, MR-004-D) to wash and blow several times, remove the embryos after the zona pellucida is degraded, and lightly rinse them with mouse embryo operating solution M2. Gently blow and inhale, clean and place in step 1 to culture in the well-balanced culture drop containing Activin A, and set the culture drop without adding Activin A working solution as a control; when the embryo develops to the late blastocyst (4.5dpc), Embryos were collected and tested as follows:

[0042]...

Embodiment 3

[0059] Embodiment 3, SB431542 promotes the development of EPI

[0060] 1. Use mouse embryo in vitro culture medium KSOM to prepare SB431542 (Selleck, S1067) into SB431542 working solution with a concentration of 10 μM, and make microdrops with a standard of 10-20 μl per drop, and cover with paraffin oil (Sigma, M8410) Afterwards, it was placed in a 37° C. incubator and equilibrated for at least 2 hours to obtain culture drops containing SB431542.

[0061] 2. Place the embryos cultured to the 4-cell stage in Example 1 in acidic bench-top solution (Millipore, MR-004-D) to wash and blow several times, remove the embryos after the zona pellucida is degraded, and lightly rinse them with mouse embryo operating solution M2. Gently blow and inhale, clean and culture in the well-balanced culture drop containing SB431542 in step 1, and set the culture drop without SB431542 working solution as a control; when the embryo develops to the late blastocyst (4.5dpc), collect the embryo , usin...

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Abstract

The invention discloses a method for improving the chimeric capability of ES cells of a mouse to epiblasts of early embryos. The invention provides receptor-embryo culture solution for preparing chimeras. The receptor-embryo culture solution contains activin A with the concentration of 500-1000ng / ml. Discovered by studies, Activin A can block the development of the epiblasts (EPI) of the embryos effectively. Compared with a control group, when the embryos (treated to a blastula stage by 4 cells) treated by Activin A is used as the receptor embryos, ES cells are easier for chimerism into the EPI, so that the chimerism rate of the ES cells into the chimeras is increased; and the method is beneficial to optimizing a chimera production system and plays a certain role in promoting expansion of the production scale.

Description

technical field [0001] The invention relates to a method for improving the chimerism ability of mouse ES cells to early embryonic epiblast. Background technique [0002] Since the successful establishment of mouse embryonic stem cells (ES cells) in 1981, obtaining transgenic mice from genetically modified embryonic stem cells has become an important means of gene function research. To produce mice based on ES cells, ES cells are usually injected into normal diploid mouse blastocysts, so that chimeric mice that develop together from ES cells and recipient embryos can be obtained. However, chimera mice usually need to spend a lot of time for passage to obtain homozygous ES cell-derived mice before performing phenotypic analysis and functional verification. In view of the shortcoming of blastocyst injection to produce mice, after long-term research, people finally found that injecting ES cells into earlier mouse embryos (such as 4-cell embryos, 8-cell embryos) can produce 0 T...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N15/877
CPCC12N5/0603C12N15/8775C12N2501/16
Inventor 韩建永相金柱曹素英王寒凝
Owner CHINA AGRI UNIV
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