Establishment method and application of sheep endometrial epithelial cell line

A technology of endometrium and epithelial cells, applied in the biological field, can solve problems affecting the physiological state of cells, abnormal expression of target genes, silencing of vectors and foreign genes, etc.

Active Publication Date: 2020-10-23
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 g

Method used

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  • Establishment method and application of sheep endometrial epithelial cell line
  • Establishment method and application of sheep endometrial epithelial cell line
  • Establishment method and application of sheep endometrial epithelial cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0043] 1. Collect sheep uterine horns, isolate and culture endometrial epithelial cells by tissue block culture method, and store them at 37°C under 5% CO. 2 Culture in the incubator for 7 to 12 days.

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

[0045] 1) Before transfection, the sheep endometrial epithelial cells of the third generation of primary culture were inoculated into a 24-well plate, and transfection was performed when the cell confluence was 70-90%.

[0046] 2) Dilute 1.5 μL of Lipofectamine 3000 reagent with 25 μL of Opti-MEM medium according to the amount per well, and mix thoroughly to prepare DNA premix.

[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...

Embodiment 2

[0053] Example 2 Identification of Cell Morphological Characteristics

[0054] After transfection, the morphological changes of the cells were observed under a phase contrast microscope, and they were 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 ) was similar in shape to primary cells, with distinct central nucleus and clearly visible nucleoli. Most of the cells were polygonal or oval in shape, and the cells were arranged in a typical cobblestone paving stone 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] According to the published reference sequence of human telomerase reverse transcriptase gene in GenBank (accession number NM_001193376), a pair of specific detection primers was designed, and the upstream and downstream primers were Forward: 5ˊ-AGAGGTCAGGCAGCATCGG-3ˊ, see SEQID NO.1; Reverse :5'-TGCGTTCTTGGCTTTCAGG-3', see SEQ ID NO.2, the amplified fragment size is 1156bp, and the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0059] (2) Extraction of total cellular RNA

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

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

[0062] ③ The cell lysate was sucked into a 1.5 mL sterile EP tube treated with DEPC wat...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an establishment method and application of a sheep endometrial epithelial cell line. An hTERT gene is introduced into the sheep endometrial epithelial cells through a PB transposon vector, immortalization of the cells is achieved, and the sheep endometrial epithelial cell line which is easy to culture, high in growth speed and easy and convenient to operate and the establishment method of the cell line are provided. The PiggyBac transposon transgenic vector used in the invention can be inserted into a TTAA locus of a cell genome at a fixed point, so the influences of genome epigenetic modification and a position effect on a target gene can be effectively avoided, and the target gene can be stably and continuously expressed. Thus, immortalized cells can be obtained more efficiently, and the invention guarantees that the physiological characteristics of the transfected cells are closer to those of primary cells.

Description

technical field [0001] 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. Background technique [0002] Endometrial epithelial cells play an extremely important role in the process of "dialogue" between the embryo and the mother, and are essential for animal reproduction. As the target tissue of ovarian hormones, the endometrium is closely related to embryo implantation. During the process of embryo implantation and the establishment of endometrial receptivity, important morphological and functional changes have occurred, which play 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 remains unclear, so an in vitro model is needed to study its complex endometrial functions. [0003] The concept of immortalized cell line provides a theoreti...

Claims

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Application Information

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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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