Application of human IGFL3 gene and related product
A technology of genes and uses, applied in medical preparations containing active ingredients, genetic engineering, plant genetic improvement, etc., can solve problems such as the absence of IGFL3 gene, and achieve the effects of inhibiting proliferation, controlling the growth process, and inhibiting the growth of gastric cancer.
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Embodiment 1
[0097] Example 1 Preparation of RNAi lentivirus against human IGFL3 gene
[0098] 1. Screening for effective siRNA targets against the human IGFL3 gene
[0099] Retrieve IGFL3 (NM_207393) gene information from Genbank; design effective siRNA targets for IGFL3 gene. Table 1-1 lists the screened effective siRNA target sequences against the IGFL3 gene.
[0100] Table 1-1 is targeted at the siRNA target sequence of human IGFL3 gene
[0101] SEQ ID NO TargetSeq(5'-3') 1 TGAGGGTTCTGGGTATGAA 2 GTGGGAACAAGATCTACAA
[0102] 2. Preparation of lentiviral vector
[0103] Synthesize double-stranded DNA Oligo sequences (Table 1-2) containing Age I and EcoR I restriction sites at both ends for siRNA targets (taking SEQ ID NO: 1 and 2 as examples); restrict with Age I and EcoR I The endonuclease acted on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to make it linear, and the digested fragment was identified by agarose ge...
Embodiment 2
[0122] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0123] Human gastric cancer AGS 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 value (MOI, AGS: 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 reverse tran...
Embodiment 3
[0130] Example 3 Detection of proliferation ability of tumor cells infected with IGFL3-shRNA lentivirus
[0131] Human gastric cancer AGS 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, AGS: 20), 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 d...
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