shRNA (short hairpin ribonucleic acid) targeted interfering YB-1 gene human lung adenocarcinoma a549 cell strains capable of stably expressing GFP (green fluorescent protein)

A stable expression, YB-1 technology, applied to cells modified by introducing foreign genetic material, recombinant DNA technology, using vectors to introduce foreign genetic material, etc., can solve the problems of time and low transfection efficiency, and shorten the research cycle , the effect of improving stability

Inactive Publication Date: 2015-09-30
FIRST AFFILIATED HOSPITAL OF DALIAN MEDICAL UNIV
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Problems solved by technology

However, siRNA-mediated gene silencing can only be maintained f

Method used

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  • shRNA (short hairpin ribonucleic acid) targeted interfering YB-1 gene human lung adenocarcinoma a549 cell strains capable of stably expressing GFP (green fluorescent protein)
  • shRNA (short hairpin ribonucleic acid) targeted interfering YB-1 gene human lung adenocarcinoma a549 cell strains capable of stably expressing GFP (green fluorescent protein)
  • shRNA (short hairpin ribonucleic acid) targeted interfering YB-1 gene human lung adenocarcinoma a549 cell strains capable of stably expressing GFP (green fluorescent protein)

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Experimental program
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Effect test

Embodiment 1

[0029] Establishment of cell lines:

[0030] 1. Construction of shRNA eukaryotic expression vector: the shRNA eukaryotic expression vector is pGPU6 / GFP / Neo, which is synthesized by shRNA guided by U6 promoter, and has neomycin resistance selection marker and green fluorescent protein GFP selection marker. The reverse complementary hairpin sequence specifically targeting different sequences of YB-1 mRNA (YBX1-homo-74: GGTTCCCACCTTACTACAT; YBX1-homo-326: AGAAGGTCATCGCAACGAA) was cloned into pGPU6 / GFP / Neo vector, the sequence was correct, purchased from Haiji mar company.

[0031] 2. Cell culture: 1 day before transfection, 2x10 5 A549 cells were plated (6-well plate), and the cell confluency was 70%-80% the next day.

[0032] 3. Expression plasmid transfection: Refer to the instructions of the Lipofectamine 2000 transfection kit, mix 2.5ug shRNA eukaryotic expression vector and 10ul Lipofectamine 2000 in 250ul medium respectively, then mix the two into 1.5ml medium to transfec...

Embodiment 2

[0035] Stable GFP-expressing shRNA targeted to interfere with YB-1 gene in human lung adenocarcinoma A549 cell line (pGPU6 / GFP / Neo-YBX1-homo-746-A549; pGPU6 / GFP / Neo-YBX1-homo-326-A549) -1 Gene expression level detection.

[0036] 1. The shRNA stably expressing GFP targets and interferes with the YB-1 gene in the human lung adenocarcinoma A549 cell line to detect YB-1 gene expression by reverse transcription PCR: extract RNA according to the instructions of the total RNA extraction kit, and use reverse transcription reagents Box instructions for reverse transcription PCR. Reverse transcription PCR YB-1 primer sequence: Forward: 5'-ACCACAGTATTCCATCCTCCTG-3'; Reverse: 5'-ATCTTCTTCATTAGCCGTCCTCTC-3'; PCR product length is 176bp. Internal reference gene β-Actin primer sequence: Forward: 5'-CATGTACGTTGCTATCCAGGC-3'; Reverse: 5'-CTCCTTAATGTCACGCACGAT-3'; PCR product length is 250bp. The PCR amplification conditions were: 94°C, 2min, 94°C, 30s, 55°C, 30s, 72°C, 10s, 35 cycles; 72°C,...

Embodiment 3

[0040] Thiazolium blue (MTT) plotted cell growth curves to detect the proliferation of human lung adenocarcinoma A549 cell lines stably expressing shRNA targeting YB-1 gene

[0041] 1. Take normal A549 cells in the logarithmic growth phase, and stably express GFP shRNA targeting YB-1 gene interference human lung adenocarcinoma A549 cells (pGPU6 / GFP / Neo-YBX1-homo-746-A549; pGPU6 / GFP / Neo -YBX1-homo-326-A549) and the cells of the negative control group were digested with trypsin and blown into a single cell suspension.

[0042] 2. 5,000 cells per well were seeded in a 96-well plate, with 6 wells in each group, and 3 plates in parallel, placed at 37°C, 5% CO 2 They were cultivated in the incubator for 24, 48, and 72 hours respectively.

[0043] 3. Discard the medium, add 20ul MTT (5mg / ml) to each well, and continue culturing for 3h.

[0044] 4. The medium was discarded, and 150ul of dimethyl sulfoxide (DMSO) was added to each well to terminate the reaction.

[0045] 5. Place th...

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Abstract

The invention relates to a shRNA (short hairpin ribonucleic acid) targeted interfering YB-1 gene human lung adenocarcinoma A549 cell strain A549/YBX1-homo-746 capable of stably expressing GFP (green fluorescent protein) and a shRNA (short hairpin ribonucleic acid) targeted interfering YB-1 gene human lung adenocarcinoma A549 cell strain A549/YBX1-homo-326 capable of stably expressing GFP, which are prepared by the following steps: constructing a targeted interfering YB-1 gene shRNA eukaryon expression vector, transfecting a human lung adenocarcinoma A549, and carrying out G418 screening. The collection numbers are respectively CCTCC C201522 and CCTCC C201523. The cell strains provide convenience for later research in correlation between in Yb-1 gene and cytobiological behavior change of lung adenocarcinoma, effectively shorten the research period and enhance the research result stability. The cell strains can stably express GFP, thereby providing convenience for later research in cell migration and invasion capacities and other cytobiological functions.

Description

technical field [0001] The invention relates to a shRNA stably expressing green fluorescent protein targeting interference YB-1 human lung adenocarcinoma A549 cell line. Background technique [0002] The incidence of lung cancer has been increasing in recent years, and its mortality rate has taken the first place in malignant tumors. About 1.1 million patients die of lung cancer in the world every year, accounting for 17.8% of cancer deaths. Among them, the incidence of lung adenocarcinoma has increased significantly, accounting for more than 40% of non-small cell lung cancer (NSCLC), and it is prone to early metastasis. Tumor invasion and metastasis directly affect the prognosis and survival of patients, and finding specific targets to inhibit tumor invasion and metastasis has become a reliable way to improve the survival rate of patients. Human Y box-binding protein-1 (YB-1) is an important nuclear transcription factor that specifically binds the internal Y-box sequence o...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85
Inventor 顾春东郭涛李锦绣
Owner FIRST AFFILIATED HOSPITAL OF DALIAN MEDICAL UNIV
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