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Method for studying lin28 in regulating Blimp1 expression in process of differentiation from ESCs to PGCs of chickens

An overexpression and targeting technology, applied in the biological field, can solve the problem of not finding the key let-7microRNA, and achieve the effect of clear thinking and high operability

Pending Publication Date: 2019-09-20
YANGZHOU UNIV
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Problems solved by technology

However, so far, it has not been reported in poultry, and the key let-7 microRNA involved in the production of poultry PGCs has not yet been found

Method used

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  • Method for studying lin28 in regulating Blimp1 expression in process of differentiation from ESCs to PGCs of chickens
  • Method for studying lin28 in regulating Blimp1 expression in process of differentiation from ESCs to PGCs of chickens

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Experimental program
Comparison scheme
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Embodiment Construction

[0013] 1. Lin28A / B targeted gga-let7s screening

[0014] Using the online software http: / / mirdb.org / cgi-bin / search.cgi to predict chicken micRNA Let7s, a total of 17 gga-let7s were screened.

[0015] DF1 cells, ESCs and PGCs in good growth state were respectively selected and divided into 3 groups, one group was transfected with siLin28A / B, one group was transfected with oeLin28A / B, and the other group was not treated. DF1 cells were transfected in complete medium, and ESCs and PGCs were transfected in factor medium. At 37°C, 5% CO 2 After culturing for 48 h under saturated humidity, samples were taken to extract total RNA.

[0016] Total RNA was extracted according to the operating procedures of the miRNA extraction and isolation kit, cDNA was synthesized by reverse transcription according to the operating procedures of the reverse transcription kit, quantitative primers for micRNA-let7s were designed, and U6 was used as an internal reference to detect the relative expressi...

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Abstract

The invention relates to a method for studying lin28 in regulating Blimp1 expression in the process of differentiation from ESCs to PGCs of chickens and belongs to the technical field of biology. Lin28 is expanded to the field of poultry by studying micRNA-let7 indirectly regulated PGCs marker gene Blimp1 and accurately positioned on gga-let-7a-2-3p, so that functional range of Lin28-let7-Blimp1 regulating chains of different species is refreshed. The method is simple and easy to implement, clear in idea and high in operation feasibility and can be completed in a general lab.

Description

technical field [0001] The invention relates to a method for studying the expression of Blimp1 regulated by lin28 during the differentiation process of chicken ESCs to PGCs, specifically a method for studying the expression of Blimp1 regulated by lin28 targeting gga-let7a-2-3p during the differentiation process of chicken ESCs to PGCs, which belongs to biological technology field. Background technique [0002] Since West et al. discovered that Lin28 is a potential key regulator of PGCs development in 2009, researchers have begun to study its specific regulatory mechanism. Previous studies have found that Lin28A / B, as an RNA-binding protein, can regulate the expression of other genes by regulating the maturation of let-7 microRNA and participate in various biological processes. [0003] At present, there are many studies on Lin28s in tumorigenesis and cell pluripotency, but there are few studies on the mechanism of action in germ cell development. A few studies in mammals ha...

Claims

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Application Information

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IPC IPC(8): C12N15/85G01N21/64G16B20/30
CPCC12N15/85G01N21/6428G01N21/6486G16B20/30C12N2800/106
Inventor 李碧春姜景译左其生王曼李建成张明石祥袁霞
Owner YANGZHOU UNIV
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