Using gene editing system to screen superh cell parent line of low immune cell line, its construction method and application
An immune cell and gene editing technology, applied to other methods of inserting foreign genetic materials, embryonic cells, animal cells, etc., can solve problems such as difficult to solve complex and diverse diseases
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment approach
[0029] According to a typical embodiment of the present invention, there is provided a SuperH cell parent line for screening low immune cell lines using a gene editing system. The SuperH cell parent line is formed by transferring the HESPer gene element from the H9 cell line, and the HESPer gene element includes the first promoter, HLA-E gene, Sr39tk gene, first terminator, loxP sequence, second promoter, Genes and second terminators are screened.
[0030] Preferably, wherein the first promoter is different from the second promoter, the first terminator is different from the second terminator, and the HESPer gene element also includes an operably linked enhancer, more preferably, the enhancer is a CMV enhancer, the first The promoter is EF1 promoter, the first terminator is rb globulin polyadenylate terminator, the second promoter is PGK promoter, the second terminator is SV40 terminator, and the screening gene is Bla gene.
[0031]优选的,HESPer基因元件具有如SEQ ID NO:1所示的核苷酸系列: GACATT...
Embodiment 1
[0054] In the following examples, the enzymes used were purchased from NEB, and the H9 cell line was from Saibei Biotech. The sequence information of the CMV enhancer, EF1 promoter, HLA-E gene, Sr39tk gene, rb globulin polyadenylate terminator, loxP sequence, PGK promoter, Bla gene and SV40 terminator of the HESPer gene elements were obtained from the NCBI database, The synthesis was completed by Jinweizhi Company, and it was transferred to the pUC19 plasmid (see Figure 2a ). refer to figure 1 Schematic map of HESPer for genetic elements.
[0055] The present invention obtains pHS-AAVS1-HESPer plasmid ( Figure 2b ), the exemplary process is as follows:
[0056] 300ng of DNA of plasmid pHS-AAVS1 was mixed with 0.5 μl restriction endonuclease Bst BI, digested, bathed in water at 37°C for 1 hour, and the linearized vector fragment pHS-AAVS1 was obtained after recovery. Using the HESPer-pUC19 plasmid as a template, HESPer-P1 (SEQ ID NO: 2) and HESPer-P2 (SEQ ID NO: 3) were ...
Embodiment 2
[0062] The obtaining of SuperH cell line among the present invention is through the following steps:
[0063] Cells were harvested when the H9 cell line had grown to approximately 60% confluence. The plasmid pHS-AAVS1-HESPer and pAVVS1-gRNA were transferred into H9 together, and under the action of the drug Bla, a monoclonal cell line was screened. Primers AAVS1-p1 (SEQ ID NO: 4), AAVS1-p2 (SEQ ID NO: 5) and AAVS1-p3 (SEQ ID NO: 6) were used to identify the DNA sequence inserted into the HESPer gene element in the SuperH cell line, The genome of the SuperH cell line will obtain two bands using the above primers, and it will be verified by sequencing.
[0064] SEQ ID NO: 4
[0065] CTCCTGTGGATTCGGGTCACC
[0066]SEQ ID NO: 5
[0067] GAGCCAGTACACGACATCAC
[0068] SEQ ID NO: 6
[0069] GGCTCCATCGTAAGCAAAC
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More