Screening method of different metabolites for evaluating goat transgenic cloned embryo quality based on gas chromatography-mass spectrum combined technique and morphology

A gas chromatography and embryo cloning technology, applied in the field of bioengineering, can solve the problems of lack of uniform standards, insufficient identification of high-quality embryos, embryo genetic or epigenetic defects, etc., to achieve the effect of improving efficiency

Inactive Publication Date: 2015-12-02
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

Although this method is intuitive and fast, it lacks a unified standard, and the subjectivity of the observer makes the assessment of embryo morphology and developmental potential biased.
Alternatively, embryos w...

Method used

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  • Screening method of different metabolites for evaluating goat transgenic cloned embryo quality based on gas chromatography-mass spectrum combined technique and morphology
  • Screening method of different metabolites for evaluating goat transgenic cloned embryo quality based on gas chromatography-mass spectrum combined technique and morphology
  • Screening method of different metabolites for evaluating goat transgenic cloned embryo quality based on gas chromatography-mass spectrum combined technique and morphology

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Experimental program
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Embodiment Construction

[0031] 1 Materials and methods

[0032] 1.1 Sample collection

[0033] During the breeding season of goats (October-December), the ovaries of discarded non-pregnant goats were collected from Erling Slaughterhouse in Danyang, preserved in normal saline containing gentamicin (37°C), and transported back to the laboratory within 3 hours. In the laboratory, the mesentery was removed after the ovaries were washed 5 times with normal saline.

[0034] 1.2 Main instruments

[0035] Needle pulling instrument (Narishige, PC-10), needle forging instrument (Narishige, MF-900), needle grinding instrument (Narishige, EG400), micromanipulator (Nikon, Ti-5), fusion instrument (BTX, ECM2001) , stereo microscope (Nikon, C-DS), GC chromatograph (Agilent, 7890A), mass spectrometer (LECO, ChromaTOFPEGASUS4D).

[0036] 1.3 Main reagents

[0037] M2 (Sigma, M7167), Demethocin (Sigma, D1925), Quinn's Advantage Cleavage Medium (Sage, ART-1027), Quinn's Advantage Blastocyst Medium (Sage, ART-1029) ...

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Abstract

The invention discloses a screening method of different metabolites for evaluating goat transgenic cloned embryo quality based on a gas chromatography-mass spectrum combined technique and morphology. The screening method comprises the following steps that 1, reconstructed embryos are cultured for 48 hours, the cleavage rate is checked, cleaved embryos are divided into a high-quality cleaved embryo group and a low-quality cleaved embryo group according to the morphology, the embryo groups are randomly put into blastocyst culture droplets for continuous culture; 2, the blastocyst rate statistics is performed at 7.5 d, and blastocysts and embryo metabolites of the high-quality cleaved embryo group and the low-quality cleaved embryo group are respectively collected; 3, the collected embryo metabolite samples are processed, and gas chromatography-mass spectrum detection is performed; 4, detection results are subjected to statistic analysis. The screening method is integrated with morphological evaluation of the embryos to find metabolism markers closely correlated with the goat transgenic cloned embryo quality, provides a reference for research on prediction of embryonic developmental potential and facilitates screening of high-quality transplanted embryos and improvement of transgenic cloning efficiency.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for screening differential metabolites of goat transgenic cloned embryo quality assessment based on gas chromatography-mass spectrometry technology and morphology. Background technique [0002] Somatic cell nuclear transfer (SCNT) can dedifferentiate somatic cells in vitro and develop into totipotent embryos, which has broad clinical application prospects. However, the process of SCNT technology is complicated and there are many influencing factors, and the efficiency of obtaining newborn individuals through SCNT technology is also very low, which seriously restricts the research and application of transgenic large livestock. The study found that the lack of an effective embryo evaluation system and the inability to screen out high-quality embryos for transfer are one of the main reasons for the low efficiency of SCNT. Therefore, it is very important to find a ...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12N5/073G01N30/02
Inventor 张艳丽张国敏万永杰王锋
Owner NANJING AGRICULTURAL UNIVERSITY
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