Application of human CCDC154 gene and related product
A technology of genes and uses, applied in medical preparations containing active ingredients, gene therapy, genetic engineering, etc.
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Embodiment 1
[0101] Example 1 Preparation of RNAi Lentivirus for Human CCDC154 Gene
[0102] 1. Screening of effective siRNA targets for human CCDC154 gene
[0103] Get CCDC154 (NM_001143980) gene information from Genbank; design effective siRNA targets for CCDC154 gene. Table 1-1 lists the screened effective siRNA target sequences for CCDC154 gene.
[0104] Table 1-1 siRNA target sequence targeting human CCDC154 gene
[0105] SEQ ID NO TargetSeq(5’-3’) 1 ACAAGTGCCTGCTTCATAA
[0106] 2. Preparation of lentiviral vector
[0107] For siRNA target (take SEQ ID NO:1 as an example), synthesize double-stranded DNA Oligo sequence (Table 1-2) with Age I and EcoR I restriction sites on both ends (Table 1-2); within the restriction of Age I and EcoR I Dicer acts on pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis to identify the digested fragments.
[0108] Table 1-2 Double-stranded DNA Oligo with sticky ends containing Age...
Embodiment 2
[0127] Example 2 Real-time fluorescence quantitative RT-PCR method to detect gene silencing efficiency
[0128] Human liver cancer BEL-7404 cells and SMMC-7721 cells in the logarithmic growth phase were trypsinized to prepare a cell suspension (the number of cells is about 5×10 4 / ml) inoculate in a 6-well plate and culture until the cell confluence reaches about 30%. According to the multiplicity of infection (MOI, BEL-7404: 10, SMMC-7721: 10), add an appropriate amount of the lentivirus prepared in Example 1, culture for 24 hours and then change the medium. After the infection time reaches 5 days, collect the cells . According to Invitrogen's Trizol operating instructions, total RNA was extracted. According to the M-MLV operating instructions of Promega, reverse transcription of RNA to obtain cDNA (see Table 2-1 for the reverse transcription reaction system, react at 42°C for 1 hour, and then inactivate the reverse transcriptase in a water bath at 70°C for 10 minutes).
[0129]...
Embodiment 3
[0135] Example 3 Detection of the proliferation ability of tumor cells infected with CCDC154-siRNA lentivirus
[0136] Human liver cancer BEL-7404 cells and SMMC-7721 cells in the logarithmic growth phase were trypsinized to prepare a cell suspension (the number of cells is about 5×10 4 / ml) inoculate in a 6-well plate and culture until the cell confluence reaches about 30%. According to the multiplicity of infection (MOI, BEL-7404: 10, SMMC-7721: 10), add an appropriate amount of virus, culture for 24 hours and then change the medium. After the infection time reaches 5 days, collect each experiment in the logarithmic growth phase Group of cells. Resuspend in complete medium into a cell suspension (2×10 4 / ml), inoculate a 96-well plate with a cell density of approximately 3000 cells / well. Each group has 5 duplicate wells, each well is 100μl. After laying the board, place it at 37℃, 5% CO 2 Culture in an incubator. Beginning on the second day after plating, use Celigo instrume...
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