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Application of KSOM-AA culture solution to in-vitro culture of non-obese diabetic (NOD) background mouse embryos

A KSOM-AA, in vitro culture technology, applied in the field of mouse fertilized egg culture, can solve problems such as incompatibility and differences in culture efficiency, and achieve the effects of increasing the birth rate, increasing the implantation ratio, and reducing the birth rate.

Pending Publication Date: 2020-12-08
GEMPHARMATECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the research operation, the researchers found that the conventional embryo in vitro culture technology has a significant difference in the culture efficiency between immunodeficiency mice and conventional strain mice, indicating that the existing culture system is not suitable for highly and severely immunodeficiency mice such as NOD and NCG. mouse strain

Method used

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  • Application of KSOM-AA culture solution to in-vitro culture of non-obese diabetic (NOD) background mouse embryos
  • Application of KSOM-AA culture solution to in-vitro culture of non-obese diabetic (NOD) background mouse embryos
  • Application of KSOM-AA culture solution to in-vitro culture of non-obese diabetic (NOD) background mouse embryos

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0075] Preparation example 1 Preparation of fertilization dish and capacitation dish

[0076] Fertilization dish: In a sterile ultra-clean bench, use a 200 μL pipette to draw 200 μL of HTF fertilization culture solution (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), drop a 200 μL HTF droplet into a 3.5 cm petri dish, and cover Mineral oil, transferred to a 37°C, 5% carbon dioxide incubator to equilibrate for at least 6 hours, and set aside;

[0077] Capabilization dish: In a sterile ultra-clean bench, use a 200 μL pipette to draw 200 μL of HTF fertilization culture solution (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), drop two 100 μL HTF microdrops into a 3.5 cm petri dish, and Cover with mineral oil, transfer to 37°C, 5% carbon dioxide incubator to equilibrate for at least 6 hours, and set aside.

preparation example 2

[0078] The preparation of preparation example 2 embryo culture dishes

[0079] In a sterile ultra-clean bench, use a 200 μL pipette to draw 200 μL of KSOM-AA culture solution, drop five 40 μL KSOM-AA microdrops into a 3.5 cm petri dish, cover with mineral oil, transfer to 37 ° C, 5% Equilibrate in a carbon dioxide incubator for 6 hours, and set aside;

[0080]Among them, the formulation of the KSOM-AA culture medium is shown in Table 1, and the essential amino acids are L-cystine, L-arginine hydrochloride, L-hydratine histidine, L-isoleucine, L-leucine amino acid, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine and L-valine, non-essential amino acid is L-hydrochloride Alanine, L-Asparagine Hydrate, L-Aspartic Acid, L-Glutamic Acid, L-Proline, L-Serine and Glycine; After the culture solution is prepared, filter and sterilize with a 0.22μm Millipore filter , distributed into brown ampoules, flushed with nitrogen, and stored in a ref...

preparation example 3

[0084] The preparation of preparation example 3 embryo petri dishes

[0085] In a sterile ultra-clean bench, use a 200 μL pipette to draw 200 μL of KSOM-AA culture solution, drop four 40 μL KSOM-AA microdrops into a 3.5 cm petri dish, cover with mineral oil, transfer to 37 ° C, 5% Equilibrate in a carbon dioxide incubator for 6 hours, and set aside;

[0086] Among them, the formula of KSOM-AA culture solution is shown in Table 2, and the essential amino acids are L-cystine, L-arginine hydrochloride, L-hydratine histidine, L-isoleucine, L-leucine amino acid, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine and L-valine, non-essential amino acid is L-hydrochloride Alanine, L-Asparagine Hydrate, L-Aspartic Acid, L-Glutamic Acid, L-Proline, L-Serine and Glycine; After the culture solution is prepared, filter and sterilize with a 0.22μm Millipore filter , distributed into brown ampoules, flushed with nitrogen, and stored in a refrigerat...

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Abstract

The invention provides application of a KSOM-AA culture solution to in-vitro culture of NOD background mouse embryos. By comparing with the effects of different culture solutions in culture of the NODbackground mouse embryos, the invention finds that the KSOM-AA culture solution is suitable for in-vitro culture of the NOD background mouse embryos, especially NCG background mouse embryos and NSG background mouse embryos; and with the cooperation of the constant temperature condition of 35-38 DEG C, the cleavage rate is reduced, the blastocyst formation rate is increased, the 2 cell developmentrate can reach 97.30%, and the blastocyst development rate can reach 18.92%; The KSOMAA culture solution is utilized to culture the NOD, NCG and NSG background mouse embryos, so that the implantationcapacity of the embryos is improved, thereby improving the birth rate; and the KSOM-AA culture solution has important significance in the aspect of in-vitro culture of the NOD, NCG and NSG backgroundmouse embryos.

Description

technical field [0001] The invention belongs to the technical field of mouse fertilized egg culture, and relates to the application of KSOM-AA culture solution in culturing mouse embryos with NOD background in vitro, in particular to the application of KSOM-AA culture solution in culturing mouse embryos with NOD, NCG and NSG backgrounds in vitro. Background technique [0002] In vitro culture of mammalian early embryos is a technique for forming early embryos from fertilized eggs in an artificially controlled environment. This technology is widely used in related fields such as research on early embryo development, construction of transgenic animals, and assisted reproduction. [0003] The in vitro culture technology of embryos is of great significance in scientific research and preservation of germplasm resources. The quality of embryo development is closely related to scientific research results. The main factors affecting the rate of embryo development are the choice of me...

Claims

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Application Information

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IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2501/998C12N2500/30C12N2500/12C12N2500/16C12N2500/34C12N2500/32
Inventor 薛红兰王红张嘉雯赵俊平
Owner GEMPHARMATECH CO LTD
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