Construction method of human astrocytes stably expressing OXTR (oxytocin receptors)

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 biological events and the mechanism behind them is not clear Clarification, unclear role of oxytocin, etc.

Inactive Publication Date: 2018-11-06
SOUTH CHINA NORMAL UNIVERSITY
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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 OXTR (oxytocin receptors)
  • Construction method of human astrocytes stably expressing OXTR (oxytocin receptors)
  • Construction method of human astrocytes stably expressing OXTR (oxytocin receptors)

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Embodiment 1

[0031] A method for constructing human astrocytes stably expressing oxytocin receptors, comprising the following steps:

[0032] 1) Constructing a recombinant lentiviral vector, the viral vector inserts the nucleotide sequence shown in SEQ ID NO.1 in the backbone of pCDH-CMV-MCS-EF1α-copGFP;

[0033] 2) packaging the recombinant lentiviral vector;

[0034] 3) Human astrocyte culture and screening: the packaged recombinant lentivirus was used to transduce human astrocytes to obtain human astrocytes stably expressing oxytocin receptor.

[0035] Further, the Flag-OXTR (SEQ ID NO.2) and mCherry (SEQ ID NO.3) fragments were linked together by overlapping PCR.

[0036] Further, a linker fragment (SEQ ID NO.4) was added between Flag-OXTR and mCherry to obtain a Flag-OXTR-mCherry fragment (SEQ ID NO.1),

[0037] Further, recognition sites for XbaI and EcoRI restriction endonucleases were respectively introduced at the 5' end and 3' end of the Flag-OXTR-mCherry fragment.

[0038] Fu...

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Abstract

The invention discloses a construction method of human astrocytes stably expressing OXTR (oxytocin receptors). The construction method comprises the following steps: 1) constructing recombinant lentiviral vectors; 2) transfecting packing cells with the recombinant lentiviral vectors for packaging; 3) transducing the human astrocytes with packaged recombinant lentiviruses to obtain the human astrocytes stably expressing the OXTR. According to the method, the OXTR with red fluorescent protein continuously and stably expressed on the human astrocytes are successfully constructed, and real-time monitoring of OXTR becomes possible. Meanwhile, the lentiviral vectors also carry another green fluorescent protein gene copGFP, which can mark the overall outline of astrocytes, and accordingly, morphological studies of the astrocytes are convenient.

Description

technical field [0001] The invention relates to a method for constructing hormone-expressing cells, in particular to a method for stably expressing oxytocin receptor human astrocytes. Background technique [0002] Oxytocin is an important neurohypophyseal hormone. When it binds to its specific receptor—oxytocin receptor (hereinafter referred to as OXTR), the downstream cascade signaling pathway is activated, thereby changing the physiological activities of target cells. With the deepening of research, scientists' research on the function of oxytocin has expanded from its early focus on its effect on the peripheral system to its effect on the central nervous system. 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 OXTR expression in astrocytes have not yet ...

Claims

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Application Information

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Patent Type & Authority Applications(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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