Construction method and application of ARHGAP11B gene deleted iPSC cell line
A construction method and cell line technology, applied in microorganism-based methods, genetically modified cells, artificially induced pluripotent cells, etc., can solve the problems of constructing and knocking out induced pluripotent stem cells ARHGAP11B gene, etc., and achieve gene improvement. Editing Efficiency, Efficiency Improvement, and Complete Knockout Effect
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[0047] Embodiment 1. A construction method for knocking out ARHGAP11B gene in iPSC cell line based on CRISPR / Cas9 system
[0048] 1. sgRNA design
[0049] "sgRNA", namely Single guide RNA, single guide RNA, can recognize specific genome segment during CRISPR / Cas9 gene editing (including gene knockout). According to the principle of sgRNA target sequence design, the target sequence was designed in exon 6 of the human ARHGAP11B gene (the nucleotide sequence is shown in SEQ ID No. 1), and three sgRNAs were constructed, namely sgRNA1, sgRNA2 and sgRNA3. The nucleotide sequence As shown in SEQ ID No. 2~4 respectively (see Table 1), the sequence diagrams of the three sgRNAs are as follows figure 1 shown.
[0050] Table 1
[0051] sgRNA number Nucleotide sequence sgRNA1 gcgtgtaccagactttatcc sgRNA2 ggaaaagataccagccatgt sgRNA3 gactttatcctggaaaagat
[0052] 2. Construction of CRISPR / Cas9 vector
[0053] The three sgRNAs were annealed to form dim...
Example Embodiment
[0142] Example 2. Pluripotency identification and analysis of iPSC cell lines with deletion of ARHGAP11B gene
[0143] (1) Cellular immunofluorescence
[0144] Cells (iPSC63, wild-type WT iPSCs) were passaged into three wells of a four-well plate, and immunofluorescence experiments were started after the cell density reached 70%-80%.
[0145] 1) Aspirate the medium in the four-well plate and wash it with PBS for 3 times, 10min each time;
[0146] 2) Fix the cells with 4% paraformaldehyde for 20 min, and wash with PBS for 3 times, 10 min each time;
[0147] 3) Add 0.5% Triition X-100 (prepared with PBS), permeabilize at room temperature for 20 minutes, and wash with PBS for 3 times, 10 minutes each time;
[0148] 4) Blot dry PBS, add normal goat serum dropwise, block at room temperature for 30min;
[0149] 5) Dry the blocking solution, add 200 μl of diluted primary antibody (Oct4, Sox2, Nanog respectively) to each well, and incubate at 4°C overnight;
[0150] 6) Aspirate th...
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