TOX gene and application of sgRNA thereof
A gene and cell technology, applied in the field of application in the preparation of drugs for recovering T cell exhaustion
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Embodiment 1
[0042] Example 1 Screening TOX gene
[0043] After the project passed the review of the ethics committee, the applicant systematically studied the transcriptome expression profile of infiltrating CD8+ T cells in human liver cancer tissues, discovered new genes that determine the exhaustion of CD8+ T cells, and analyzed liver cancer tissues by flow cytometry. Infiltrating CD8+ T cells were sorted according to the expression level of inhibitory surface receptors (PD-1, TIM3) on the cell surface: 3 cases of non-exhausted CD8+ T cells (PD1-TIM3-), exhausted CD8+ T cells (PD1 +TIM3+) 3 cases, a total of 6 samples (see Table 1 for specific information from 3 patients):
[0044] Table 1 Sample information 1
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[0046] Table 2 Sample information 2
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[0049] Then through high-throughput RNA sequencing, a group of differentially expressed genes were found in CD8+ T cells with different degrees of depletion, and the invasiveness of liver cancer tissues ...
Embodiment 2
[0068] Example 2 Design, synthesis, connection, transformation, and electrotransfection of sgRNA
[0069] 1) Design of TOX-sgRNA
[0070] According to the gene sequence of TOX on NCBI Gene (its sequence is shown in SEQ ID NO.1), combined with the principle of CRISPR / Cas9 gene editing, according to the PAM sequence (NGG) positioning, according to its exon 3 (its sequence is shown in SEQ ID NO.2), select 5 sites located on TOX exon3, and design sgRNA sequences 1-5, the nucleotide sequences of which are shown in sequence as SEQ ID NO.3-SEQ ID NO.7.
[0071] 2) Synthesis of TOX-sgRNA
[0072] Combining pGL3-U6-sgRNA-PGK-puromycin carrier plasmid (the carrier structure is as follows figure 1 Shown) (Carleton, J.B., et al. (2017). "Multiplex Enhancer Interference RevealsCollaborative Control of Gene Regulation by Estrogen Receptor alpha-BoundEnhancers." Cell Syst 5(4):333-344e335.) and the restriction endonuclease BsaI (NEB, R3535S), on the basis of the sgRNA1-5 designed in ste...
Embodiment 3
[0128] Example 3 Identification of the effect of target-specific knockout of TOX in CD8+ T cells
[0129] 1) RT-PCR method to detect the expression level of TOX mRNA in CD8+ T cells
[0130] a) Total RNA extraction
[0131] The TOX-knockout exhausted CD8+ T cells in Example 2 were lysed by TRIzol (Invitrogen), and then total RNA was extracted using chloroform, isopropanol and 75% ethanol.
[0132] b) cDNA preparation Prepare cDNA by reverse transcription reagent PrimeScript RT Master Mix (Takara). Reaction conditions: 37°C for 15min, 85°C for 5sec, 4°C∞
[0133] The reaction system is as follows:
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[0135] c) qPCR detection of TOX mRNA expression in exhausted and non-exhausted CD8+ T cells
[0136] According to TOX mRNA sequence situation design specific primer, sequence (SEQ ID NO.21 and SEQ ID NO.22)
[0137] Fluorescent quantitative PCR program: pre-denaturation at 95°C for 30s, followed by 40 cycles: 95°C for 5s, 60°C for 32s.
[0138] The reaction system i...
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