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Method for function verification of Nanos2 in differentiation of chicken ESCs into male germ cells

A germ cell and functional verification technology, applied in the field of functional verification of Nanos2 in the process of differentiation of chicken ESCs into male germ cells, can solve problems such as unstable induction effect, improve generation efficiency, strong reproducibility, and simple and easy method. Effect

Inactive Publication Date: 2018-12-18
YANGZHOU UNIV
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Problems solved by technology

[0006] However, because the differentiation process is still affected by other factors, the induction effect is unstable, and the induction system needs to be further improved.
(1) The effect of chicken embryo injection experiment depends on the degree of random diffusion after injection; (2) The differentiation process is still affected by other unknown factors, resulting in unstable induction effect

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  • Method for function verification of Nanos2 in differentiation of chicken ESCs into male germ cells

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Embodiment Construction

[0026] In order to illustrate the technical scheme and technical purpose of the present invention, the present invention will be further introduced below in conjunction with the description of the drawings and specific implementation methods.

[0027] (1) Detection of the expression level of Nanos2 in different chicken tissues

[0028] In order to detect the localized expression of Nanos2 in chickens, different tissues of adult male and female chickens were collected: heart, liver, spleen, lung, kidney, glandular stomach, gizzard, pancreas, duodenum, cecum, leg muscle, gonad, The relative expression level of Nanos2 was detected.

[0029] (2) Nanos2 cloning and construction of pcDNA3.0-Nanos2 vector

[0030] Aiming at the sequence of the Gallus chicken Nanos2 coding region provided by NCBI, design specific primers, amplify the CDS region of Nanos2 by PCR, and clone the fragment into the pcDNA3.0 vector digested with Kpn I / EcoR I (Takara Bio, Dalian, China) superior. The comp...

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Abstract

The invention belongs to the field of animal breeding and cell biology, and particularly relates to a method for function verification of Nanos2 in differentiation of chicken ESCs into male germ cells, and the method includes detection of the expression level of the Nanos2 in different chicken tissues, Nanos2 clone and construction of pcDNA3.0-Nanos2 vector, Nanos2 knockout vector construction andknockout activity verification, in-vitro Nanos2 function verification, in-vivo Nanos2 function verification, Quantitative Real Time PCR (quantitative RT-PCR) verification, cell morphology observationand cellular immunochemical detection, flow cell analysis, paraffin section and PAS staining. Compared with the prior art, the function verification of the Nanos2 can be accomplished relatively quickly at the chicken germ cell level. The method is simple and easy to perform, has high repeatability and can be performed in an ordinary laboratory. The method greatly enhances the efficiency of in-vitro induction differentiation of the chicken ESCs into the male germ cells. The method greatly improves the efficiency of in-vivo induction generation of chicken PGCs and SSCs.

Description

technical field [0001] The invention belongs to the fields of animal breeding and cell biology, and in particular relates to a method for functional verification of Nanos2 in the differentiation process of chicken ESCs to male germ cells. Background technique [0002] Germ cells (Germ cells, GCs) are a type of cells that can reproduce offspring in multicellular organisms, and have the function of transmitting genetic information to the next generation. Therefore, research on germ cells is of great significance to life cycle and inheritance. Among them, Male germ cells play a prominent role in the treatment of male infertility, and do not involve ethical and immune rejection issues. Therefore, it is necessary to quickly conduct research on how to induce stem cells from other tissues to differentiate into male germ cells. [0003] Embryonic stem cells (ESCs) are a type of cells isolated from early embryos or primitive gonads, which have the characteristics of unlimited prolife...

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N9/22G01N33/569
CPCC12N9/22C12N15/85C12N15/902G01N33/56966
Inventor 张亚妮张文慧李碧春王颖洁王曼程少泽汪怡临何娜娜
Owner YANGZHOU UNIV
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