PC9 cell strain knocked down or over-expressed by TAZ as well as construction method and application of PC9 cell strain

A cell line and overexpression technology, applied in the field of genetic engineering, can solve problems that have not been reported, and achieve the effects of shortened doubling time, slow cell growth, and accelerated cell growth

Inactive Publication Date: 2014-06-11
JIANGSU PROVINCE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a newly discovered oncogene, TAZ, a key factor in the Hippo pathway, may participate in the resistance of lung cancer cells to EGFR-TKIs therapy through the formation of CSCs and the TAED-AREG pathway, and its expression and function in EGFR-sensitive or drug-resistant lung adenocarcinoma research has not yet been reported

Method used

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  • PC9 cell strain knocked down or over-expressed by TAZ as well as construction method and application of PC9 cell strain
  • PC9 cell strain knocked down or over-expressed by TAZ as well as construction method and application of PC9 cell strain
  • PC9 cell strain knocked down or over-expressed by TAZ as well as construction method and application of PC9 cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Obtaining of plasmid pGPU6-GFP-Neo-TAZ-shRNA

[0055] 1. Use Designer3.0 (Genepharma) software for shRNA design and synthesis

[0056] 1.1 Design and synthesize the shRNA DNA fragment targeting the human TAZ sequence on GENE-BANK according to the above software. The loop structure in the shRNA template is TTCAAGAGA to avoid the formation of a termination signal, and the shRNA transcription termination sequence adopts the T6 structure. CACC is added to the 5' end of the sense strand template, which is complementary to the sticky end formed after digestion with BbsI; GATC is added to the 5' end of the antisense strand template, which is complementary to the sticky end formed after digestion with BamHI; if the first siRNA If a base is not G, add a G after CACC. The following are the shRNA-specific sites of 1 pair of TAZ sequences, the oligonucleotide chain sequence, and the oligonucleotide chain sequences of the negative control and positive control.

[0057] (...

Embodiment 2

[0108] Example 2. Acquisition of plasmid pEX-2-TAZ-over

[0109] 1. Obtain TAZ sequence fragments by PCR method

[0110] Oligo design, target gene TAZ upstream and downstream primers respectively add EcoRI, BamHI and protective bases for subcloning of vectors, oligo sequences such as B775-1-B775-54 in SEQ ID NO:10-63.

[0111] The primers were synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd., the above oligo was dissolved to 50 μM, and the same volume of oligo was taken into a 1.5ml centrifuge tube, mixed evenly, and made into oligo mix.

[0112] Use the prepared oligo mix for the first round of PCR reaction, the PCR system is as follows:

[0113]

[0114]

[0115] Loop condition:

[0116]

[0117] The second round of PCR reaction was carried out with B775-1 and B775-54, and the template was the product of the first round of PCR reaction.

[0118] The PCR system is as follows:

[0119] First-round PCR product 1μl 10×Pfu Buffer (+Mg...

Embodiment 3

[0132] Example 3 Plasmids pGPU6-GFP-Neo-TAZ-shRNA and pEX-2-TAZ-over respectively transfect PC9 cells

[0133] (a) The day before transfection, the logarithmic growth cells were seeded into 6-well plates, and 3×10 cells were seeded in each well according to the cell shape and growth rate. 5 Cells were placed in 10% fetal bovine serum RPMI DMEM complete medium at 37°C in 5% CO 2 Cultivate overnight in a saturation incubator for 20h-24h, so that the confluence of cells per well is about 70%-90% during transfection.

[0134] (b) Transfection according to Lipofectamine2000 Reagent reagent ①Add 5μl transfection reagent to 145μl Opti-MEM medium, mix well with pipette tip, ②Add 2.5μg plasmid to 150μl Opti-MEM medium, mix well with pipette tip, add ② In step ①, mix and let stand for 20 minutes to allow DNA-liposome complexes to form. Wash the cells once with Opti-MEM medium, add 1.7ml serum-free and antibiotic-free DMEM medium to each well, then add 300μl of the above mixture dropw...

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Abstract

The invention discloses an shRNA (Short Hairpin Ribonucleic Acid) molecule of a PC9 cell strain knocked down by a TAZ factor, a recombinant vector of the shRNA molecule, the recombinant vector of the PC9 cell strain over-expressed by the TAZ factor, and an establishment method of the PC9 cell strain knocked down and over-expressed by the TAZ. The method comprises the following steps: establishing the PC9 cell strain knocked down by the TAZ by transfecting PC9 cells via TAZ-shRNA by adopting an shRNA technology; establishing the PC9 cell strain over-expressed by the TAZ by transfecting the PC9 cells via a TAZ expression vector; detecting the TAZ in the cell strain by adopting an immune cell fluorescence technology, a RT-PCR (Reverse Transcription-Polymerase Chain Reaction) technology and a Western blot technology so as to prove the successful establishment of the cell strain; and measuring a cell growth curve and the cell doubling time. Thus, the powerful experimental material for researching the functions of the TAZ in the targeted therapy sensitivity of lung cancers is provided on the basis of the establishment of the PC9 cell strain knocked down and over-expressed by the TAZ.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a TAZ knockdown PC9 or overexpression cell line and its construction method and application. Background technique [0002] Lung cancer is a malignant tumor with the highest morbidity and mortality in the world. Non-small cell lung cancer (NSCLC) accounts for 75%-85% of lung cancers, and most of them are in the middle and advanced stages (IIIb and IV stages) when diagnosed, and the opportunity for surgery is lost. After comprehensive treatment, the 5-year survival rate is only 5%-10%. At present, tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) are not only convenient to take orally, have low toxicity, and are well tolerated, but also improve the quality of life of patients and prolong the life of patients. The survival time of selected patients plays an important role in the treatment of advanced lung cancer. Howev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N5/10
Inventor 许伟吴剑卿程雁
Owner JIANGSU PROVINCE HOSPITAL
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