Application of human UAP1L1 gene and related product
A gene and application technology, applied in the application of human UAP1L1 gene and related products, can solve the problem of lack of UAP1L1 gene
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Embodiment 1
[0107] Example 1 Preparation of RNAi lentivirus against human UAP1L1 gene
[0108] 1. Screening for effective siRNA targets against the human UAP1L1 gene
[0109] Retrieve UAP1L1 (NM_207309) gene information from Genbank; design effective siRNA targets for UAP1L1 gene. Table 1-1 lists the screened effective siRNA target sequences against the UAP1L1 gene.
[0110] Table 1-1 is targeted at the siRNA target sequence of human UAP1L1 gene
[0111] SEQ ID NO TargetSeq(5'-3') 1 CCTTCTTACTGCAAACCAT
[0112] 2. Preparation of lentiviral vector
[0113] 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.
[0114] Table 1-2 Double-stranded DNA Oligo with sticky...
Embodiment 2
[0133] Example 2 Detection of gene silencing efficiency of tumor cells infected with UAP1L1-siRNA lentivirus
[0134] Human gastric cancer AGS and MGC80-3 cells in 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, AGS:10, MGC80-3:20), an appropriate amount of the lentivirus prepared in Example 1 was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 5 days, the cells were collected.
[0135] a) Real-time fluorescent quantitative RT-PCR method
[0136] 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, an...
Embodiment 3
[0170] Example 3 Detection of proliferation ability of tumor cells infected with UAP1L1-siRNA lentivirus
[0171] Human gastric cancer AGS and MGC80-3 cells in 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, AGS:10, MGC80-3:20), add an appropriate amount of virus, and replace the medium after 24 hours of culture. After the infection time reaches 5 days, collect the cells of each experimental group in the logarithmic growth phase . The complete medium was resuspended into a cell suspension (2×10 4 / ml), inoculate a 96-well plate at a cell density of about 15,000 / well. 5 replicate wells in each group, 100 μl per well. After laying the board, place at 37°C, 5% CO 2Incubator cultivation. From the second day after plating, the plate was detected and read once a day with a Celig...
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