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sgRNA of targeting IGF-IR gene, and applications thereof

An IGF-IR, gene technology, applied in the field of genetic engineering, to achieve the effect of decreased migration ability

Inactive Publication Date: 2017-09-22
AFFILIATED HOSPITAL OF NANTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No report on directional splicing of IGF-IR gene in liver cancer

Method used

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  • sgRNA of targeting IGF-IR gene, and applications thereof
  • sgRNA of targeting IGF-IR gene, and applications thereof
  • sgRNA of targeting IGF-IR gene, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] (1) Recovery of HepG2 cell line

[0041] The cryopreservation tube of the human liver cancer HepG2 cell line was taken out from the -80°C refrigerator by rapid thawing, and immediately placed in a 37°C constant temperature water bath to thaw quickly with shaking for 1 min. Disinfect with ethanol first in the ultra-clean bench, and then turn it on. Use a pipette to suck the HepG2 cell suspension into a centrifuge tube with nine times the volume of complete culture medium (referred to as complete culture, DMEM containing 10% fetal bovine serum), mix well and centrifuge at a low speed (1000rpm×5min) in a centrifuge. Discard the supernatant, then add 5mL of complete culture solution, pipette and mix well, then inhale into a culture bottle, place at 37°C, 5% CO 2 And cultivate in an incubator with saturated humidity, observe the cell growth the next day, and replace the culture medium.

[0042] (2) HepG2 cell line culture

[0043] When the cell confluence is greater than 80...

Embodiment 2

[0049] Construction, infection and screening of Crispr / cas9-sgRNA dual vector lentivirus

[0050] (1) Construction of lentivirus targeting IGF-IR gene

[0051] According to the human IGF-IR sequence (NCBI: NM000875) and the principle of Crispr / cas9-sgRNA technology, three sgRNA sequences were designed:

[0052] sgRNA-1: 5'-TCAGTACGCCGTTTACGTCA-3; (interference-1)

[0053] sgRNA-2: 5'-TGTTTCCGAAATTTACCGCA-3'; (interference-2)

[0054] sgRNA-3: 5'-GGCTCTCTCCCCCGTTGTTCC-3'; (interference-3)

[0055] And construct the plasmid vector Lenti-CAS9-puro and Lenti-sgRNA-EGFP.

[0056] (2) Target cell lentivirus infection and screening

[0057] Liver cancer HepG2 cells not infected with lentivirus were inoculated in 6-well plates to achieve 70-80% confluence. After the cells were attached to the wall, 1 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL of puromycin (puromycin) drug, observe the cell morphology after 48h to carry out the screening of the lowest lethal concentration, so as to obtai...

Embodiment 3

[0062] Cell proliferation detected by CCK-8 method

[0063] According to the operation steps of the instructions provided by the CCK-8 kit, a blank control group, a negative control group, and an experimental group were set up. Cells in the logarithmic growth phase in good growth state were taken, digested with trypsin, prepared into a cell suspension, and then inoculated in 96-well plate (n=3), about 100 μL per well, put the 96-well plate in the incubator for pre-incubation (37°C, 5% CO 2 Under the conditions), take it out at a predetermined time point and change the medium, add 10 μL of CCK-8 reagent to each well, continue to incubate in the incubator for 1-4 hours, and then detect the A450 value with a microplate reader.

[0064] After the cells successfully infected with the lentivirus were collected, the protein was extracted for Western detection to analyze the gene knockout efficiency at the protein level ( Figure 4 , Figure 5 ), the expression of IGF-IR protein in ...

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Abstract

The invention belongs to the technical field of gene engineering, and discloses sgRNA of targeting IGF-IR gene. The nucleotide sequence of the sgRNA of targeting IGF-IR gene is represented by SEQ ID NO:2. According to applications, a Crispr / cas9-sgRNA lentiviral vector system is adopted, knockout or modification of human hepatoma cell line IGF-IR gene on cellular level is realized successfully, hepatoma carcinoma cell IGF-IR gene transcriptional level is reduced obviously, hepatoma carcinoma cell proliferation, invasion, and migration capacities are reduced, a novel method is provided for treatment of hepatic carcinoma, and clinical application prospect is promising.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a sgRNA targeting IGF-IR gene and its application. Background technique [0002] Recent studies have found that IGF-IR, a key signaling molecule in the IGF signaling pathway, is carcinoembryonic and closely related to the progression of liver cancer. However, whether its gene transcription or activation intervention affects biological functions and therapeutic value is unclear. The IGF family (IGFs) consists of IGF-I, IGF-II, IGF-IR, IGF-IIR and six binding proteins. IGFs and insulin have a similar molecular structure, and its functions include promoting cell proliferation, differentiation and the survival of various types of cells and maintaining various functions of cells, which play an important role in growth regulation in vivo. The main function of IGF-II is to produce growth factors, which play a key role in many growth and development processes and are widely ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N15/90
CPCC07K14/65C12N9/22C12N15/1136C12N15/86C12N15/907C12N2310/10C12N2740/15043
Inventor 董志珍姚敏王理姚登福郑文杰蔡胤
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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