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Analytical method for excavating key lncRNA in process of differentiating chick embryo stem cells into male germ cells

An embryonic stem cell and germ cell technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of cumbersome operation, low safety, long cycle, etc., and achieve strong operability, high efficiency and safety. High performance, short cycle effect

Pending Publication Date: 2016-08-10
YANGZHOU UNIV
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Problems solved by technology

[0005] Aiming at the problems of long period, low efficiency, cumbersome operation and low safety of many current methods for inducing ESCs into male germ cells in vitro, the present invention establishes an analysis method for mining key lncRNAs, aiming to establish A new method to improve the efficiency of ESCs in vitro directed differentiation into male germ cells

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  • Analytical method for excavating key lncRNA in process of differentiating chick embryo stem cells into male germ cells
  • Analytical method for excavating key lncRNA in process of differentiating chick embryo stem cells into male germ cells
  • Analytical method for excavating key lncRNA in process of differentiating chick embryo stem cells into male germ cells

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Embodiment Construction

[0030] (1) Isolation and purification of chicken ESCs, PGCs and SSCs

[0031] ①Isolation of chicken ESCs

[0032] Newborn fertilized eggs are first cleaned with bromogeramine, and then sterilized again with 75% alcohol for later use. The separation experiment of chicken embryo disc was carried out in ultra-clean bench. First, hold the blunt end of the chicken embryo upwards in your hand, use the tip of the tweezers to gently drill a small hole in the middle of the fertilized egg, then insert one end of the tweezers into the small hole (do not insert too deep, 2-5mm is appropriate), along the equator In the direction of the equator line, use tweezers to crush the eggshells one by one (do not use too much force, just crush the eggshell in the middle of the tweezers), separate the eggshells along the equator line, and gently peel off the blunt end of the eggshell. The chicken embryo should be kept as horizontal as possible to prevent the yolk from flowing out with the egg white...

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Abstract

The invention discloses an analytical method for excavating key lncRNA in a process of differentiating chick embryo stem cells into male germ cells. The method comprises the following steps: firstly, separating chick ESCs, PGCs and SSCs from an embryonic disc of a fresh fertilized egg of a Rugao yellow chicken, a germinal ridge of the 19th stage (72h) and a testicle of the 18th day, performing sex determination by using a PCR amplification method according to a design specificity of a CDH1 gene, sorting antibody labeled ESCs, PGCs and SSCs, extracting cell total RNA from positive cells, detecting the quality of RNA after purification, and performing RNA-Seq sequencing after passing the detection; screening a transcript of assembled RNA from a sequencing result; separating chicken ESCs, PGCs and SSCs, extracting total RNA, and reversely transcribing the total RNA into cDNA; performing predictive analysis of Trans and Cis target genes on candidate lncRNA, searching a GO function item of the lncRNA by virtue of a DAVID database, annotating functions of the lncRNA by virtue of GO analysis, and performing functional analysis on the lncRNA; and drawing an interaction network diagram of the relationship between the key lncRNA, a target gene thereof and related signal channels.

Description

Technical field: [0001] The invention relates to an analysis method for mining key lncRNA in the differentiation process of chicken embryonic stem cells to male germ cells, and belongs to the fields of stem cell engineering, preservation of rare species, tissue and cell engineering, and the like. technical background: [0002] Germ cells can transmit biological genetic information to the next generation and maintain the transmission of life, which is of great significance to the study of germ cell differentiation. Therefore, germ cells can replace embryonic stem cells in the treatment of infertility and other diseases, 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. At present, the research on inducing ESCs into germ cells in vitro has been carried out for many years. On the one hand, exogenous genes are transferred into ESCs to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2537/165C12Q2535/122
Inventor 李碧春李东张亚妮纪艳芹王飞汪怡临王颖洁路镇宇靳锴何娜娜
Owner YANGZHOU UNIV
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