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Establishment and application of genetically engineered cell lines and high-throughput drug screening models for the anti-obesity drug target ucp1

A cell line and target cell technology that can be used in genetic engineering, compound screening, and cells modified by the introduction of foreign genetic material, which can solve problems such as weight rebound and poor compliance.

Active Publication Date: 2022-07-19
INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • 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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  • Establishment and application of genetically engineered cell lines and high-throughput drug screening models for the anti-obesity drug target ucp1
  • Establishment and application of genetically engineered cell lines and high-throughput drug screening models for the anti-obesity drug target ucp1
  • Establishment and application of genetically engineered cell lines and high-throughput drug screening models for the anti-obesity drug target ucp1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Design of Cas9 / sgRNA targeting the UCP1 locus of immortalized brown adipocytes

[0066] 1. Sequencing confirmation of UCP1 sequence

[0067] Different cell lines may have different UCP1 gene sequences. 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 immortalized brown adipocyte DNA showed that the UCP1 gene sequence of immortalized brown adipocyte was completely consistent with the UCP1 gene sequenc...

Embodiment 2

[0085] Example 2. Construction and preparation of recombinant exogenous gene vector

[0086] Synthesize the 5'-homologous arm (LR) and the knock-in fragment luciferase-T2A-tdTomato-WPRE-pA (A), and clone the 3'-homologous arm (RR) fragment , and connect the LR-A and RR fragments with the vector LScKO-4G to form a recombinant exogenous gene vector UCP1-LScKO-4G-Neo-LR-A-RR. After identification and sequencing, the construction of the recombinant exogenous gene vector was confirmed. .

[0087] 1. Foreign gene cloning

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

[0089]

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

[0091]

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

[0093] Experimental results: The gel electrophoresis image of exogenous gene RR fragment ampli...

Embodiment 3

[0105] Example 3. Screening of positive clones

[0106] 1. Cell transfection

[0107] Experimental method: UCP1-sgRNA1, UCP1-sgRNA6 and recombinant exogenous gene vector were electroporated into immortalized brown adipocytes. For the electrotransformation method, please refer to the instructions of neon transfection.

[0108] 2. Drug Screening

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

[0110] 3. PCR screening of mixed clones

[0111] Experimental method: The mixed clones were screened by PCR method. The specific process is as follows:

[0112] (1) PCR screening mixed clone 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] reaction component Volume(μL) Final concentration ddH2O 19 — 2×...

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Abstract

The invention discloses a method for constructing a genetically engineered cell line of anti-obesity drug target UCP1, and at the same time discloses the establishment and application of a high-throughput screening model for anti-obesity drugs. It mainly involves using the CRISPR / Cas9 system to design two unique sgRNAs to knock in luciferase‑T2A‑tdTomato‑WPRE‑pA after the N-terminal ATG of the UCP1 gene in cells, especially knocking luciferase and tdTomato into immortalized brown adipocyte UCP1 The first stably transfected brown adipocyte cell line with luciferase and tdTomato inserted into the UCP1 promoter region was formed, and it was used in high-throughput screening of anti-obesity drugs, evaluation of anti-obesity drugs, and anti-obesity active substances in the body. evaluation and development of UCP1 detection kits. UCP1 is an uncoupling protein specifically expressed in brown adipose tissue and a new target for anti-obesity. The construction of this stable transgenic engineered cell line is suitable for the application of reporter genes and high-content methods, sensitive, accurate and efficient. Through flux screening, it is of great significance to obtain anti-obesity drugs that promote thermogenesis and reduce body weight.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a CRISPR / Cas9 system for knocking in luciferase-T2A-tdTomato-WPRE-pA at a cell uncoupling protein 1 (UCP1) gene site, replacing exon1 and part of intron1, Construction of stable transgenic cell lines, especially the gene-directed editing method and application of knocking luciferase and tdTomato into the gene locus of immortalized brown adipocytes UCP1, and the use of positive clones to establish a high-throughput screening model 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, innovations in site-directed design of nucleases, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have led to dramatic advances in genome editing technology. However, difficulties in d...

Claims

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Application Information

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Patent Type & Authority Patents(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 CHINESE ACAD OF MEDICAL SCI
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