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Bmal1 gene mutant cell strain as well as construction method and application thereof

A construction method and cell line technology, applied in the field of genetic engineering, can solve problems such as controversial conclusions

Pending Publication Date: 2022-05-27
ANHUI UNIVERSITY
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  • Claims
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Problems solved by technology

Although the research on the effect of Bmal1 gene on tumors has achieved certain results, the conclusions are still controversial

Method used

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  • Bmal1 gene mutant cell strain as well as construction method and application thereof
  • Bmal1 gene mutant cell strain as well as construction method and application thereof
  • Bmal1 gene mutant cell strain as well as construction method and application thereof

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Embodiment Construction

[0019] A method for constructing a Bmal1 gene mutant cell line. The present invention is explained in detail below, but does not mean that the present invention is limited thereto.

[0020] (1) Design sgRNA primer sequences: find the mBmal1 exon sequence found on the NCBI website, use the sgRNA design website of Zhang Feng's laboratory (http: / / crisprp.mit.edu) to find the sgRNA sequence in the exon, The sgRNA sequence with the highest score and the lowest off-target rate was selected according to the sgRNA sequence score. The final designed and synthesized sgRNA of Bmal1 gene was ACCCGTATTTCCCCGTTCGCTGG.

[0021] (2) Construction of px459-mBmal1 vector: digestion of px459 plasmid. px459 5.5μL (1μg), FastDigestBbs1 1uL, 10xFastDigest Buffer 2μL, ddH 2 O, 11.5 μL. Digestion at 37°C for 30 min. Phosphorylation and annealing of both strands of synthetic complementary sgRNA. Oligol (100 mM) 1 μL, Oligo2 (100 mM) 1 μL, 10xT4Ligation Buffer 1 μL, T4 PNK 0.5 μL, ddH 2 O 6.5 μL. ...

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Abstract

The invention discloses a Bmal1 gene mutation cell strain as well as a construction method and application thereof, the construction method comprises the following steps: extracting a genome from a screened mutation Bmal1 gene cell strain, amplifying a Bmal1 genome editing region, purifying and recovering an amplified product by PCR (Polymerase Chain Reaction), and sequencing. A sequencing result shows that the 92478th site to the 92490th site of the Bmal1 genome are edited, the corresponding exon is the 156th site to the 165th site of the 13th exon, and a CCACAACCAGCGA sequence is edited into a TCGC sequence. Other positions are not edited through off-target detection. The Bmal1 gene mutation cell strain constructed by the invention has an influence on the circadian rhythm and the cell cycle of cells. Experiments show that compared with wild type cells, the cell line NIH3T3 B1-B10G5 has the advantages that the cell cycle is stagnated in the G2 / M period, and the difference is extremely remarkable.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a Bmal1 gene mutant cell line and a construction method and application thereof. Background technique [0002] Most living things in nature are rhythmic. The molecular basis of biological rhythms consists of transcriptional / translational feedback loops, of which Bmal1 and Clock are two core clock transcription factors. The human Bmal1 gene was cloned in 1997. It consists of 20 exons and is located on the short arm (11p15) of chromosome 11. It encodes the protein BMAL1 and belongs to the structure of bHLH (basic helix-loop-helix)-PAS (PER-ARNT-SIM) The domain transcription factor family can generate circadian rhythm through its own expression regulation and the formation of positive and negative feedback regulatory pathways, and finally make the activities at the organ tissue level and the cell level highly coordinated. A large number of studies in recent years have ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N15/12C12N15/10C12N5/10
CPCC12N15/85C12N15/65C07K14/4702C12N15/102C12N5/0656C12N2800/107C12N2510/00C12Q2521/327C12Q2525/151C12Q2525/161
Inventor 秦曦明沈隆庚田田殷凡凡周琴
Owner ANHUI UNIVERSITY
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