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

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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

Example Embodiment

[0039] Example 1

[0040] (1) HepG2 cell line recovery

[0041] Using a rapid thawing method, the cryopreservation tube of the human liver cancer HepG2 cell line was taken out of the -80°C refrigerator, and immediately placed in a 37°C constant temperature water bath to shake and thaw quickly for 1 min. In the ultra-clean bench, disinfect with ethanol first, then turn on. Use a pipette to suck the HepG2 cell suspension into a centrifuge tube containing nine times the volume of the complete culture medium (abbreviated as "end culture, DMEM containing 10% fetal bovine serum"), mix well and centrifuge at low speed (1000rpm×5min) with a centrifuge. Discard the supernatant, then add 5 mL of complete culture solution, mix by pipetting, and suck it into a culture flask, place it 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 fusion degre...

Example Embodiment

[0048] Example 2

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

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

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

[0052] SgRNA-1: 5’-TCAGTACGCCGTTTACGTCA-3; (Interference-1)

[0053] SgRNA-2: 5’-TGTTTCCGAAATTTACCGCA-3’; (Interference-2)

[0054] SgRNA-3: 5’-GGCTCTCTCCCCGTTGTTCC-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] Inoculate HepG2 cells that are not infected with lentivirus in a 6-well plate to achieve 70-80% confluency. After the cells adhere to the wall, add 1μg / mL, 2μg / mL, 2.5μg / mL, 3μg to the 6-well plate, respectively. / mL of puromycin (puromycin) drug, cell morphology was observed after 48h to screen for the lowest lethal concentration, and the lowes...

Example Embodiment

[0061] Example 3

[0062] CCK-8 method to detect cell proliferation

[0063] According to the operation steps provided by the CCK-8 kit, set up a blank control group, a negative control group, and an experimental group. Take cells in the logarithmic growth phase in good growth condition, add trypsin for digestion, prepare a cell suspension, and then inoculate it in 96-well plate (n=3), about 100μL per well, place the 96-well plate in an incubator for pre-culture (37℃, 5% CO 2 Under the conditions), take out and change the medium at a predetermined time point, 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 in the microplate reader.

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

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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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