Application of human DNAJC24 gene and related products
A gene and application technology, applied in the field of application of human DNAJC24 gene and related products, can solve problems such as the absence of DNAJC24 gene
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Embodiment 1
[0114] Example 1 Preparation of RNAi lentivirus against human DNAJC24 gene
[0115] 1. Screening for effective siRNA targets against human DNA JC24 gene
[0116] Retrieve DNAJC24 (NM_181706) gene information from Genbank; design effective siRNA targets for DNAJC24 gene. Table 1-1 lists the screened effective siRNA target sequences against the DNAJC24 gene.
[0117] Table 1-1 is targeted at the siRNA target sequence of human DNAJC24 gene
[0118] SEQ ID NO TargetSeq(5'-3') 1 AAGTACAGATGTACCAGCA
[0119] 2. Preparation of lentiviral vector
[0120] 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.
[0121] Table 1-2 Double-stranded DNA Oligo with sti...
Embodiment 2
[0139] Example 2 Detection of Silencing Efficiency of Tumor Cells Infected with DNAJC24-siRNA Lentivirus
[0140] The human lung cancer A549 and NCI-H1299 cells in the logarithmic growth phase were trypsinized 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, A549:10, NCI-H1299:10), an appropriate amount of the lentivirus prepared in Example 1 was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 5 days, the cells were collected.
[0141] a) Detection of gene silencing efficiency by real-time fluorescent quantitative RT-PCR
[0142] 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 r...
Embodiment 3
[0177] Example 3 Detection of proliferation ability of tumor cells infected with DNAJC24-siRNA lentivirus
[0178] a) Celigo experiment
[0179]The human lung cancer A549 and NCI-H1299 cells in the logarithmic growth phase were trypsinized 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, A549: 10, NCI-H1299: 10), add an appropriate amount of virus, replace the medium after 24 hours of culture, and collect cells in each experimental group in the logarithmic growth phase after the infection time reaches 5 days . The complete medium was resuspended into a cell suspension (2×10 4 / ml), at a cell density of about 1500 / well (A549), 1200 / well (NCI-H1299), inoculate a 96-well plate. 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 d...
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