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Targeting vector, recombinant Huh7 cell line, construction method and application

A targeting carrier and cell line technology, applied in the field of genetic engineering, can solve problems such as the application of drugs for liver cancer without the MFSD2A gene

Pending Publication Date: 2022-02-25
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no records and reports on how to apply MFSD2A gene in the preparation of drugs for the treatment of liver cancer in the prior art

Method used

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  • Targeting vector, recombinant Huh7 cell line, construction method and application
  • Targeting vector, recombinant Huh7 cell line, construction method and application
  • Targeting vector, recombinant Huh7 cell line, construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] 1. Design scheme of CL-YK2-001-A knock-in Huh7 cell line

[0101] 1.1 Background introduction

[0102] 1.1.1 The MFSD2A gene is on the positive strand of chromosome 1, with a total length of about 14.9kb. NCBI ID: 84879, see the following URL: https: / / www.ncbi.nlm.nih.gov / gene / ? term=84879.

[0103] 1.1.2 The MFSD2A gene has 10 transcripts, see the following website: http: / / uswest.ensembl.org / Homo_sapiens / Gene / Summary? db=core; g=ENSG00000168389; r=1:39955112-39969968.

[0104] 1.1.3 Structure domain see the following website: https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001136493.3? report=graph.

[0105] 1.1.4 Regulatory regions refer to the following website: http: / / uswest.ensembl.org / Homo_sapiens / Gene / Summary? db=core; g=ENSG00000168389; r=1:399551 12-39969968.

[0106]1.1.5 Conservation see the following URL: http: / / genome.ucsc.edu / cgi-bin / hgTracks? db=hg19&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position...

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Abstract

The invention provides a targeting vector, a recombinant Huh7 cell line and a construction method and application thereof, and belongs to the technical field of gene engineering. The targeting vector comprises a gene MFSD2A, and the nucleotide sequence of the targeting vector is as shown in SEQ ID NO. 1. The invention also provides a method for constructing a recombinant Huh7 cell line based on the targeting vector CL-YK2-001-A in the scheme, and the method comprises the following steps: transfecting a Huh7 cell with the targeting vector CL-YK2-001-A and a Cas9 / sgRNA plasmid to obtain the recombinant Huh7 cell line; the nucleotide sequence of the sgRNA is as shown in SEQ ID NO. 2. According to the method, the positive clone of the recombinant Huh7 cell line can be successfully obtained and further can be used for preparing a medicine for treating liver cancer; the sgRNA has the advantage of good comprehensive performance such as activity and specificity.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a targeting vector, a recombinant Huh7 cell line, a construction method and application. Background technique [0002] MFSD2A (Major facilitator superfamily domain containing 2a, MFSD2A), also known as sodium-dependent LPC symporter 1 (Sodium-dependent LPC symporter1, NLS1), is a member of the major facilitator superfamily (MFS) in mammals one. [0003] In recent years, there have been more and more studies on MFSD2A in tumors. Studies have shown that MFSD2A is a novel tumor suppressor gene in non-small cell lung cancer, which plays an important role in the inhibition of cell cycle and matrix attachment (Spinola et al.2010). Shi et al. detected MFSD2A and CD34 expression by immunohistochemistry on tissue microarrays of 170 cases of gastric cancer and adjacent normal tissues, and also investigated the relationship between MFSD2A and microvessel density and clinicopat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/65C12N15/12C12N5/10C12Q1/02C12R1/91
CPCC12N15/85C12N15/907C12N15/65C07K14/4705C12N5/0693C12N5/067G01N33/5011G01N33/5067C12N2800/107C12N2510/00C12N2503/02G01N2500/10
Inventor 荣维淇肖汀冯林程书钧吴健雄安松林
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI