Human cell with low immunogenicity and preparation method thereof
An immunogenic, human pluripotent stem cell technology, applied in the direction of genetically modified cells, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problem of prone to immune rejection, human adult cells or human The clinical application of stem cells is limited and other issues
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0210] Example 1. Design of TALEN target sequences
[0211] 1. Download the human β2-microglobulin genome sequence (Gene ID: 567) and TAP1 genome sequence (Gene ID: 100507463) from NCBI.
[0212] 2. Design primers, PCR amplify the target site fragments on the genome and sequence them. The PCR primers and sequencing primers are shown in Table 1-1 and Table 1-2:
[0213] Table 1-1
[0214]
[0215] Table 1-2
[0216]
[0217] 3. Design TALEN recognition sequence (target sequence):
[0218] According to the sequence obtained by sequencing, the TALEN recognition sequence was determined according to the following principles:
[0219] (1) The 0th base is T (the base before the first in the recognition sequence is the 0th);
[0220] (2) The last base is T;
[0221] (3) The length of the recognition sequence is between 13-19;
[0222] (4) The length of the spacer sequence (Spacer) between the two recognition sequences is controlled between 13-21 (12 is also possible, but...
Embodiment 2
[0229] Example 2. Verification of targeting efficiency by transfecting 293T cells with TALENs plasmids
[0230] 1. Aspirate the culture medium in the T25 bottle of 293T cells, wash it once with PBS, add 1ml of 0.25% trypsin, shake back and forth to make it evenly cover the bottom of the bottle, and place it at 37°C, 5% CO 2 5min in the incubator.
[0231] 2. After the digestion is completed, use 2ml of DMEM medium containing 10% FBS to stop the reaction, pipette the cells into single cells and transfer them to a centrifuge tube for counting.
[0232] 3. Place 300,000 cells in one well of a 24-well plate, and add 0.5ml of fresh medium to each well.
[0233] 4. After 24h, the transfection was carried out.
[0234] 5. Transfect the cells with the constructed B2M TALEN plasmids according to the paired combinations shown in Table 3, a total of 16 combinations.
[0235] table 3
[0236] L86&R102
L87&R105
L165&R181
L244&R260
L87&R102
L88&R105
...
Embodiment 3
[0252] Example 3. Human pluripotent stem cells transfected with TANLENs plasmid
[0253] 1. Add 100 μl Matrigel to each well of a 6-well plate, shake it back and forth to make it cover the bottom of the entire well, and place it at 37°C, 5% CO 2 30min in the incubator.
[0254] 2. Aspirate the medium in the T25 bottle for culturing human pluripotent stem cells, wash it once with PBS, add 4ml type IV collagenase, shake back and forth to make it evenly cover the bottom of the bottle, and place it at 37°C, 5% CO 2 30min in the incubator.
[0255] 3. After the digestion is completed, use human pluripotent stem cell culture medium to terminate the reaction, pipette into small pieces, transfer to a centrifuge tube, and centrifuge at 1200rpm for 5min.
[0256] 4. After removing the supernatant, use the medium to resuspend the cells, take 500,000 cells and place them in a 6-well plate that has been covered with Matrigel, and add 2ml of fresh medium.
[0257] Transfection was carr...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More 
PatSnap Eureka turns technology decisions into work you can execute. Powered by our Innovation Knowledge Graph, it runs expert workflows across engineering, life sciences, materials and intellectual property. Get your review-ready output in minutes.
