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Application of DPBS (Dulbecco's phosphate buffered saline) free of calcium and magnesium ions in mouse embryo blastomere biopsy

A calcium-magnesium ion, mouse embryo technology, applied in the field of genetics, can solve the problem of non-disclosure, etc., and achieve the effects of fast segmentation, wide application prospects, and high fertilized egg birth rate.

Inactive Publication Date: 2021-02-09
GEMPHARMATECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It can be seen that there is no disclosure in the prior art that there will be no Ca 2+ -Mg 2+ DPBS solution for blastomere biopsy and blastomere segmentation, validated low-cost Ca-free 2+ -Mg 2+ Whether the DPBS solution is suitable for blastomere biopsy and blastomere segmentation has become an urgent problem to be solved.

Method used

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  • Application of DPBS (Dulbecco's phosphate buffered saline) free of calcium and magnesium ions in mouse embryo blastomere biopsy
  • Application of DPBS (Dulbecco's phosphate buffered saline) free of calcium and magnesium ions in mouse embryo blastomere biopsy

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Acquisition of 8-cell embryos

[0034] The present embodiment adopts the in vivo direct acquisition method to obtain cell embryos, and the steps are as follows:

[0035] C57 female mice aged 3.5 to 4 weeks were selected and injected intraperitoneally with 5IU of pregnant horse serum gonadotropin (PMSG), and 44 to 48 hours later, intraperitoneally injected with 5IU of chorionic gonadotropin (HCG). The cages were closed overnight, and the emboli were checked before 10:00 am the next day, and the mice with embolism were recorded as 0.5 days of pregnancy;

[0036] 63-65 hours after the intraperitoneal injection of HCG in female mice, the donor mice were euthanized, the 2.5-day-old mouse uterus was excised, and the embryos were flushed out of the uterine horns using a dulled punching needle, washed with DPBS, and 8-cell embryos with clear blastomeres were selected;

[0037] The 8-cell embryos were transferred to the pre-equilibrated M16 medium and placed at 37°C,...

Embodiment 2

[0038] Example 2 Acquisition of 8-cell embryos

[0039] In this embodiment, cell embryos are obtained by in vitro culture method, and the steps are as follows:

[0040] Select C57 female mice aged 3.5-4 weeks, inject 5IU pregnant horse serum gonadotropin (PMSG) intraperitoneally into each mouse, and inject 5IU chorionic gonadotropin (HCG) intraperitoneally after 44-48 hours, and kill them by dislocation of the neck 15 hours later to collect eggs use;

[0041] The male mice were killed by neck dislocation, and the sperm from the tail of the epididymis was put into the c-TYH drop balanced for half an hour for capacitation for 1 hour; the egg cells of the female mouse were placed in the well-balanced fertilization drop HTF, and 3-10 μL of capacitated sperm was added , fertilized and equilibrated overnight;

[0042] After 12-15 hours of IVF, obtain 1-cell embryos, transfer them to pre-balanced M16 culture medium, and place them at 37°C, 5% CO 2 8-cell embryos were obtained by c...

Embodiment 3

[0043] Example 3 Blastomere Biopsy

[0044] The prepared 8-cell embryos were placed in calcium and magnesium ion-free DPBS (sodium chloride 8g, potassium chloride 0.2g, disodium hydrogen phosphate 1.15g, potassium dihydrogen phosphate 0.2g, glucose 1g, sodium pyruvate 0.036g, penicillin 0.06 g, streptomycin sulfate 0.05g, BSA3g, 4-hydroxyethylpiperazineethanesulfonic acid 5g, dissolved in 1L water, pH=7.4) and incubated for 10min, then the embryos were transferred to the strip of M2 operating solution, placed in The biopsy operation is performed in an inverted microscope with the fixation needle and egg transfer needle installed: first use the Piezo system to punch a hole at the position of the zona pellucida between the two blastomeres, avoid directly punching the zona pellucida aligned with the blastomere, To prevent damage to blastomeres;

[0045] Insert a biopsy needle with a diameter of 15 μm into the punched hole after punching, slightly adjust the angle of the needle t...

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Abstract

The invention provides application of DPBS (Dulbecco's phosphate buffered saline) free of calcium and magnesium ions in mouse embryo blastomere biopsy. The DPBS free of calcium and magnesium ions is used for processing eight cell embryos of a mouse for 5-10 minutes, so that the viscosity among the mouse embryo blastomeres is weakened, the blastomere forms are miniaturized, and the perivitelline space is obviously enlarged. During blastomere segmentation, the damage to other blastomeres caused by the operation of obtaining the blastomeres in the blastomere biopsy process is avoided, the embryobirth rate is high, and the blastomere segmentation method has important significance in the technical field of embryo biopsy.

Description

technical field [0001] The invention relates to the technical field of genetics, in particular to the application of calcium-magnesium ion-free DPBS in mouse embryo blastomere biopsy. Background technique [0002] Embryos are the embryos of multicellular organisms in the initial stage of the individual development process starting from fertilized eggs. After fertilization, the eggs form fertilized eggs, and begin to divide and develop to form embryos. The embryo formed first is a morula, and then a blastocyst is formed, which is implanted in the endometrium, absorbs the nutrition of the mother, and continues to develop. The stage in which the fertilized egg develops to the morula is called the cleavage stage, and the cells in the cleavage stage embryo are called blastomeres. Preimplantation genetic diagnosis (PGD) is mainly used in clinical practice to provide embryo biopsy technology for women with assisted reproduction. The specimens tested for PGD are 1-2 single cells (b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2509/00C12N2509/10
Inventor 赵俊平王红薛红兰
Owner GEMPHARMATECH CO LTD
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