New application of human MED19 genes in tumor therapy
A gene, tumor technology, applied in the field of new use of human MED19 gene in tumor therapy
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Embodiment 1
[0044] Example 1: Preparation of human MED19 gene efficient interference lentivirus
[0045] The gene information of MED19 (NM_153450) was retrieved from genbank; an effective siRNA target for MED19 was designed by using the design software genechem accumulated and summarized by Jikai Gene Chemical Technology Co., Ltd. for many years. The siRNA target sequence is: GGTGAAGGAGAAGCTAAGT.
[0046] Synthesize double-stranded DNA oligo sequences with sticky ends containing Hpa I and Xho I restriction sites at both ends for the siRNA target, as shown in the table below;
[0047] The double-stranded DNA oligo with sticky ends containing Hpa I and Xho I restriction sites at both ends is:
[0048] No.
5’
STEM
Loop
STEM
3’
1-F
T
GGTGAAGGAGAAGCTAAGT
TTCAAGAGA
ACTTAGCTTCTCCTTCACC
TTTTTTC
1-R
TCGAGAAAAAAA
GGTGAAGGAGAAGCTAAGT
TCTCTTGAA
ACTTAGCTTCTCCTTCACC
A
[0049] Construction of lentivir...
Embodiment 2
[0053] Example 2: The method of immunohistochemistry detects the expression difference of MED19 in normal hyperplastic tissue and cancerous tissue (taking prostate tissue as an example)
[0054] There were 30 patients with prostate tumors and 10 patients with benign prostatic hyperplasia. Paraffin sections of surgically resected prostate tissue were dewaxed and rehydrated with xylene and graded ethanol, boiled in citrate buffer for antigen retrieval, blocked with 10% BSA at 37°C for 1 hr, added primary antibody to MED19, the working concentration of the primary antibody was 1 μg / ml , and incubated overnight at 4°C with the primary antibody. The corresponding secondary antibody was added for staining, the secondary antibody was HRP-labeled antibody against the species of the primary antibody, and the working concentration was diluted 1:100. 37 degrees Celsius, one hour. After washing with PBS, develop color with DAB solution, room temperature for 3-5 minutes, rinse with tap w...
Embodiment 3
[0057] Example 3: MTT method to detect the proliferation ability of tumor cells infected with GCG-973-siRNA
[0058] pko (colorectal cancer cell line) cells were trypsinized and seeded in a 12-well plate at a cell density of 10-15%. The next day, replace with fresh medium containing 5ug / ml polybrene. Add GCG-973-siRNA lentivirus to the culture plate according to MOI 10, and replace with fresh medium after 12-24 hours of infection. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.
[0059] Trypsinize the virus-infected cells in the logarithmic growth phase, resuspend the complete medium into a cell suspension; inoculate in a 96-well plate, 100 μl per well; culture in a 5% CO2 incubator at 37°C; before the termination of the culture Add 10 μl of 5 mg / ml MTT at 4 hours; add 100 μl DMSO to stop the reaction after discarding the culture medium; detect the OD value with a microplate reader at 570 n...
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