Application of human DGKZ gene and related drugs thereof
A gene and drug technology, applied in the field of the use of human DGKZ gene and related drugs, can solve the problem of few reports on DGKZ
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Embodiment 1
[0082] Example 1: Preparation of RNAi lentivirus against human DGKZ gene
[0083] 1. Screening for effective siRNA targets against the human DGKZ gene
[0084] Retrieve DGKZ (NM_003646.3) gene information from Genbank; design effective siRNA targets for DGKZ gene. Table 1 lists 13 effective siRNA target sequences for the DGKZ gene.
[0085] Table 1 is targeted at the siRNA target sequence of human DGKZ gene
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[0088] 2. Preparation of lentiviral vector
[0089] Aim at the siRNA target (taking SEQ ID NO: 1 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 2) with Age I and EcoR I restriction endonucleases at both ends; use Age I and EcoR I restriction enzyme Act on the pGCSIL-GFP carrier (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to linearize it, and identify the digested fragments by agarose gel electrophoresis.
[0090]Table 2 Double-stranded DNA Oligo with sticky ends containing Age I and Eco...
Embodiment 2
[0110] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of DGKZ gene
[0111] The human glioma U87 cells in the logarithmic growth phase were trypsinized to make a 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, U87:10) value, an appropriate amount of the virus prepared in Example 1 was added, the culture medium was replaced after 24 hours of cultivation, 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 7 for the reverse transcription reaction system, react at 42° C. for 1 h, and then bathe in a water bath at 70° C. for 10 min to inactivate reverse transcriptase)....
Embodiment 3
[0119] Example 3: Detection of proliferation ability of tumor cells infected with DGKZ-siRNA lentivirus
[0120] The human glioma U87 cells in the logarithmic growth phase were trypsinized to make a 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, U87:10), an appropriate amount of virus was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 5 days, the cells of each experimental group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2×10 4 / ml), inoculate a 96-well plate at a cell density of about 2000 / well. 5 replicate wells in each 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 plate was detected and read once a day with a Cello...
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