Application of human SHCBP1 gene and related product
A technology of gene and use, applied in the field of use of human SHCBP1 gene and related products, can solve problems such as functions that have not been reported yet
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Embodiment 1
[0126] Example 1 Preparation of RNAi lentivirus against human SHCBP1 gene
[0127] 1. Screening for effective siRNA targets against the human SHCBP1 gene
[0128] Retrieve SHCBP1 (NM_024745) gene information from Genbank; design effective siRNA targets for SHCBP1 gene. Table 1-1 lists the screened effective siRNA target sequences against the SHCBP1 gene.
[0129] Table 1-1 is targeted at the siRNA target sequence of human SHCBP1 gene
[0130] SEQ ID NO TargetSeq(5'-3') 1 TGGTGAAACCTACAATCTT
[0131] 2. Preparation of lentiviral vector
[0132] Aim at the siRNA target (take SEQ ID NO: 1 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 1-2) with Age I and EcoR I restriction sites at both ends; Dicer acts on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis identifies the digested fragment.
[0133] Table 1-2 Double-stranded DNA Oligo with sticky...
Embodiment 2
[0152] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0153] Human thyroid cancer TPC-1 cells and K1 cells in the logarithmic growth phase were trypsinized to make cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, TPC1:20; MOI, K1:20), an appropriate amount of the lentivirus prepared in Example 1 was added, the culture medium was replaced after 24 hours of culture, and the cells were collected after the infection time reached 5 days. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see Table 2-1 for the reverse transcription reaction system, react at 42°C for 1 hour, and then bathe in a water bath at 70°C for 10 minutes to inactivate the rev...
Embodiment 3
[0160] Example 3 Detection of proliferation ability of tumor cells infected with SHCBP1-siRNA lentivirus
[0161] Human thyroid cancer TPC-1 cells and K1 cells in the logarithmic growth phase were trypsinized to make cell suspension (the number of cells was about 2×10 5 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, TPC-1:20; MOI, K1:20), add an appropriate amount of virus, and replace the medium after 16 hours of culture. After the infection time reaches 3 days, collect each experiment in the logarithmic growth phase. group of cells. The complete medium was resuspended into a cell suspension (1.5×10 4 / ml), inoculate a 96-well plate at a cell density of about 1500 cells / well. Three replicate wells per group, 100 μl per well. After laying the board, place at 37°C, 5% CO 2 Incubator cultivation. From the second day after plating, the Tecan infinite microplate instrument was ...
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