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Method for constructing TAP gene-deleted pig T2 cell by using CRISPR/Cas9 system

A tap2-sgRNA-2, tap1-sgRNA-2 technology, applied in urinary tract/kidney cells, genetically modified cells, cells modified by the introduction of foreign genetic material, etc. The establishment of porcine-derived T2 cell lines and epitope screening are time-consuming and labor-intensive, achieving the effect of easy transformation, convenient operation and less workload

Active Publication Date: 2020-11-24
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are the following problems in the in vitro binding experiment for screening porcine virus CTL epitopes: first, the combination of SLA-I and polypeptides in vitro cannot fully guarantee that the polypeptides have biological functions in vivo; Both involve protein expression, purification, renaturation, etc., making epitope screening time-consuming and laborious
It should be pointed out that the research level of pigs is different from that of humans and mice, and no tumor tissue model has been established yet, so it is impossible to establish a pig-derived T2 cell line by screening for the natural deletion of TAP1 and TAP2

Method used

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  • Method for constructing TAP gene-deleted pig T2 cell by using CRISPR/Cas9 system
  • Method for constructing TAP gene-deleted pig T2 cell by using CRISPR/Cas9 system
  • Method for constructing TAP gene-deleted pig T2 cell by using CRISPR/Cas9 system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Recovery and Karyotype Detection of PK15 Cells

[0074] Take out the cryopreservation tube from the liquid nitrogen and immediately place it in a 37°C water bath for shaking to melt the frozen cells rapidly. Transfer the thawed frozen cells into a 15mL centrifuge tube with 9mL of fresh medium, centrifuge at 1500rpm for 3min, discard the supernatant, add preheated fresh DMEM culture medium and resuspend. Add the resuspended PK15 cells to a 25 cm medium containing 4 mL of DMEM complete medium 2 In the cell culture flask, shake gently to mix, in CO 2 cultured in an incubator. After 24 hours of cultivation, it can grow to 25cm 2 Cell culture flasks for subsequent experiments. Cells were typed using spectral karyotyping techniques:

[0075] Karyotype analysis:

[0076] Freshly cultured cells were treated with colchicine to obtain more metaphases, and then treated with hypotonicity and fixation. After the specimens were made, they were stained with Giemsa sta...

Embodiment 2

[0084] Example 2 Construction of TAP2 Knockout PK15 Cell Line Using CRISPR / Cas9 Technology

[0085] 1 Construction of pUC57-T7-sgRNA vector

[0086] (1) Design and synthesis of TAP2 gene target site

[0087] The TAP2 gene information was obtained through the Ensembl genome database in NCBI, and it has been found that there are two transcripts of this gene, both of which encode proteins. Log on to the GenBank website, download the TAP2 gene sequence of pig PK15 cells and obtain the second exon sequence (Gene ID: 733650), and design TAP2 according to the target prediction website CCtop (http: / / crispr.cos.uni-heidelberg.de) 3 pairs of sgRNA sequences of the second exon of the gene. CACC was added to the 5' end of the upstream primers of the two pairs of oligonucleotide sequences, and AAAC was added to the 5' end of the downstream primers. The TAP2-sgRNA design is shown in Table 1.

[0088] Table 1 sgRNA sequence

[0089]

[0090] Send 3 pairs of designed sgRNA sequences to...

Embodiment 3

[0141] Example 3 Construction of TAP1 Knockout PK15 Cell Line Using CRISPR / Cas9 Technology

[0142] 1 PUC57-sgRNA vector construction

[0143] (1) Design and synthesis of TAP1 gene target site

[0144] The porcine TAP1 gene transcript information was obtained through the Ensembl database in NCBI, TAP1ENSSSCG00000025618, and there are two transcripts of the TAP1 gene, namely TAP1-201 and TAP1-202. Using the second exon of the TAP1 gene as the knockout sequence, 4 pairs of oligonucleotide target recognition sequences were designed using the sgRNA prediction website (http: / / crispr.cos.uni-heidelberg.de). Add CACC to the 5' end of the upstream primer of the four pairs of oligonucleotide sequences, and add AAAC to the 5' end of the downstream primer. See Table 2 for the 4 pairs of sgRNA sequences.

[0145] Table 2 sgRNA sequence

[0146]

[0147] Send 4 pairs of designed sgRNA sequences to the company for synthesis, and anneal the synthesized sgRNA sequences to form double s...

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Abstract

The invention relates to a method for constructing a TAP gene-deleted pig T2 cell by using a CRISPR / Cas9 system, and belongs to the technical field of animal genetic engineering and genetic modification. The technical scheme mainly comprises the following steps: firstly, carrying out TAP2 gene knockout, connecting sgRNA for identifying TAP2 to a pUC57-sgRNA vector, transforming competent cells toobtain a pUC57-sgRNA plasmid containing a targeted recognition sequence, and screening positive clones through sequencing; co-transfecting a PK15 cell with a recombinant plasmid pUC57-sgRNA and a Cas9plasmid; and knocking out TAP1 on the TAP2 gene-knocked cell by using the same method. According to the method, a TAP gene-knocked PK15 cell line is constructed by using CRISPR / Cas9, a cell model isestablished for screening multiple pig-derived virus CTL epitopes, and meanwhile, a cell model is established for researching a TAP non-dependent presentation mechanism of exogenous antigens.

Description

technical field [0001] The invention relates to the technical field of animal genetic engineering and genetic modification, in particular to a method for constructing TAP gene-deleted pig T2 cells using a CRISPR / Cas9 system. Background technique [0002] In vivo, almost all cells express major histocompatibility complex (MHC) class I molecules and present antigenic peptides to CD8+ T lymphocytes. These antigenic peptides originate from the degradation of endogenous proteins or antigenic proteins of intracellular pathogens by the proteasome. Peptides produced in the cytosol need to be transported to the endoplasmic reticulum (ER) through the Transporter associated with antigen processing (TAP), and in the molecular chaperones of the peptide transport complex such as calreticulin, tapasin and With the help of ERp57, it assembles with MHC I heavy chain and light chain β2m into a pMHC complex, which is processed by the Golgi apparatus and presented to the cell surface to activa...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N5/10C12N9/22
CPCC12N15/113C12N15/85C12N5/0686C12N9/22C07K14/47C12N2310/20C12N2510/00C12N2800/107
Inventor 高凤山
Owner DALIAN UNIV
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