Slow virus green fluorescence autophagy report vector and building method thereof

A green fluorescence and lentivirus technology, applied in the field of cell research, can solve the problems of high price, limited use, and low stability, and achieve the effect of short screening cycle, low cost, and high stability

Inactive Publication Date: 2017-10-10
FIRST HOSPITAL OF SHANXI MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, there are some commercial green fluorescent vectors for autophagy detection, but there are some limitations: 1. The vector backbone is a common plasmid vector, and it is difficult to obtain stable expression cell lines; 2. The price is expensive and the use is limit

Method used

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  • Slow virus green fluorescence autophagy report vector and building method thereof
  • Slow virus green fluorescence autophagy report vector and building method thereof
  • Slow virus green fluorescence autophagy report vector and building method thereof

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Embodiment Construction

[0027] The invention provides a method for constructing a lentiviral green fluorescent autophagy reporter carrier, including the construction of inserting the target vector pLenti-puro-GFP-LC3, the verification of inserting the target gene, and the verification of transfected cells observed by fluorescence microscope for autophagosomes. In the following, the present invention will be further described in detail in conjunction with specific experimental procedures and experimental results, which are explanations rather than limitations of the present invention.

[0028] A method for constructing a lentiviral green fluorescent autophagy reporter carrier, comprising the following steps:

[0029] First provide the source or preparation method of the plasmid and main reagents:

[0030] The pLenti-puro vector and pEX-GFP-hLC3WT vector were provided by our laboratory. DH5α competent cells were purchased from Beijing Quanshijin Biotechnology Company. Lipofectamine 3000 transfection ...

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Abstract

The invention belongs to the field of cell research, and relates to a lentiviral green fluorescent autophagy reporter carrier based on a lentiviral carrier skeleton, which is used for screening stable strains and observing changes in autophagosomes in cells and a construction method. The invention discloses a lentiviral green fluorescent autophagy reporter carrier, which comprises ampicillin resistance, puromycin selection marker and green fluorescent autophagy marker protein LC3 gene sequence. The green fluorescent autophagy marker protein LC3 plasmid of the present invention uses lentivirus as the backbone, which is conducive to the establishment of stably expressing green fluorescent cell lines, and due to the wide applicability of lentivirus to mammalian cells, the green fluorescent plasmid of the present invention can be used for various cells. Compared with the expensive price and specificity of using common vectors, the cost is very low.

Description

technical field [0001] The invention belongs to the field of cell research, and relates to a lentiviral green fluorescent autophagy reporter carrier based on a lentiviral carrier skeleton, which is used for screening stable strains and observing changes in autophagosomes in cells and a construction method. Background technique [0002] Autophagy is a phenomenon of "self-eating" in cells, and apoptosis is a phenomenon of "suicide". The two share the same stimulating factors and regulatory proteins, but the induction thresholds and thresholds are different. How to switch and coordinate is still unclear . Autophagy refers to the membrane wrapping part of the cytoplasm and the organelles and proteins that need to be degraded in the cell to form autophagosomes, and finally fuse with lysosomes to form autophagy lysosomes, which degrade the contents wrapped by them, so as to achieve cell homeostasis and Cellular renewal. [0003] Since the autophagy process is very fast, dynamic ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12N15/66
CPCC12N15/86C12N15/65C12N15/66C12N2740/15043
Inventor 吴勇延郭慧娜王斌全高伟温树信赵沁丽赵均朱美霞洛鸿洁相彩霞
Owner FIRST HOSPITAL OF SHANXI MEDICAL UNIV
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