Application and related products of human EME1 gene
A gene and application technology, applied in the application of human EME1 gene and related products, can solve the problem of no report of EME1 function
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Embodiment 1
[0119] Example 1 Preparation of RNAi lentivirus against human EME1 gene
[0120] 1. Screening for effective siRNA targets against the human EME1 gene
[0121] Retrieve EME1 (NM_152463) gene information from Genbank; design effective siRNA targets for EME1 gene. Table 1-1 lists the screened effective siRNA target sequences against the EME1 gene.
[0122] Table 1-1 is targeted at the siRNA target sequence of human EME1 gene
[0123] SEQ ID NO TargetSeq(5'-3') 1 GATAAAGAACGCCAGAATT 2 CTGAGAAGACAGGAAAGAA
[0124] 2. Preparation of lentiviral vector
[0125] For siRNA targets (take SEQ ID NO: 1 and 2 as examples), synthesize double-stranded DNA Oligo sequences (Table 1-2) containing Age I and EcoR I enzyme cleavage sites at both ends; Restriction endonuclease 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 fragments.
[0126] Table ...
Embodiment 2
[0144] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0145] Human nasopharyngeal carcinoma CNE-2Z cells in logarithmic growth phase were digested with trypsin 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 value (MOI=10, the following examples are all 10), 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 mi...
Embodiment 3
[0164] Example 3 Celigo assay to detect the proliferation ability of tumor cells infected with EME1-shRNA lentivirus
[0165] Human nasopharyngeal carcinoma CNE-2Z cells in logarithmic growth phase were digested with trypsin 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, 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...
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