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Method for performing high-throughput screening on high-quality oocytes

A high-throughput technology for oocytes, applied in the field of cell engineering, can solve the problem of low efficiency of oocyte screening and achieve the effect of improving the efficiency of in vitro maturation

Active Publication Date: 2018-05-08
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In view of the deficiencies in existing technologies, the purpose of the present invention is to provide a method for high-throughput screening of high-quality oocytes that is simple and convenient to operate, so as to solve the problem of low efficiency of oocyte screening at present

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: the preparation of solution

[0019] The method for judging the quality of oocytes by treating porcine oocytes with osmotic pressure and the solution formula group method of the present invention have a total of five formulas of sequential hypertonic solution and isotonic solution, which are respectively as follows:

[0020] Maturity solution Ⅰ, the formula is: TCM199 + 3.05mM glucose + 0.91mM sodium pyruvate + 0.57mML-cysteine ​​+ 100IU / mL penicillin + 100μg / mL streptomycin sulfate + 10% follicular fluid + 10IU / mL pregnancy Horse serum gonadotropin (PMSG) + 10IU / mL human chorionic gonadotropin (hCG).

[0021] Maturation solution II, the formula is: TCM199+3.05mM glucose+0.91mM sodium pyruvate+0.57mML-cysteine+100IU / mL penicillin+100μg / mL streptomycin sulfate+10% follicular fluid.

[0022] Cucumulus removal solution, the formula is: PVA-TL-Hepes+20mg / mL hyaluronidase+12mg mannitol+0.2mgBSA.

[0023] Isotonic hormone-free solution, the formula is: TCM199+...

Embodiment 2

[0025] Example 2: High-throughput screening of porcine oocytes

[0026] The test steps are as follows:

[0027] (1) Prepare the vacuum pump and its accessories, and adjust the negative pressure to 0.02 atmospheres. Use a 16-gauge needle to extract follicles with a diameter of 3-6 mm on the ovary. Cumulus-oocyte complexes (COCs) with intact granulosa cells and uniform cytoplasm were picked under a microscope, and the selected COCs were cultured in maturation solution I for 24 hours at 39°C, 5% CO 2 , saturated humidity. After 24 hours, it was replaced with maturation solution II, and then cultivated for 16-24 hours.

[0028](2) After culturing for 40h-48h, the oocytes were transferred into the decumulus solution, treated on a vortex shaker for 1 min, and then the oocytes with perivitelline space were transferred with an egg transfer needle for the next step. It should be noted that if the cytoplasm of the oocyte cannot shrink, it means that the membrane structure of the ooc...

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Abstract

The invention provides a method for performing high-throughput screening on high-quality oocytes. The method comprises the following steps: enabling oocytes in an in-vitro culture environment to be mature through mature liquid I and II, removing cumulus cells with a cumulus removing solution, performing initial selection on the integrity of the cytomembrane structure of the oocytes, enabling perivitelline space not to occur in the oocytes with an incomplete semipermeable membrane, eliminating one part of inferior oocytes in the process, and performing re-selection in an isosmotic hormone-freesolution, wherein if the cytomembranes of the initially selected oocytes cannot recover to the state before treatment with the cumulus removing solution, the oocytes must be eliminated, so that high-quality oocytes can be screened rapidly and with high throughput and are applied to development of productive cloning work.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a method for high-throughput screening of high-quality oocytes through osmotic pressure treatment technology. Background technique [0002] At present, in nuclear transfer research, there are basically only the following two methods for screening the oocytes used: the first is blind selection, no matter what the quality of the oocyte is, and it is done directly; the second is to use bright The oocytes were stained with cresyl blue, the stained oocytes were judged to be of good quality, and the unstained oocytes were judged to be of poor quality. Document retrieval disclosure: 1.Theriogenology, 68 (2007) 1299-1304 author: B.M.Manjunatha etc., published " select the buffalo oocyte that has developmental potential with bright cresyl blue before maturation " article content is, place oocyte In the BCB staining solution for 90 minutes, the selection rate of diffe...

Claims

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Application Information

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IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2500/12C12N2500/32C12N2500/34C12N2500/35C12N2501/31C12N2501/73
Inventor 肖红卫张立苹华再东任红艳李莉华文君毕延震刘西梅郑新民李会锋
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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