Verification method of key transcription factors in core region of Nanos2 promoter

A core region and transcription factor technology, applied in the fields of animal breeding and cell biology, can solve the problems of unstable induction effect and unstable transfection efficiency, and achieve high repeatability, simple method and high efficiency. Effect

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

However, due to the unstable transfection efficiency of ESCs in vitro, the induction effect is unstable, and the induction s

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  • Verification method of key transcription factors in core region of Nanos2 promoter
  • Verification method of key transcription factors in core region of Nanos2 promoter
  • Verification method of key transcription factors in core region of Nanos2 promoter

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Embodiment Construction

[0031] 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.

[0032] (1) Qualitative detection of the activity of the Nanos2 promoter region

[0033] The Nanos2 5' flanking region -2bp~-1189bp was amplified by PCR, and the amplified product was subjected to 1% agarose gel electrophoresis, recovered by cutting the gel, and constructed into the pEGFP-N1 vector after double digestion with AseI and KpnI, and recombined Plasmid pNanos2-EGFP was identified by single and double enzyme digestion and bacterial liquid PCR, and positive samples were sent for testing. Nucleic acid sequencing and comparison showed that the recombinant vector was successfully constructed.

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Abstract

The invention discloses a verification method of key transcription factors in the core region of the Nanos2 promoter, comprising the following steps: firstly, carrying out PCR amplification of the sequence of the 5' flanking region of Nanos2 to construct an eukaryotic expression vector pNanos2-EGFP, and respectively transfecting pNanos2-EGFP, pEGFP-N1 and pLinker-EGFP into DF-1 cells to qualitatively analyze the promoter region of Nanos2; respectively constructing vectors pGL3-1187, pGL3-959, pGL3-788, pGL3-493 and pGL3-155 with different lengths deleted at 5' end of the promoter and co-transfecting the vectors and pRL-SV40 into DF-1 cells, carrying out luciferase reporter gene assay for identification of the core region of the Nanos2 promoter, constructing a vector with the core region deleted, and performing luciferase activity analysis to observe whether the promoter activity is lost or remarkably reduced after the core region is deleted; carrying out transcription factor binding site prediction on the core region of the Nanos2 promoter, and performing Dual luciferase reporter assay and CHIP test to determine the key transcription factors. Male germ cell differentiation is detected by methods of cell morphology observation, qRT-PCR, cellular immunochemistry and flow cytometry.

Description

technical field [0001] The invention belongs to the fields of animal breeding and cell biology, and specifically relates to a method for verifying key transcription factors in the core region of a Nanos2 promoter. Background technique [0002] Embryonic stem cells (ESCs) are a type of totipotent stem cells isolated from early embryos or primitive gonads. They have the potential to differentiate into various types of cells in the body, and their differentiation into male germ cells has always been one of the current research hotspots. one. It is well known that the differentiation of ESCs into male germ cells is regulated by factors such as inducing factors, key genes, and signaling pathways. Therefore, it is only necessary to elucidate the regulatory mechanism of these regulatory factors in the differentiation of male germ cells and establish an effective in vitro induction differentiation system. , to fundamentally improve the in vitro generation efficiency of male germ ce...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/66G01N33/68
CPCC12Q1/66C12Q1/6851G01N33/68C12Q2531/113C12Q2563/107C12Q2561/113
Inventor 张亚妮张文慧李碧春王颖洁王曼
Owner YANGZHOU UNIV
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