Application of human CSPG5 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 the problem of lack of CSPG5 gene
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0106] Example 1 Preparation of RNAi lentivirus for human CSPG5 gene
[0107] 1. Screening for effective siRNA targets against the human CSPG5 gene
[0108] Retrieve CSPG5 (NM_006574) gene information from Genbank; design effective siRNA targets for CSPG5 gene. Table 1-1 lists the screened effective siRNA target sequences against CSPG5 gene.
[0109] Table 1-1 is targeted at the siRNA target sequence of human CSPG5 gene
[0110] SEQ ID NO TargetSeq(5'-3') 1 TCCCAAGTTACTGTCACAA
[0111] 2. Preparation of lentiviral vector
[0112] 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.
[0113] Table 1-2 Double-stranded DNA Oligo with sticky ends containi...
Embodiment 2
[0131] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0132] Human liver cancer SMMC-7721 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, SMMC-7721:20, the following examples are based on this multiplicity of infection), add an appropriate amount of the lentivirus prepared in Example 1, and replace the medium after 24 hours of cultivation, and wait until the infection time reaches 5 Days later, cells were collected. 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 ...
Embodiment 3
[0140] Example 3 Celigo detects the proliferation ability of tumor cells infected with CSPG5-shRNA lentivirus
[0141] Human liver cancer SMMC-7721 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, an appropriate amount of virus was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 3 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 2500 / 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 day wi...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap