Application and related pharmaceuticals of human ubiquitin-protein ligase 138 (RNF138) gene
A gene and drug technology, applied in the use of human RNF138 gene and related drug fields, can solve the problems to be further studied and so on
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Embodiment 1
[0071] Example 1: Preparation of RNAi lentivirus for human RNF138 gene
[0072] 1. Screening effective siRNA targets against human RNF138 gene
[0073] Select the coding region sequence of the human RNF138 (NM 016271) gene from Genbank, and obtain a sequence of 21 bases starting from every other base; use the design software of Shanghai Jikai Gene Chemical Technology Co., Ltd., and use the human RNF138 mRNA sequence as a template , to determine 13 effective siRNA target sequences (SEQ ID NO: 1-13), as shown in Table 1:
[0074] Table 1 is targeted at the siRNA target sequence of human RNF138 gene
[0075]
[0076] Aim at the siRNA target (take SEQ ID NO: 8 as an example) to synthesize a double-stranded DNA Oligo sequence (see Table 2) with Age I and EcoR I restriction sites at both ends; The enzyme acts on the pGCSIL-GFP carrier (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to make it linearized (the reaction system is shown in Table 4, 37° ...
Embodiment 2
[0098] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of RNF138 gene
[0099] Human pancreatic cancer Panc-1 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 value: 10), an appropriate amount of the lentivirus 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 operation manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see Table 7 for the reverse transcription reaction system), reacted at 42°C for 1h, and then bathed in a water bath at 70°C for 10min to inactivate the reverse tr...
Embodiment 3
[0107] Example 3: Detection of proliferation ability of tumor cells infected with siRNF138-Lentivims lentivirus
[0108] Human pancreatic cancer Panc-1 cells in the logarithmic growth phase were digested with trypsin 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 value is 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 ...
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