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Culture-in-vitro method for mouse early embryo

An in vitro culture and embryo technology, applied in embryo cells, culture process, tissue culture, etc., can solve the problem of unclear changes in transcriptomics, and achieve the effect of improving the rate of high-quality embryos

Pending Publication Date: 2019-12-31
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the changes that occur in the whole transcriptome of embryos after light exposure are not clear

Method used

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  • Culture-in-vitro method for mouse early embryo
  • Culture-in-vitro method for mouse early embryo
  • Culture-in-vitro method for mouse early embryo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] 1. We collected mouse embryos at the 2-cell stage for different light treatment times. The specific numbers are as follows: no light treatment (0 hours) 227, 0.5 hours 102, 1 hour 98, 2 hours 103, 4 hours 104 and 118 in 6 hours.

[0018] 2. Treat mouse embryos at the 2-cell stage according to different light times, and continue to culture in the dark in a 37-degree Celsius incubator after the treatment is completed.

[0019] 3. Counting the blastocyst generation rate of mice treated with each light time, we found that the blastocyst rate decreased significantly after 6 hours of treatment. The results are as follows figure 1 shown.

Embodiment 2

[0021] 1. We collected mouse embryos at the 2-cell stage for different light intensities, and the specific numbers were as follows: no light treatment (0 lux) 332, 2000 lux 131, 3000 lux 152, 5000 lux 191.

[0022] 2. Treat mouse embryos at the 2-cell stage according to different light intensities, and continue to culture in the dark in a 37-degree Celsius incubator after the treatment is completed.

[0023] 3. Counting the blastocyst formation rate of mice treated with each light intensity, we found that the blastocyst rate decreased significantly after 6 hours of treatment. The results are as follows: figure 2 shown.

[0024] 4. Statistics on the morphology of mouse blastocysts treated with each light intensity, the results are as follows image 3 shown.

[0025] 5. Count the diameter of mouse blastocysts treated with each light intensity, and the results are as follows Figure 4 as shown,

Embodiment 3

[0027] 1. We collected the light group (5000 lux light for 6 hours at the 2-cell stage mouse embryo) and the non-light group mouse embryo cells at various stages, and used the Smart-seq2 single-cell transcriptome high-throughput sequencing method, and compared the sequences, Expression calculation Obtain single-cell transcriptome expression data.

[0028] 4. Comparing the principal components of single-cell transcriptomes in the light group and the non-light group in each period, it was found that there were large differences in the transcriptomes of the light group and the non-light group in each period, and they gathered in different positions, such as Figure 5 shown.

[0029] 5. Comparing the differential genes in the transcriptome of single cells in each period between the light group and the non-light group, and performing enrichment analysis of the G0 signaling pathway on these differential genes ( Figure 6 A-E), it was found that the transcriptional regulation (DNA t...

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Abstract

The invention discloses a culture-in-vitro method for a mouse early embryo. The method is characterized in comprising the following step of processing the mouse early embryo for 0.5-6h by a 2000-5000Lux light source, wherein the mouse early embryo includes embryo single cells of 2-cell, 4-cell, 8-cell, Morula and Blastocyst. The culture-in-vitro method has the advantages that percentage blastulation and a blastula diameter are effectively improved.

Description

technical field [0001] The invention relates to a method for culturing mouse early embryos in vitro. Background technique [0002] Assisted reproductive technology usually means that embryos are exposed to visible light during examination and transfer. Exposure to light is an unnatural stressor for embryos during IVF because the internal environment of the embryo is much darker than its in vitro environment. Such high-intensity or prolonged light exposures are harmful to embryos during embryo manipulation because they directly induce many stress-related metabolic processes or activate responses that lead to embryo apoptosis, such as the generation of intracytoplasmic reactive oxygen species. generate etc. However, changes in the entire transcriptome of embryos following light exposure are unknown. Because the preimplantation process of different mammalian embryos is similar in many ways, current studies generally consider mouse embryos, as a substitute for human embryos, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N13/00
CPCC12N5/0604C12N13/00C12N2529/10
Inventor 薛志刚曾桥
Owner TONGJI UNIV
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