Cell line with gene YTHDF1 knocked out and construction method thereof
A cell and gene technology, applied in the field of knockout gene YTHDF1 cell line and its construction, can solve the problem of unclear function and achieve the effect of avoiding cell death
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Embodiment 1
[0035] Example 1 Determination of YTHDF1 Gene Knockout Target Site and gRNA
[0036] The 240bp-266bp sequence corresponding to the first exon of the YTHDF1 gene (Gene ID: 54915) coding region was selected as the knockout target site, and multiple gRNAs were designed. The specific sequences are as follows:
[0037] gRNA-1: TACCTGGGTGTCCACGCTGGTGG (SEQ ID NO: 1);
[0038] gRNA-2: CGCTACCTGGGTGTCCACGCTGG;
[0039] gRNA-3: GTAACCCGGCCCCGCTACCTGGG;
[0040] gRNA-4: GGCCACCAGCGTGGACACCCAGG.
Embodiment 2
[0041] Example 2 Construction of gRNA expression vector
[0042] 1. Experimental method
[0043] (1) According to the gRNA designed in Example 1, design the DNA oligos of the synthetic gRNA, specifically cited
[0044] The object sequence is as follows:
[0045] gRNA1-FP: CACCGTACCTGGGTGTCCACGCTGG (SEQ ID NO: 2);
[0046] gRNA1-RP: AAACCCAGCGTGGACACCCAGGTA (SEQ ID NO: 3);
[0047] gRNA2-FP: CACCGCGCTACCTGGGTGTCCACGC;
[0048] gRNA2-RP: AAACGCGTGGACACCCAGGTAGCG;
[0049] gRNA3-FP: CACCGGTAACCCGGCCCCGCTACCT;
[0050] gRNA3-RP: AAACAGGTAGCGGGGCGGGTTAC;
[0051]gRNA4-FP: CACCGGGCCACCAGCGTGGACACCC;
[0052] gRNA4-RP: AAACGGGTGTCCACGCTGGTGGCC.
[0053] (2) The DNA oligos of the 8 gRNAs synthesized above were used ddH 2 O was dissolved and the concentration was adjusted to 100 μM.
[0054] The annealing reaction system is: gRNA1-FP or gRNA2-FP or gRNA3-FP or gRNA4-FP, 4 μL; gRNA1-RP or gRNA2-RP or gRNA3-RP or gRNA4-RP, 4 μL; 10×NEB Buffer, 1 μL; ddH 2 O, 1 μL.
[0055] Th...
Embodiment 3
[0064] Example 3 Construction of YTHDF1 Gene Knockout Cell Line
[0065] 1. Experimental method
[0066] (1) Construction of HeLa-GFP-LC3B cells that can inducibly express Cas9
[0067] build as figure 2 For the Phage-tet-N-3×flag-SpCas9-DEST-UBC-neo1 vector shown, the specific method is: clone the Cas9 sequence on the PX330 vector into the pENTR-D-TOPO vector by enzyme digestion and ligation, and then pass LR reaction The Cas9 sequence was cloned into the pHAGE-Tet-N-3×FLAG-DEST-UBC-neo vector (the nucleotide sequence is shown in SEQ ID NO: 4). That is, the vector expressing Cas9 was obtained, which was named Phage-tet-N-3xflag-SpCas9-DEST-UBC-neo1 (the nucleotide sequence is shown in SEQ ID NO: 5).
[0068] HEK293T cells were seeded into 6-well cell culture plates, and when the cell density reached 70% the next day, the transient transfection experiment could be carried out.
[0069] The specific method of the transient transfection experiment is as follows: Add 1.2 μg ...
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