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A method for establishing a sheep endometrial epithelial cell line and its application

A technology of endometrium and epithelial cells, applied in the biological field, can solve problems such as abnormal expression of target genes, silencing of vectors and foreign genes, and influence on the physiological state of cells, and achieve low nutritional requirements, shortened cell passage time, and high efficiency. The effect obtained

Active Publication Date: 2021-08-24
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of common commercial vectors to transfect cells not only has low integration efficiency, but also the vector and foreign genes are easily silenced, resulting in the abnormal expression of the target gene and affecting the normal physiological state of the cells.

Method used

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  • A method for establishing a sheep endometrial epithelial cell line and its application
  • A method for establishing a sheep endometrial epithelial cell line and its application
  • A method for establishing a sheep endometrial epithelial cell line and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Establishment of Sheep Endometrium Epithelial Cell Line

[0043] 1. Collect sheep uterine horns, separate and culture endometrial epithelial cells by tissue block culture method, at 37 ℃, 5% CO 2 Cultivate in the incubator for 7-12 days.

[0044] 2. Using the liposome transfection method, the universal PB transposon carrier pTP-hTERT carrying the hTERT gene was introduced into the primary cultured sheep endometrial epithelial cells for transfection. The specific steps are as follows:

[0045] 1) Before transfection, inoculate the third-generation primary cultured sheep endometrial epithelial cells in a 24-well plate, and perform transfection when the cell confluence is 70-90%.

[0046] 2) According to the amount of each well, dilute 1.5µL Lipofectamine 3000 reagent with 25µL Opti-MEM medium, mix well, and prepare Lipofectamine 3000 dilution.

[0047] 3) Add 25 µL of Opti-MEM medium, 2 µL of 1 µg / µL plasmid and 1 µL of P3000 reagent into a new EP tube, and mi...

Embodiment 2

[0053] Example 2 Cell Morphological Characterization Identification

[0054] After the cells were transfected, the morphological changes were observed under a phase-contrast microscope, and compared with primary sheep endometrial epithelial cells (such as figure 1 )Compare.

[0055] The results showed that the transfected sheep endometrial epithelial cells ( figure 2 ) is similar to the primary cell morphology, the central nucleus of the cell is obvious, and the nucleolus is clearly visible. Most of the cells were polygonal or oval in shape, and the cells were arranged in a typical cobblestone-like arrangement, and there was contact inhibition between cells.

Embodiment 3

[0056] Example 3 RT-PCR detection of hTERT expression in transfected cells

[0057] (1) Design and synthesis of primers

[0058] Design a pair of specific detection primers according to the human telomerase reverse transcriptase gene reference sequence published in GenBank (accession number NM_001193376), the upstream and downstream primers are Forward: 5ˊ-AGAGGTCAGGCAGCATCGG-3ˊ, see SEQ ID NO.1; Reverse: 5ˊ-TGCGTTCTTGGCTTTCAGG-3ˊ, see SEQ ID NO.2, the size of the amplified fragment is 1156 bp, and the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0059] (2) Extraction of total cellular RNA

[0060] ① Inoculate the transfected cells and primary cells in a 6-well plate. After the cells are confluent, discard the culture medium and wash twice with PBS.

[0061] ②Add 750 μL Trizol reagent to each well, mix well by pipetting, and let stand at 4°C for 5 minutes.

[0062] ③Pipe the cell lysate into a 1.5 mL sterilized EP tube treated with DEPC water, cent...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a method and application for establishing a sheep endometrial epithelial cell line. The invention introduces the hTERT gene into the sheep endometrial epithelial cells through the PB transposition vector, realizes the immortalization of the cells, and provides a sheep endometrial epithelial cell line that is easy to culture, has a fast growth rate and a simple operation method and a method for establishing the same. . The PiggyBac transposon transgenic vector used in the present invention can be inserted into the "TTAA" site of the cell genome at a fixed point, which can effectively avoid the influence of genome epigenetic modification and position effect on the target gene, so that the target gene can be expressed stably and continuously. As a result, immortalized cells can be obtained more efficiently, and the physiological characteristics of the transfected cells and the primary cells are guaranteed to be closer.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for establishing a sheep endometrium epithelial cell line and its application. Background technique [0002] Endometrial epithelial cells play an extremely important role in the "dialogue" between the embryo and the mother, and are crucial to animal reproduction. As the target tissue of ovarian hormones, the endometrium is closely related to embryo implantation. During the process of embryo implantation and establishment of endometrial receptivity, important morphological and functional changes have taken place, and it plays an important role in the study of reproductive physiology. important position. The complex mechanism by which the endometrium is regulated by various factors during pregnancy is still not well understood, so an in vitro model is needed to study its complex endometrial function. [0003] The concept of immortalized cell lines provides a theo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/54C12R1/91
CPCC12N5/0682C12N9/1276C12N15/85C12N2510/04C12N2800/107C12N2800/90C12Y207/07049
Inventor 胡广东谷新利郝科兴王静凌芳黄涛龙德智陈慧慧
Owner SHIHEZI UNIVERSITY
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