Construction method of human astrocytes stably expressing oxytocin receptor

A technology of astrocytes and oxytocin receptors, applied in botany equipment and methods, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve the unclear role of oxytocin, biological events and The underlying mechanism has not been clearly elucidated and other issues, to achieve the effect of convenient research

Inactive Publication Date: 2021-10-22
SOUTH CHINA NORMAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

Astrocytes are a large group of very important specialized glial cells. Although it has been reported earlier that astrocytes express OXTR, the effect of oxytocin on astrocytes is still unclear.
In addition, the biological events and underlying mechanisms that regulate the expression of OXTR in astrocytes have not yet been clearly elucidated.

Method used

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  • Construction method of human astrocytes stably expressing oxytocin receptor
  • Construction method of human astrocytes stably expressing oxytocin receptor
  • Construction method of human astrocytes stably expressing oxytocin receptor

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Experimental program
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Embodiment 1

[0031] A construction method of stabilizing the expression of oxytocin receptor human beech colloidal cells, including the following steps:

[0032] 1) Construct a recombinant lentiviral vector, which inserts a nucleotide sequence shown in SEQ ID NO.1 in the PCDH-CMV-MCS-EF1α-COPGFP skeleton;

[0033] 2) Package the recombinant slow viral vector;

[0034] 3) Human star colloidal cell culture and screening: use a packaging recombinant slow virus to transduction human star-collabore cells to obtain a stable expression of oxytocin receptor human astrocytes.

[0035] Further, a method of overlapping PCR connects Flag-OXTR (SEQ ID No. 2) and the mcherry (SEQ IDNO. 3) clip.

[0036] Further, the LINKER fragment (SEQ ID NO.4) is added between Flag-OXTR and MCHERRY to get the flag-oxtr-mcherry fragment (SEQ ID NO.1),

[0037] Further, the identification site of XBaI and EcoRI limiting endonuclease is introduced into the 5 'end and 3' end of the Flag-Oxtr-mCherry fragment.

[0038] Further...

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Abstract

The invention discloses a construction method for human astrocytes stably expressing oxytocin receptors, comprising: 1) constructing a recombinant lentiviral vector; 2) transfecting the recombinant lentiviral vector into packaging cells for packaging; 3) using the packaged Human astrocytes were transduced with recombinant lentivirus to obtain human astrocytes stably expressing oxytocin receptor. The method of the present invention successfully constructs and obtains the oxytocin receptor with red fluorescent protein continuously and stably expressed on human astrocytes, making it possible to monitor OXTR in real time. At the same time, the lentiviral vector also carries another green fluorescent protein gene, copGFP, which can mark the overall outline of astrocytes and facilitate morphological research on astrocytes.

Description

Technical field [0001] The present invention relates to a method of constructing a hormone expression cell, and more particularly to the construction method of stabilizing the expression of oxytocin receptor human beetrocytes. Background technique [0002] Oxytocin is an important nerve pituitary hormone. When it is combined to its specific receptor-oxytomerin receptor (Oxytocin RecePtor, the downstream cascading signal path is activated, and the physiological activity of the target cells is changed. With the in-depth study, scientists have focused on their role in the peripheral system, and expanded to the role of the Chinese nervous system. Starped colloidal cells are a very important specialized glial cells. Although there have been reported that astrocytes express OXTR, the effect of oxytocin on astrocytes is still unclear. In addition, the biological events and the mechanism behind the expression of OXTR in astrocytes have not been clearly clarified. Inventive content [00...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12Q1/02
CPCC12N15/86C12N2740/15043C12Q1/02
Inventor 吴冈义何耀俊霍清伟
Owner SOUTH CHINA NORMAL UNIVERSITY
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