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.
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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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