Application of human DEPDC1 gene, and related product
A DEPDC1 gene technology, applied in medical preparations containing active ingredients, genetic engineering, plant genetic improvement, etc., can solve problems such as lack of DEPDC1 gene
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Embodiment 1
[0102] Example 1 Preparation of RNAi lentivirus against human DEPDC1 gene
[0103] 1. Screening for effective siRNA targets against the human DEPDC1 gene
[0104] Retrieve DEPDC1 (NM_017779) gene information from Genbank; design effective siRNA targets for DEPDC1 gene. Table 1-1 lists the screened effective siRNA target sequences against DEPDC1 gene.
[0105] Table 1-1 siRNA target sequence targeting human DEPDC1 gene
[0106] SEQ ID NO TargetSeq(5'-3') 1 tatccagtaaggctatcat
[0107] 2. Preparation of lentiviral vector
[0108] 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.
[0109] Table 1-2 Double-stranded DNA Oligo with sticky ends containing...
Embodiment 2
[0128] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0129] Human tongue squamous cell carcinoma CAL27 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, CAL27:10), 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 minutes to inactivate the...
Embodiment 3
[0136] Embodiment 3Western Blotting method detects the silencing efficiency of gene
[0137] 1. Extraction of total cell protein
[0138] 1) Infect target cells with control virus and DEPDC1-siRNA lentivirus targeting DEPDC1 interference target according to the multiplicity of infection (MOI, CAL27:10).
[0139] 2) After 5 days of infection, collect the cell samples, take an appropriate amount of RIPA lysate (Beiyuntian, P0013C), and add PMSF within a few minutes before use, so that the final concentration of PMSF is 1mM.
[0140] 3) Add an appropriate amount of RIPA lysate, and lyse on ice for 10-15 minutes. Scrape the cells and transfer them into a new EP tube, and then ultrasonically disrupt the cells (20 times at 40W, 1 s each time, 2 s apart).
[0141] 4) Centrifuge at 12000g for 15min at 4°C, take the supernatant and use BCA Protein Assay Kit (manufacturer: Biyuntian, article number: P0010S) to measure the protein concentration.
[0142] 5) Add new lysate to adjust th...
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