Method for transfection of bone mesenchymal stem cells by BDNF

A bone marrow mesenchymal and stem cell technology, applied in the biological field, can solve the problems of plasmids not carrying labels, inability to observe transduction efficiency intuitively, cumbersome operation, etc., and achieve the effect of optimizing centrifugation speed and time

Pending Publication Date: 2020-05-12
JILIN UNIV
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Problems solved by technology

[0005] The existing transfection methods have the following problems: because the transfection is performed when the confluence of 293T cells is lo...

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  • Method for transfection of bone mesenchymal stem cells by BDNF

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Embodiment Construction

[0018] Introduce technical scheme of the present invention in detail below by embodiment:

[0019] A transfection method for BDNF transfected bone marrow mesenchymal stem cells, comprising the following steps:

[0020] (1) Requirements for transfection: transfect when the degree of confluence (100 mm dish) of 293T cells reaches 70%-80%;

[0021] (2) Incubation of the plasmid: 500 μl of the plasmid, lentiviral transfection reagent and serum-free MEM were incubated at room temperature for 5 minutes, then mixed and incubated for 20 minutes, and the EP tube was gently shaken every 5 minutes during the incubation. The ratio of packaging plasmid PAX2:PMD2G is 3:1, the amount of packaging plasmid and target plasmid is 1:1, the total amount of plasmid is 40ug, and the amount of lentiviral transfection reagent is 40μl.

[0022] (3) Cell transfection: each dish of 293T cells was replaced with half an amount of fresh medium, and the mixture was slowly added to the culture dish after the...

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Abstract

The invention discloses a method for transfection of bone mesenchymal stem cells by brain derived neurotrophic factors (BDNF). Transfection is performed when the fusion degree of 293T cells reaches 70-80%, and the density of 293T cells during transfection is increased; filtering and concentrating are performed by using an ultrafiltration tube during transfection to improve the virus titer; the centrifugal rotation speed and time are optimized; and rat bone mesenchymal stem cells capable of efficiently secreting BDNF factors is prepared by using lentivirus as a vector. A green fluorescent label(GFP) is added to a target fragment, and a milder transfer aid is used to reduce cytotoxicity during transduction, so that transduction efficiency is improved.

Description

technical field [0001] The invention relates to a novel lentivirus packaging method, which belongs to the field of biotechnology, and in particular to a transfection method for transfecting bone marrow mesenchymal stem cells with BDNF. Background technique [0002] Bone marrow mesenchymal stem cells (BMSCs) are the earliest discovered MSCs. Bone marrow mesenchymal stem cells are adult stem cells derived from bone marrow stroma, with self-renewal ability and multi-directional differentiation potential. As well as the advantages of being able to differentiate into various types of cells, it is a new method for clinical treatment of diseases. [0003] BDNF (brain derived neurotrophic factor, brain-derived neurotrophic factor), as a member of the neurotrophic factor family, is widely distributed in the central and peripheral nervous systems, and is essential for the growth, differentiation and repair of neurons in the central system. [0004] The existing transfection method i...

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Application Information

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IPC IPC(8): C12N15/867C12N15/12C12N5/10
CPCC07K14/475C12N15/86C12N2740/15043
Inventor 郭丽李安培张智丽尹飞
Owner JILIN UNIV
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