Genetic engineering cell strain of obesity-resistant medicine target point UCP1, establishing and application of high-flux medicine screening model

A technology of target cells and cell lines, applied in genetic engineering, screening of compounds, and cells modified by introducing foreign genetic material, etc., can solve problems such as poor compliance and weight rebound, and reduce the mismatch rate and false positive rate. , the effect of high detection sensitivity

Active Publication Date: 2020-05-05
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Non-drug treatment of obesity, such as increasing exercise and controlling diet, often has poor compliance, while weight loss drug treatment achieves the purpose of restricting energy intake and then losing weight by inhibiting appetite or intestinal lipid absorption. Long-term use will lead to depression and steatorrhea And other side effects, once the drug is stopped, there will be obvious weight rebound

Method used

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  • Genetic engineering cell strain of obesity-resistant medicine target point UCP1, establishing and application of high-flux medicine screening model
  • Genetic engineering cell strain of obesity-resistant medicine target point UCP1, establishing and application of high-flux medicine screening model
  • Genetic engineering cell strain of obesity-resistant medicine target point UCP1, establishing and application of high-flux medicine screening model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1, Cas9 / sgRNA design targeting the UCP1 gene locus of immortalized brown adipocytes

[0066] 1. Sequencing confirmation of UCP1 sequence

[0067] The sequence of UCP1 gene may be different in different cell lines. In order to ensure the efficiency of the designed Cas9 / sgRNA, the UCP1 gene sequence of immortalized brown adipocytes was first amplified by PCR and sequenced to verify that the sgRNA recognition sequence was completely consistent with the UCP1 gene sequence of immortalized brown adipocytes.

[0068] Experimental method: (1) PCR primers are as follows:

[0069]

[0070] (2) The PCR reaction system is as follows:

[0071]

[0072]

[0073] (3) PCR reaction conditions are as follows:

[0074] Enzyme: KOD-FX

[0075] Program: Touchdown PCR

[0076]

[0077] Experimental results: PCR and sequencing of the DNA of the immortalized brown adipocytes showed that the UCP1 gene sequence of the immortalized brown adipocytes was completely consistent...

Embodiment 2

[0085] Embodiment 2, construction and preparation of recombinant exogenous gene carrier

[0086] Synthesize the 5' homologous arm (5'-homologous arm, LR) and the knock-in fragment luciferase-T2A-tdTomato-WPRE-pA (A), clone the 3' homologous arm (3'-homologous arm, RR) fragment , and the LR-A and RR fragments were connected with the carrier LScKO-4G to form a recombinant exogenous gene carrier UCP1-LScKO-4G-Neo-LR-A-RR. After enzyme digestion identification and sequencing, it was confirmed that the recombinant exogenous gene carrier was constructed .

[0087] 1. Exogenous gene cloning

[0088] Experimental method: (1) UCP1-LR-A was prepared by direct synthesis method

[0089]

[0090] (2) UCP1-RR is prepared by PCR method, and the primer sequences are as follows:

[0091]

[0092] (3) The LR-A (EcoRI+XhoI) and RR (NotI+NheI) fragments were connected to the vector LScKO-4G by enzyme digestion and ligation to form a recombinant exogenous gene vector UCP1-LScKO-4G-Neo-LR-...

Embodiment 3

[0105] Embodiment 3, positive clone screening

[0106] 1. Cell transfection

[0107] Experimental method: UCP1-sgRNA1, UCP1-sgRNA6 and recombinant exogenous gene vectors were electroporated to immortalize brown adipocytes. For electroporation methods, refer to the neon transfection instructions.

[0108] 2. Drug screening

[0109] Experimental method: 24 hours after transfection, 600 μg / mL G418 was added for drug screening, and 7 days later, enrichment was carried out to obtain mixed clones.

[0110] 3. PCR screening of mixed clones

[0111] Experimental method: Screening of mixed clones by PCR method, the specific process is as follows:

[0112] (1) PCR screening hybrid cloning primer design strategy:

[0113] See details Figure 9 .

[0114] (2) PCR screening primer sequences are as follows:

[0115]

[0116] (3) The PCR reaction system is as follows:

[0117] React components Volume(μL) final concentration ddH2O 19 — 2×KOD FX buffer 10 ...

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Abstract

The invention discloses a method for constructing a genetic engineering cell strain of an obesity-resistant medicine target point UCP1, and besides, discloses establishing and application of an obesity-resistant medicine high-flux screening model. Mainly a CRISPR/Cas9 system is related and utilized, two unique sgRNAs are designed, luciferase-T2A-tdTomato-WPRE-pA is knocked in after N end ATG of aUCP1 gene of cells, particularly luciferase and tdTomato are knocked in gene sites of immortalization brown fat cells UCP1, a first stably-transfected brown fat cell strain in which luciferase and tdTomato are inserted in a promoter region of the UCP1 is formed, and the first stably-transfected brown fat cell strain is applied to high-flux screening of obesity-resistant medicines, evaluation of the obesity-resistant medicines, evaluation of obesity-resistant active substances of organisms and development of a UCP1 detection reagent kit. The UCP1 is uncoupling protein specifically expressed atbrown fat tissue, and is an obesity-resistant new target point. The construction of the stably-transfected genetic engineering cell strain has important significance in the aspects of applying reportgenes and a high-connotation method, performing high-flux screening on a compound library acutely, accurately and efficiently, and obtaining obesity-resistant medicines capable of promoting thermogenesis and reducing weight.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a method of applying CRISPR / Cas9 system to knock in luciferase-T2A-tdTomato-WPRE-pA at the gene site of cell uncoupling protein 1 (UCP1), replacing exon1 and part of intron1, Construction of stable transgenic cell lines, especially gene-directed editing method and application of knocking luciferase and tdTomato into the gene locus of immortalized brown adipocyte UCP1, and using positive clones to establish high-throughput screening models for anti-obesity drugs and evaluate anti-obesity drugs , Evaluation of the body's anti-obesity active substances and the development of UCP1 detection kits. Background technique [0002] In the past two decades, genome editing technologies have been greatly advanced by innovations in site-directed design of nucleases, such as zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs). However, difficul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/113C12N15/90C12Q1/02
CPCC12N15/113C12N15/907C12N9/0069C07K14/43595G01N33/5044C12N2510/00C12N2310/10C12N2310/20G01N2500/10
Inventor 杜冠华强桂芬何萍刘俊成杨秀颖张莉侯碧玉徐春阳
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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