Construction method of stable cell line realizing FTH1 gene controlled expression

A construction method and cell line technology, applied in the field of cell line construction, can solve problems such as cell damage, iron metabolism disorder, and reduced cell proliferation activity, and achieve the effect of precise control and controllable expression

Inactive Publication Date: 2016-02-24
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it has also been reported that in some specific cells such as cervical cancer Hela cells and nasopharyngeal carcinoma cells, the overexpression of FTH1 can significantly reduce the proliferation activity of cells.
In addition, the long-term and continuous expression of th

Method used

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  • Construction method of stable cell line realizing FTH1 gene controlled expression
  • Construction method of stable cell line realizing FTH1 gene controlled expression
  • Construction method of stable cell line realizing FTH1 gene controlled expression

Examples

Experimental program
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Effect test

Embodiment 1

[0055] A method for preparing a stable cell line with controlled expression of FTH1 gene, comprising the following steps:

[0056] (1) Construction of viral vector:

[0057] ◆Acquisition of the target gene FTH1: Synthesize the cDNA sequence of the FTH1 gene according to the information of FTH1 (BC000857) in Genebank, design primers, and the upstream and downstream primers have AgeI and EcoRI restriction sites respectively;

[0058] FTH1-F:

[0059] 5'-AACCGTCAGATCGC ACCGGT GCCACCATGACGACCGCGTCCACCTC-3'

[0060] FTH1-R:

[0061] 5'-TCCTTGTAGTCCAT GAATTC GCTTTCATTATCACTGTCTC-3'

[0062] PCR reaction system:

[0063]

[0064] PCR amplification conditions: 94°C, 5min; 94°C, 30s; 55°C, 30s; 72°C, 2min; 30cycle; 72°C, 10min; 4°C storage;

[0065] ◆FTH1 gene product digestion: use AgeI and EcoRI for double digestion;

[0066] Enzyme digestion reaction system:

[0067]

[0068] 37 ℃, reaction 4h; 70 ℃ fire extinguishing 10min;

[0069] ◆Agarose gel electrophoresis of ...

Embodiment 2

[0145] Detection of the SK-N-SH / Tet-on / FTH1 stable cell line constructed in Example 1

[0146] 1. Western blot assay for the induced expression of FTH1 protein in SK-N-SH cells

[0147] ◆Doxycycline (Dox) induces the expression of FTH1 in cells

[0148] 1) Divide SK-N-SH / Tet-on / FTH1 cells into 8×10 5 Density inoculation into 75ml culture flask, after the cells adhered to the wall, discard the original medium and divide into 2 groups;

[0149] 2) Group 1 needs 8 75ml culture flasks, add complete medium containing doxycycline at different concentrations (0, 0.02, 0.2, 0.6, 1, 2, 5, 10 μg / ml) respectively, and extract the whole cells after culturing for 72 hours. protein;

[0150] 3) The second group needs six 75ml culture flasks, add complete medium containing 0.6μg / ml doxycycline, and extract the whole protein of the cells at 0, 24, 48, 72, 96, and 120 hours respectively;

[0151] ◆Preparation of cell protein samples

[0152] 1) Pour off the original medium in the culture ...

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Abstract

The invention provides a construction method of a stable cell line realizing FTH1 gene controlled expression. The construction method comprises the following steps: (1) acquiring FTH1 genes; (2) transferring the FTH1 genes into a Tet-On vector to obtain recombinant plasmids; (3) constructing the recombinant plasmids into a lentiviral vector; (4) inoculating human neuroblastoma cells, after the cell fusion reaches 40-50%, adding a safe medium containing lentivirus liquid 0.8 [mu]l/ml (the titer is 1X10<9>TU/ml) and polybrene 5 [mu]g/ml, and polyethylene glycol 0.5-1 [mu]g/ml, and placing the safe medium into an incubator for culturing for 20-24 h; and (5) after cell passage, and the cell fusion reaches 50-60%, adding a medium containing 8 [mu]g/ml puromycin and treating for 7d, then adding a medium containing 4 [mu]g/ml puromycin and screening for 3d, and thus obtaining the stable cell line capable of inducing expression of the FTH1 genes, and the stable cell line is named SK-N-SH/Tet-on/FTH1. With the adoption of the construction method, the expression level and the expression time of FTH1 are accurately regulated, so that FTH1 is expressed when MR imaging is needed, and is turned off when MR imaging is not needed.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to the construction of cell strains. Background technique [0002] Clinically, magnetic resonance imaging (MRI) is widely used in the diagnosis, staging, follow-up and prognosis of diseases; in the field of molecular imaging, MRI has great applications because of its high spatial resolution, good soft tissue contrast and no radiation damage. Potential, especially for studies on transplanted cell tracking. MRI tracks transplanted cells mainly through magnetic labeling imaging and reporter gene imaging. The former uses MRI contrast agents such as metal chelates or superparamagnetic iron oxide particles (SPIO) to directly label cells. Due to the high transverse relaxation rate of SPIO, it is widely used in cell tracking imaging. However, this method of using MRI contrast agent to directly label cells has a big defect, that is, the contrast agent will gradually degrade with the divi...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10
Inventor 蔡金华贺小娅秦勇
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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