A kind of sgRNA targeting sequence specifically targeting human abcg2 gene and its application
A kind of specific and genetic technology, applied in the field of sgRNA guide sequence specifically targeting human ABCG2 gene
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Embodiment 1
[0037] (1) sgRNA design
[0038] According to the genome sequence of human ABCG2 gene (gene ID: 9429), a sgRNA targeting human ABCG2 gene was designed. The 20nt oligonucleotide sgRNA guide sequence is: Sg1: 5'-GCTGCAAGGAAAGATCCAAG-3' (located in the third exon of gene ABCG2) ( figure 1 ); add CACCG to its 5' end to obtain a forward oligonucleotide (Forward oligo) (see Sg1-F in Table 1); obtain its corresponding DNA complementary strand according to the guide sequence, and add Add C to AAAC and its 3' end to obtain a reverse oligonucleotide (Reverse oligo) (see Sg1-R in Table 1). Synthesize the above-mentioned forward oligonucleotides and reverse oligonucleotides respectively, denature the Forward oligo and Reverse oligo of the synthesized sgRNA oligonucleotides in pairs, and anneal; At the same time, the designed gRNA target sequence was blasted to exclude non-specific target cutting sites. The specific oligonucleotide sequence is shown in Table 1.
[0039] Oligonucleotide ...
Embodiment 2
[0046] (1) Co-transfection of core plasmid and packaging plasmid pMD2.G and psPAX2 into 293T cells
[0047] Cultivate 293T cells until the confluence rate of 293T cells reaches 50% to 60%, 12 to 18 hours after seeding is the best time for transfection; replace fresh culture medium before transfection, add 3mL medium to a 60mm dish; The amount used is 4 μg of core plasmid (lentiCRISPRv2-hABCG2-Sg1 prepared in Example 1, and lentiCRISPRv2 empty vector as a control), 3 μg of psPAX2, 1 μg of pMD2.G, 24 μL of PEI, supplemented with DMEM to a total volume of 200 μL, and the order of addition DMEM, PEI, and plasmid DNA (core plasmid, pMD2.G, and psPAX2) respectively; then stand at room temperature for 30 minutes to fully polymerize PEI and plasmid DNA, and add the transfection system dropwise to the above-mentioned small dish with 293T cells after polymerization , shake gently and place in 37°C, 5% CO 2 Continue culturing in the incubator;
[0048] (2) Virus harvest and concentrati...
Embodiment 3
[0061] Example 3 In vitro cell experiments to identify the knockout effect of the human gene ABCG2 on the cells after screening
[0062] (1) western blot
[0063] The ABCG2 protein expression level of the cell line S1M1-80sg1, which successfully knocked out the ABCG2 gene in Example 2, was identified by western blot experiments. The cell line S1M1-80 successfully transfected with the empty vector lentiCRISPRv2 was used as a blank control, S1 was used as a positive control, and S1M1-80 was used as a positive control. 80 as a negative control; among them, the primary antibody is Anti-ABCG2 (MM SC-58222, purchased from santacruze), the secondary antibody is Anti-mouse IgG, HRP-linked Antibody (product number 7076, cell signaling), and the specific method is conventional western blot operation process.
[0064] The result is as Figure 4 As shown, in S1M1-80 cells, there is a significant difference in the expression of ABCG2 between the cells transformed into sg1 and the cells o...
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