Method for constructing EPB41 gene knockout THP-1 cell line based on CRISPR-Cas9 system

A THP-1, gene knockout technology, applied in the biological field, can solve the problems of low fertility and long reproductive cycle of mice

Pending Publication Date: 2021-03-12
ZHENGZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In view of the important role of protein 4.1R in the immune response, it is very important to obtain a cell line that knocks out the EPB41 gene for the study of the function of protein 4.1R. Currently, 4.1R deletion mice have low fertility and long reproductive cycles.
Human 4.1R-deficient cell lines have not been reported. In order to better study t

Method used

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  • Method for constructing EPB41 gene knockout THP-1 cell line based on CRISPR-Cas9 system
  • Method for constructing EPB41 gene knockout THP-1 cell line based on CRISPR-Cas9 system
  • Method for constructing EPB41 gene knockout THP-1 cell line based on CRISPR-Cas9 system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 A method for constructing a cell line that knocks out the EPB41 gene based on CRISPR-Cas9 editing technology

[0044] 1. Design of sgRNA and synthesis of oligonucleotide chains. Three sgRNAs were designed for the No. 1 exon, No. 4 exon and No. 7 exon of human EPB41 gene, and the oligonucleotide chains were synthesized. synthesis. Target sequences are shown in Seq-No.1, Seq-No.2 and Seq-No.3.

[0045] 2. LentiCRISPR v2 digestion

[0046] (1) LentiCRISPRv2 digestion and terminal dephosphorylation:

[0047]

[0048] Cut at 37°C for 30 minutes.

[0049] (2) 1% agarose gel electrophoresis detection (results see figure 1 ); TIANgel Midi Purification Kit gel recovery LentiCRISPRv2 large fragment, concentration determination.

[0050] 3. Construction of LentiCRISPR v2 vector

[0051] (1) Annealing and phosphorylation of gRNA primers:

[0052] Oligo 1 (100 μM) 1 μL

[0053] Oligo 2 (100 μM) 1 μL

[0054] 10×T4 Ligation Buffer 1μL

[0055] T4 PNK 0.5 μL

...

Embodiment 2

[0077] Example 2. Gene level identification of THP-1 cell line with EPB41 gene knockout

[0078] (1) Genomic DNA in three cell lines THP-1-KO-EPB41-1, THP-1-KO-EPB41-2 and THP-1-KO-EPB41-3 were extracted. (Tiangen Genome Extraction Kit catalog number: DP304)

[0079] (2) Three pairs of identification primers were designed for the target sites of the three sgRNAs. The forward and reverse identification primer sequences of the cell line THP-1-KO-EPB41-1 are Seq-No.10 and Seq-No.11 respectively ;, cell line THP-1-KO-EPB41-2 forward and reverse identification primer sequences such as Seq-No.12 and Seq-No.13 respectively; cell line THP-1-KO-EPB41-3 forward and reverse The primer sequences for reverse identification are Seq-No.14 and Seq-No.15, respectively, and ranh extracts genomic DNA from cells for PCR amplification.

[0080] (3) PCR amplified product (about 200bp) and carry out 1% agarose gel electrophoresis detection (results see attached image 3 ), cut out the target band...

Embodiment 3

[0085] Example 3 Western Blot detection of 4.1R protein expression in 4.1R+ / + THP-1 and 4.1R- / -T HP-1 cells

[0086] (1) Extraction of total cell protein

[0087] Collect THP-1 cells in good logarithmic growth phase, wash twice with 1×PBS, centrifuge at 800rpm for 5min, discard the supernatant; prepare cell lysate according to the ratio of Western lysate: protein inhibitor (PMSF) at 100:1 , add to the collected cells, pipette and mix well, lyse on ice for 30 minutes, shake once every 5 minutes to fully lyse; centrifuge at 12,000 rpm for 10 minutes at 4°C, pipette the supernatant into a 1.5mL centrifuge tube, take a small part of the protein, and use To detect the protein concentration, add 5×SDSLoading Buffer to the rest, cook in boiling water for 5 minutes, store in a -80°C refrigerator for later use.

[0088] (2) Determination of protein concentration

[0089] The protein concentration was determined according to the BCA protein concentration determination kit, and the spe...

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Abstract

The invention discloses a method for constructing an EPB41 gene knockout THP-1 cell line on the basis of a CRISPR-Cas9 system. The method comprises the following steps: taking 'N18 or N20' of a fragment conforming to 5'-G-N18-NGG-3 or 5' -G-N20-NGG-3' or 5' -CCN-N18-C-3' or 5'-CCN-N20-C-3' sequence regular arrangement in a protein coding sequence in a first exon, a fourth exon and a seventh exon of a human EPB41 gene as a target sequence; the THP-1 cell line with the EPB41 gene knocked out is stably formed by transferring the cells to the next generation along with the division and proliferation of the cells. Experiments prove that the THP-1 cell line with the EPB41 gene knocked out cannot correctly express 4.1R protein. Influence of protein 4.1R deletion on cell membrane functions is studied, and direct or indirect interaction between protein 4.1R and some immunoreactions or other signal pathway protein molecules can also be studied.

Description

Technical field: [0001] The invention relates to a THP-1 cell line for constructing a knockout EPB41 gene based on a CRISPR-Cas9 system, belonging to the field of biotechnology. Background technique [0002] THP-1 is a mononuclear cell line from human peripheral blood, originally derived from patients with acute monocytic leukemia. It belongs to suspension cells and is suitable for transfection or infection experiments. Its surface antigen HLA types are: A2, A9, B5, DRw1, DRw2. Protein 4.1R (EPB41 gene) is a membrane-cytoskeleton crosslinker and adapter that binds the cytoplasmic fibrin filament complex and various transmembrane proteins. In addition, protein 4.1R in CD4 + Negative regulation of the initiation phase of T lymphocyte activation and signal transduction. CD8 + Negative regulation of LAT and ERK protein phosphorylation levels in T cells and thus negative regulation of CD8 + T cell activation and proliferation. Protein 4.1R inhibits CD4 + T cell activation ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/12C12N5/10C12R1/91
CPCC12N15/86C07K14/4703C12N5/0645C12N2740/15043C12N2800/107C12N2510/00
Inventor 乔凯旋郑永唐刘鑫刘丰亮范雪刚
Owner ZHENGZHOU UNIV
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