Use of human ifitm3 gene and related medicines
A technology of use and gene expression, applied in the use of human IFITM3 gene and its related pharmaceutical fields, and can solve the problem that the expression situation has not been reported.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0077] Example 1 Preparation of RNAi lentivirus against human IFITM3 gene
[0078] 1. Screening for effective siRNA targets against the human IFITM3 gene
[0079] Get IFITM3 (NM021034.2) gene information from Genbank; design effective siRNA targets for IFITM3 gene by using the design software Genechem of Shanghai Jikai Gene Chemical Technology Co., Ltd. In the coding sequence (CDS) region of the IFITM3 gene, a sequence of 21 bases was obtained starting from every other base, and Table 1 lists 12 effective siRNA target sequences for the IFITM3 gene.
[0080] Table 1 siRNA target sequence targeting human IFITM3 gene
[0081] SEQ ID NO
TargetSeq
1
CCTGTTCAACACCCCTCTTCAT
284
2
GCTTCATAGCATTCGCCTACT
322
3
CCTGAACATCTGGGCCCTGAT
416
4
CCTCATGACCATTCTGCTCAT
446
5
CAGTGCTGATCTTCCAGGCCT
475
6
GTGCTGATCTTCCAGGCCTAT
477
7
GATCTTCCAGGCCTATGGATA
482
8
ATCTTCCAGGC...
Embodiment 2
[0105] Example 2 Real-time fluorescent quantitative RT-PCR method to detect the silencing efficiency of IFITM3 gene
[0106] Human breast cancer MCF-7 cells in the logarithmic growth phase were trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml) were seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, MCF-7:20) value, an appropriate amount of the virus 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 Promega's M-MLV operating instructions, RNA was reverse-transcribed to obtain cDNA (see Table 6 for the reverse transcription reaction system, react at 42°C for 1h, and then bathe in a water bath at 70°C for 10min to inactivate reverse transcriptase).
[0107] Real-t...
Embodiment 3
[0113] Example 3 Detection of proliferation ability of tumor cells infected with IFITM3-siRNA lentivirus
[0114] Human breast cancer MCF-7 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 seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, MCF-7:20), 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 in 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 cells / 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 ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More